FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

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
Services
Right arrow Email this article to a friend
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 Maurer, M.
Right arrow Articles by von Stebut, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maurer, M.
Right arrow Articles by von Stebut, E.
(The FASEB Journal. 2006;20:2460-2467.)
© 2006 FASEB

Skin mast cells control T cell-dependent host defense in Leishmania major infections

Marcus Maurer*,{dagger}, Susanna Lopez Kostka*, Frank Siebenhaar*,{dagger}, Katharina Moelle*, Martin Metz*, Jürgen Knop* and Esther von Stebut*,1

* Department of Dermatology, University Hospital Mainz, Mainz, Germany; and

{dagger} Department of Dermatology and Allergy, Allergie-Centrum-Charité, Charité-Universitätsmedizin Berlin, Berlin, Germany

1Correspondence: Department of Dermatology, Johannes Gutenberg-University, Langenbeckstrasse 1, D-55131 Mainz, Germany. E-mail: vonstebu{at}mail.uni-mainz.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells (MCs) initiate protective immunity against bacteria. Here we demonstrate that MCs also contribute to the control of parasitic skin infections by Leishmania major. L. major-infected MC-deficient KitW/KitW-v mice developed markedly larger skin lesions than did normal Kit+/+ mice (>2-fold), and cutaneous reconstitution with MCs resulted in normalization of lesion development. KitW/KitW-v lesions contained significantly more parasites, and infections resulted in enhanced spreading of parasites to the spleens as compared to controls. In addition, recruitment of proinflammatory neutrophils, macrophages, and dendritic cells (DCs) in infected mice was MC dependent. In the absence of MCs, reduced numbers of lesional DCs were associated with decreased production of Th1-promoting interleukin (IL)-12. Antigen-specific T cell priming was delayed in KitW/KitW-v mice and cytokine responses were skewed towards Th2. Notably, local skin MC reconstitution at sites of infection was sufficient for the induction of systemic protection. Thus, MC-mediated control of L. major infections is not limited to the induction of local inflammation. Instead, MCs contribute significantly to local DC recruitment, which mediates protective immunity. These findings extend the view of MCs as salient sentinels of innate immunity to complex host defense reactions against intracellular pathogens.—Maurer, M., Lopez Kostka, S., Siebenhaar, F., Moelle, K., Metz, M., Knop, J., von Stebut, E. Skin mast cells control T cell-dependent host defense in Leishmania major infections.


Key Words: leishmaniasis • Th1/Th2 • dendritic cell • cytokine release


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MAST CELLS (MCS) are key effector cells in the induction of inflammation and are crucial to the pathogenesis of inflammatory disorders, including allergic and autoimmune diseases (1) . In addition, MCs provide protection from the pathology caused by bacterial pathogens at least in part by inducing inflammatory responses (2) . For example, MC-dependent innate immune responses reduce the morbidity and mortality associated with acute septic peritonitis, where bacteria released from the gut into the peritoneal cavity are cleared by neutrophils recruited by activated MCs (3 , 4) .

MCs have also been suggested to contribute to protective immunity against nonbacterial pathogens. This notion is supported by several independent lines of evidence: 1) parasitic intestinal infections are known to result in MC hyperplasia, MC activation, and MC release of host defense-related mediators (1 , 5 6 7 8 9) ; 2) parasite-specific IgE contributes to protective immune responses against intestinal parasites, presumably by activating local MC populations (5) , and 3) mucosal MCs reportedly contribute to the expulsion of various intestinal parasites (5 6 7 8 9) . However, few studies have attempted to provide direct evidence for MC-dependent protective effects in settings of parasite infections of the skin (10 11 12) . Thus, the role of MCs as players in antiparasitic cutaneous immunity remains to be characterized in detail.

Leishmaniasis, a vector-borne infectious disease caused by protozoan parasites of the genus Leishmania, is endemic in tropical and subtropical regions around the world. Currently, ~12 million patients worldwide are believed to suffer from leishmaniasis and some 500,000 new cases and 59,000 deaths are reported each year. We hypothesized that MCs are involved in the immune responses raised against Leishmania major (L. major), a parasite strain that induces cutaneous leishmaniasis (CL). This hypothesis is based on findings from numerous studies regarding the role of MCs in intestinal parasite infections in patients and animal models (9) as well as on the following observations. 1) Large numbers of MCs are found in the skin, predominantly in the superficial dermis, where L. major is encountered after the bite of infected sand flies (13 , 14) . MCs are markedly more numerous at skin regions that are often infected by L. major and other cutaneous parasites (e.g., face, hands, and feet) (14) . 2) MCs are critically involved in the induction of innate immune responses against bacteria; previous studies have determined that MCs are also involved in the regulation of immunity against various Leishmania sp. (10 , 11) . Specifically, L. major has been described to activate MCs to induce the release of proinflammatory mediators, and to be phagocytosed by MCs (10 , 12 , 15) . 3) Finally, we recently showed that skin MCs are required for the recruitment of macrophages (M{Phi}) during cutaneous granuloma formation, a hallmark feature of parasite-induced inflammatory responses (16) .

In murine experimental L. major infection as well as in human CL, successful control of infection is crucially dependent on two subsequent events: granulomatous inflammatory responses that contribute to parasite containment at sites of L. major inoculation, and subsequent Th1-dominated systemic antigen-specific immune responses that control and eliminate the parasite (17) . Hypothesizing that MCs contribute to both of these processes, we subjected genetically MC-deficient KitW/KitW-v mice to L. major skin infections and found CL to be markedly pronounced in the absence of MCs. We also prove for the first time that MCs critically control CL, and we show that MC-dependent protection is not limited to effects during the initial local innate immune response after infection. Notably, MCs turn out to be crucial for recruiting effector cells of innate and adaptive immunity to sites of L. major infection and for inducing systemic protective dendritic cell (DC) -dependent adaptive immune responses that ultimately control L. major infection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
C57BL/6 mice, genetically MC-deficient WBB6F1-KitW/KitW-v (KitW/KitW-v) mice, and congenic wild-type (WT) WBB6F1-Kit+/+ (Kit+/+) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were kept in community cages at the Animal Care Facilities at the University of Mainz. All animal care and experimentation were conducted in accordance with current federal, state, and institutional guidelines.

Leishmania major infection and determination of lesion size
Metacyclic promastigotes of L. major clone VI (MHOM/interleukin/80/Friedlin) were prepared as described previously (18) . Isolated parasites were opsonized with 5% normal mouse serum. Levels of lipopolysaccharide (LPS) in parasite stock preparations were below the limit of detection. Mice were infected with 2 x 105 infectious stage metacyclic promastigotes by injection of ~10 µl of PBS containing parasites into the ventral dermis of the ears. Lesion size was measured weekly in 3 dimensions (ear thickness, width, and length of granuloma) using a caliper and calculated (in mm3) as ellipsoids [(a/2x b/2x c/2) x 4/3 x {pi}]. Parasites in lesional tissue were enumerated at the indicated times (≥3 animals/group) using a limiting dilution assay as described (19) .

Histomorphometric analysis of mast cell degranulation
Metacyclic promastigotes of L. major (2x105 or 103) or PBS were injected intradermally (in 20 µl) into ventral sides of ears, and ear skin was processed for histochemistry 6 h later. Plastic-embedded 1 µm sections were stained with alkaline Giemsa and MC activation was assessed by quantitative histomorphometry at 400x. MCs were classified as "extensively degranulated" (>50% of cytoplasmatic granules exhibiting staining alterations, fusion, and/or exteriorization), "moderately degranulated" (10–50% of granules affected), or "not degranulated" (<10% granules affected) (20) . Sections were scored by a blinded observer.

Reconstitution of mast cell-deficient KitW/KitW-v mice
The MC deficiency of KitW/KitW-v mice was repaired by local injection of bone marrow-derived MCs (BMCMCs) into the ears. Bone marrow cells were obtained from normal Kit+/+ mice and cultured in vitro for ~4 wk in IL-3-containing media until MCs represented >95% of the total cells according to staining by Giemsa. BMCMCs (106 in 50 µl 0.9% NaCl) were injected intradermally and mice were used for experiments, together with sex- and age-matched MC-deficient KitW/KitW-v and Kit+/+ mice, 4 wk after adoptive transfer (16 , 21) . Reconstitution of cutaneous MC populations was confirmed by histomorphometric analyses of paraffin-embedded, Giemsa-stained sections of injected skin.

Flow cytometry and antibodies
Ears of MC-deficient KitW/KitW-v, normal Kit+/+, and MC-reconstituted KitW/KitW-v + MCs mice were obtained at specified time points after Leishmania major infection. Inflammatory cells were isolated from ear skin as described (22) . In brief, ears were split and placed dermis down on media containing 2 mg/ml liberase (Boehringer Ingelheim, Pearl River, NY, USA). After 2 h, cells were dissociated mechanically, counted, and the frequency of leukocytes was assessed using flow cytometry. Cells were stained for surface Ag expression as described previously (18) . Paraformaldehyde (1% in PBS) -fixed cells were analyzed using a FACScan® flow cytometer equipped with CellQuest® software (Becton Dickinson, Mountain View, CA, USA). The following antibodies were used for this study: anti-CD16/CD32 (2.4G2), IgG2a{kappa} (R2a), anti-I-Ad/I-Ed (2G9), anti-CD11c/CD18 (HL3), anti-CD45RB (B220, 16A), anti-CD4 (L3T4), and anti-CD8 (Ly2) were purchased from BD PharMingen (San Diego, CA, USA). Anti-7/4, and anti-F4/80 (CI:A3–1) were obtained from Caltag Laboratories (Burlingame, CA, USA).

Quantification of antigen-specific priming and cytokine release
The frequency of daughter cells of proliferating antigen-reactive compared with nonproliferating LN T cells was estimated using flow cytometry (23 24 25) . Six weeks postinfection, LN cells were harvested and 5 x 106 cells/ml were labeled with 1 µM CFSE (Molecular Probes, Eugene, OR, USA). LN cells were subsequently plated at 1 x 106/200 µl media in a 96-well U-bottom plates and left untreated or stimulated with SEB (10 µg/ml, Sigma, St. Louis, MO, USA) or soluble Leishmania antigen (SLA) (19) . After 5 days, proliferation was determined using flow cytometry. T cells were selected for analysis using mAbs against CD4 (L3T4, RM4–5), CD8 (Ly2, 53–6.7), or isotype control monoclonal antibody (mAb) (all from PharMingen). For each mouse, the percentage of Leishmania-reactive cells compared with nonproliferating cells was calculated.

To assess cytokine release of T cells, LN cells were harvested and plated at 106 cells/200 µl. Cells were stimulated with SLA for 48 h and antigen-specific release of IFN-{gamma}, IL-4, and IL-10 was determined by ELISA (BD PharMingen, San Diego, CA, USA).

Determination of IL-12 release
Inflammatory cells were isolated from infected ears of MC-deficient KitW/KitW-v and normal Kit+/+ mice at the time points indicated. Cells from two ears of each mouse were resuspended in PBS supplemented with protease inhibitor cocktail (Roche, Nutley, NJ, USA). Cell suspensions were homogenized for 1–2 min using a pellet pestle and centrifuged at 4°C for 15 min (30,000 g). IL-12p40 content was determined in supernatants of homogenates of inflammatory ear cells by ELISA (BD PharMingen).

Statistical analysis
All data were tested for statistical significance using the unpaired 2-tailed Student’s t test (lesion volumes, Fig. 1 ), ANOVA test for repeated measurements (lesion volumes; see Fig. 3 ), Wilcoxon signed rank test (cytokine release), or X2 test (MC degranulation in situ) and are expressed as means ±SEM.


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		Figure 1. Increased lesion volumes and parasite loads in L. major-infected mast cell-deficient KitW/KitW-v mice. Mast cell-deficient KitW/KitW-v and normal Kit+/+ mice were infected with 2 x 105 (A, upper panel; B, C) or physiological low doses (1000, A, lower panel) of L. major metacyclic promastigotes. In some experiments, locally MC-reconstituted KitW/KitW-v mice were used for infections (C). Lesion development was monitored 3-dimensionally over the course of 3 months and calculated as ellipsoid (A, C). Data are expressed as mean ± SEM (n≥5; ***P≤0.005, *P≤0.05). In wk 3, lesional and systemic parasite loads were estimated at different time points in infected MC-deficient KitW/KitW-v and normal Kit+/+ mice (B) using limiting dilution assays (n≥5 mice per group). Dots represent numbers of parasites per organ; bars show the arithmetic mean of all mice/group. Data are expressed as mean ± SEM (n≥3; ***P≤0.002, **P≤0.005, *P≤0.05). One of ≥2 experiments with similar results is shown.


Figure 3
View larger version (22K):
[in this window]
[in a new window]

  		Figure 3. Increased recruitment of MHC class II+ cells into L. major lesions of MC-deficient mice. The inflammatory responses in KitW/KitW-v, Kit+/+, and KitW/KitW-v + BMCMC ears infected with 2 x 105 L. major were characterized by flow cytometry at respective time points. Data are expressed as mean ± SEM (n≥3/group; ***P≤0.002, **P≤0.005, *P≤0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
L. major-infected, mast cell-deficient mice exhibit increased lesion volumes and impaired parasite containment
To determine the functional significance of MCs in the control of L. major skin infections, genetically MC-deficient KitW/KitW-v mice and normal Kit+/+ mice were subjected to intradermal injections of standard inocula (2x105, Fig. 1A , upper panel) or physiological low doses (103, Fig. 1A , lower panel) of metacyclic promastigotes of L. major. Skin lesion development was followed in 3 dimensions for ≥ 3 months. KitW/KitW-v mice infected with L. major developed markedly larger skin lesions compared with normal Kit+/+ mice. In high-dose infections in the absence of MCs, lesion volumes were significantly larger in wk 1 to 12 postinfection. Most dramatic differences were found during wk 3 to 6 postinfection, with up to ~2-fold increased lesion volumes compared with WT controls (44±4 vs. 20±2 mm3 at wk 5, n≥5, P≤0.002). Similar differences between MC-competent and MC-deficient mice were observed using physiological low-dose infections mimicking natural transmission of the parasite by the bite of the sand fly. Increased lesion sizes in KitW/KitW-v mice were paralleled by increased lesional parasite loads (Fig. 1B , left panel). For instance, the ears of MC-deficient mice contained significantly greater numbers of parasites, reaching a peak load of 108 parasites/ear at wk 4 (wild-type Kit+/+ mice: 2x105 parasites/ear).

Impaired granuloma formation is generally associated with increased pathogen spreading into other organs. After inoculation of 2 x 105 parasites into the ear skin of KitW/KitW-v mice, we analyzed the numbers of parasites present in the spleen at different time points. L. major infections in KitW/KitW-v mice resulted in enhanced systemic disease (i.e., spreading of parasites to the spleens), whereas Kit+/+ mice exhibited only occasional and transient systemic infections (Fig. 1B , right panel). Dramatic differences were found between wk 1 and 3 postinfection, reaching up to ~20-fold increased parasite burdens in MC-deficient mice compared with Kit+/+ controls (wk 3, n≥5, P≤0.05).

To test whether impaired granuloma formation as well as increased lesion volumes and parasite spreading in MC-deficient KitW/KitW-v mice are due to the lack of MCs in these mice, we infected KitW/KitW-v mice that had been repopulated with skin MCs by local adoptive transfer of Kit+/+ BMCMCs 4 wk prior to infections. MC reconstitution resulted in the complete normalization of lesion volumes (Fig. 1C ). In parallel, lesional parasite loads were lower in MC-reconstituted KitW/KitW-v mice than in KitW/KitW-v mice (0.9±0.5x104 vs. 2.7±1.7x104 parasites/lesion at wk 3) and comparable to those of WT Kit+/+ mice (0.9±0.4x104 parasites/lesion at wk 3, n≥8). These data suggest that MCs are required for the development of efficient inflammatory responses to L. major infection that prevent parasite spreading and systemic disease.

Cutaneous L. major infection leads to MC activation
Several earlier studies have documented extensive MC degranulation at sites of L. major infection (10 , 12 , 26) . In addition, in vitro studies using a human MC line (HMC-1) or murine BMCMCs demonstrated activation of MCs after coincubation with L. major promastigotes (15 , 27) . We also detected pronounced MC degranulation as assessed by histomorphometric analyses of infected skin sites obtained 6 h after L. major inoculation in vivo (Fig. 2 A). In addition, measurements of skin thickness after the intradermal injection of C57BL/6 mice with 2 x 105 metacyclic L. major promastigotes demonstrated pronounced and transient inflammatory responses, which were comparable in their extent and kinetics to maximal MC-dependent, immediate hypersensitivity-type skin responses (Fig. 2B ) (28) .


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		Figure 2. Intradermal injection of L. major leads to activation of MCs. 2 x 105 metacyclic promastigotes of L. major or PBS were injected intradermally (in 20 µl) into ventral sides of C57BL/6 ears and ear skin was processed for histochemistry 6 h later (A). Plastic-embedded 1 µm sections were stained with alkaline Giemsa and MC activation was assessed by quantitative histomorphometry at 400x. MC degranulation was classified as "extensive" (>50% of cytoplasmatic granules exhibiting staining alterations, fusion, and/or exteriorization), "moderate" (10–50% of granules affected), or "none" (<10% granules affected). Data derived from 2 independent experiments (n≥9, ***P≤0.005, **P≤0.01, *P≤0.05 compared with vehicle-injected control). B) Ear swelling after skin infection was measured for 6 h using a caliper. (n≥3, ***P≤0.005).

Activated skin mast cells modulate local inflammatory responses to cutaneous L. major infection
To determine whether MCs also contribute to long-term inflammatory events in CL, we studied recruitment of proinflammatory cells induced by the injection of both MC-containing and MC-deficient mice with 2 x 105 metacyclic L. major promastigotes. KitW/KitW-v mice exhibited significantly reduced inflammatory responses as assessed by total numbers of locally infiltrating cells unless they had been locally reconstituted with adoptively transferred MCs (Fig. 3 ). In addition, 7/4+ neutrophils as well as F4/80+ M{Phi} were significantly reduced in MC-deficient mice. These findings are in line with our earlier observation that cytokine release from activated MCs (i.e., TNF{alpha}) regulates M{Phi} recruitment via modulation of neutrophil influx to sites of granulomatous inflammation (16) . The total numbers of antigen-presenting (MHC class II+) cells were also found to be significantly decreased in MC-deficient mice 3 wk after injection of L. major. Specifically, CD11c+ DC levels were dramatically reduced (P≤0.002). Finally, the numbers of infiltrating CD8+ T cells and of B220+ B cells were reduced in KitW/KitW-v mice compared with wild-type mice. In contrast, CD4+ lymphocyte recruitment to sites of infection was normal in MC-deficient mice.

To assess whether or not the lack of MCs is responsible for the impaired recruitment of inflammatory cells to L. major lesions observed in KitW/KitW-v mice, we adoptively transferred the skin of these mice with BMCMCs 4 wk prior to infection. In 3-wk-old lesions of MC-reconstituted and L. major-infected KitW/KitW-v mice, the numbers of recruited neutrophils, DCs, B cells, and T cells were normalized to at least control levels (Fig. 3 , bar graphs). Thus, impaired granuloma formation in KitW/KitW-v mice secondary to L. major infection is accompanied by a failure to effectively induce and coordinate the influx of M{Phi} and DCs into developing lesions.

Impaired antigen-specific T cell priming in KitW/KitW-v mice
Earlier studies from several groups, including our own, have shown that CD11c+-infected DCs are critically involved in the initiation of protective Th1-dependent immunity in mice (18 , 19 , 29 , 30) . Consequently, impaired MC-dependent local recruitment of DCs could be responsible for a more progressive disease in KitW/KitW-v mice. Alternatively, a study has identified several mechanisms by which MCs can influence T cell proliferation and cytokine production directly (31) . Thus, we assessed Leishmania-specific T cell priming over the course of the initial 3 wk postinfection with 2 x 105 parasites [i.e., CFSE-labeled lymph node (LN) cells were restimulated with soluble Leishmania lysate for 4–5 days and the percentage of responding CD4+ and CD8+ T cells was determined] (Fig. 4 ). Decreased numbers of Leishmania-reactive CD4 and CD8 cells were found in MC-deficient KitW/KitW-v mice compared with control Kit+/+ mice during the first weeks postinfection. These differences were most pronounced in the CD8 compartment, with an almost absent MHC class I-dependent T cell priming in KitW/KitW-v mice until day 13 (0.3±0.1 vs. 7.2±3% proliferating cells in Kit+/+ mice at day 8, n≥9, P≤0.005). No quantitative difference in the capacity of naive CD4+ and CD8+ T cells to respond to superantigen (staphylococcal enterotoxin B, SEB) was found between KitW/KitW-v and Kit+/+ mice (Fig. 4) .


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		Figure 4. MC-deficient KitW/KitW-v mice show diminished antigen-specific T cell priming. KitW/KitW-v and Kit+/+ mice were infected with 2 x 105 metacyclic promastigotes of L. major. LN cells were harvested at different time points, labeled with CFSE, and subcultured with soluble Leishmania lysate (SLA) or SEB for 4 days. T cells were pregated using staining for CD4 or CD8. For each mouse, the relative number of Leishmania-reactive cells (in % of total CD4+ or CD8+ T cells) was calculated in SLA-stimulated compared with untreated control cultures (mean±SEM, n=9, **P≤0.005, *P≤0.05).

Because L. major-specific, IFN{gamma}-producing Th1 and Tc1 cells are necessary to control Leishmania infections (13 , 17) , we next analyzed the quality of T cell priming in MC-deficient KitW/KitW-v mice. To determine whether MCs are involved in Th1 development during CL, antigen-dependent cytokine production was assessed in lesion-draining LN cells 1, 3, and 5 wk after infection with L. major. As early as 1 wk after infection, the IFN-{gamma}/IL-4 ratio in KitW/KitW-v mice was skewed toward Th2 compared with Kit+/+ mice (i.e., antigen-specific IFN-{gamma} release by lymph node cells obtained from infected KitW/KitW-v mice was reduced by >50%, whereas Th2 cytokine levels of IL-4 and IL-10 were increased by ~100% compared with Kit+/+ mice) (Fig. 5 A). Differences in cytokine levels between KitW/KitW-v and control mice were most pronounced at early time points (wk 1 and 3).


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		Figure 5. Impaired Th1/Tc1 education in MC-deficient mice as a result of decreased IL-12 production. A) LN cells of MC-deficient KitW/KitW-v and normal Kit+/+ mice were harvested at wk 1, 3, and 5 postinfection and plated at 106 cells/200 µl. Cells were stimulated with soluble Leishmania antigen (SLA) for 48 h and antigen-specific release of IFN-{gamma}, IL-4, and IL-10 was determined by ELISA. Data are expressed as mean ±SEM. Data shown are representative of 3 independent experiments with similar results (*P<0.05, ***P<0.002, n=4 mice/group). B) IL-12p40 content was measured in homogenates of lesional inflammatory cells isolated from L. major-infected KitW/KitW-v and Kit+/+ mice at different time points. IL-12p40 production was determined by ELISA (mean±SEM, n=9, *P≤0.05).

Efficient Th1 priming has been shown to result from IL-12 release by infected DCs (17) . Thus, we tested whether the impaired early Th1 education in MC-deficient KitW/KitW-v mice is the result of altered levels of IL-12. To this end, homogenates of inflammatory cells isolated from L. major-infected KitW/KitW-v or Kit+/+ mice were assayed for the presence of IL-12p40 at several time points by ELISA. As shown in Fig. 5B , we found significantly decreased levels of IL-12 in KitW/KitW-v mice as compared with control mice. Differences were most pronounced during wk 1 and 2 postinfection (P≤0.05). IL-12p70 protein was not detectable, which was to be expected because only a small proportion of IL-12p40 (factor 1000–10,000) released from infected DCs is released as part of the bioactive heterodimer (18) .

MC-dependent regulation of Leishmania major infection sites acts systemically on T cells
Our observations that the priming of T cells as well as their protective responses against Leishmania are impaired in the absence of MCs suggested that MCs regulate systemic adaptive immunity against L. major. To test this, KitW/KitW-v mice that had been reconstituted locally with MCs only in their left ears were simultaneously infected in both ears. These MC-reconstituted KitW/KitW-v mice exhibited not only normalized lesion volumes in the MC-reconstituted left ears but also in the MC-deficient right ears (Fig. 6 ), indicating that skin MC populations at sites of infection are sufficient and required for normal systemic, long-lasting protection against Leishmania.


Figure 6
View larger version (16K):
[in this window]
[in a new window]

  		Figure 6. Mast cell-dependent regulation of L. major infection sites acts systemically on T cells. Left ears of KitW/KitW-v mice were reconstituted with MC and mice were infected in both ears 4 wk after reconstitution. Lesion development in MC-reconstituted and MC-deficient ears was assessed independently in the same mouse. Data are representative of 3 independent experiments (n≥4) and expressed as mean ± SEM.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The control of infection in experimental murine cutaneous leishmaniasis is dependent on the development of antigen-specific, IFN{gamma}-producing T cell subsets (Th1 and Tc1) (17) . However, local inflammatory processes have also been shown to be important for successful generation of protective immunity (32) . In an earlier study we determined that local skin MCs contribute to early neutrophil and M{Phi} recruitment to sites of inflammation by means of TNF{alpha} release (16) . In this study, using an important human pathogen we demonstrate that MCs regulate both the local inflammatory response as well as resulting adaptive immune responses by using MC-deficient KitW/KitW-v mice. We found that L. major-induced lesion volumes and parasite loads were increased due to impaired granuloma formation and that DC-associated T cell priming and Th education toward Th1/Tc1 were decreased in the absence of MCs. Both effects proved to be MC dependent, as KitW/KitW-v mice that had been repaired for their skin MC deficiency by adoptive transfer before L. major infection showed regular granuloma formation, followed by normal adaptive immune responses and control of infection.

In the present study, MC-deficient KitW/KitW-v mice developed larger skin lesions after infection with L. major over a period of >2 months. This was true for experimental high-dose infections with 2 x 105 parasites, as well as when a more physiological infection model was used. In the latter model, L. major lesions are induced by intradermal inoculation of only 103 infectious-stage L. major metacyclic promastigotes, mimicking the bite of a sand fly.

Of note, an earlier study by Wershil et al. concluded that MCs augment rather than control lesion size during L. major skin infections (10) , which is explained by the different methods used in this and our study for measuring the size of CL lesions. L. major skin infections are controlled and contained by the formation of granulomas, which are ideally assessed in 3 dimensions (i.e., by measuring their volumes, as done in the present study). In contrast, 1-dimensional measurements of CL lesions (i.e., by quantifying skin thickness) as performed by us (data not shown) and Wershil et al. fail to detect that skin granuloma formation is impaired in L. major-infected KitW/KitW-v mice. In our study, higher lesion volumes in KitW/KitW-v mice were associated with increased lesional parasite loads, as expected. Wershil and co-workers also found higher parasite loads in MC-deficient mice infected with L. major at certain time points, although these differences were not statistically significant (10) . We also found more parasites in the spleens of infected MC-deficient mice, which may reflect the inability of KitW/KitW-v skin granulomas to limit the infection to the site of inoculation.

Earlier studies have convincingly demonstrated the activation of MCs by L. major both in vivo at the site of parasite inoculation (10 , 12 , 26) and in vitro (15 , 27) . In line with these observations, we found significantly more degranulated MCs in ear skin injected with L. major metacyclic promastigotes 6 h postinfection. In addition, measurements of skin ear thickness induced by intradermal injection of L. major demonstrated increased inflammatory responses. The kinetics of this response was similar to that of MC mediator release-dependent, immediate hypersensitivity-type skin responses (28) . These data confirmed that infection with L. major leads to MC activation and mediator release in vivo.

We recently found that recruitment of inflammatory cells, including neutrophils and M{Phi}, to the skin of MC-deficient KitW/KitW-v mice is impaired during the induction of foreign body granuloma formation (16) . Specifically, we found that MC-derived TNF{alpha} was responsible for neutrophil immigration and that release of MIP-1{alpha} from neutrophils induced subsequent M{Phi} influx. We now demonstrate a similar MC-dependent recruitment of inflammatory cells to the skin after injection of a physiologically relevant infectious agent such as L. major. In the absence of MCs we noted decreased immigration of neutrophils and M{Phi}. In contrast to a foreign body granuloma, cell recruitment after infection with L. major is not limited to these two cell types. The total numbers of recruited MHC class II+ cells as well as CD11c+ DCs were dramatically reduced in MC-deficient mice infected with L. major (Fig. 3) . DC recruitment was MC dependent, as shown in reconstitution experiments, which has not been demonstrated before.

Earlier studies have shown that infected M{Phi} and DCs contribute to MHC class II-dependent presentation of Leishmania antigens to CD4+ T cells (13 , 17 , 18 , 33) . Infection of M{Phi} soon after parasite inoculation appears to be a silent process and does not lead to M{Phi} activation (17 , 34 , 35) . In contrast, infection of DCs is reportedly associated with strong cell activation, up-regulation of MHC class I and II, and migration to draining lymph nodes (18) . In this study, we observed impaired MHC class II-dependent Leishmania-specific priming in KitW/KitW-v mice during the first week postinfection. This is most likely the result of decreased recruitment of inflammatory DCs (and M{Phi}) early after infection.

It was recently shown that besides CD4+ cells, CD8+ T cells also play an essential role for protective immunity against L. major (33) . Using KitW/KitW-v mice, we determined that MCs regulate MHC class I-dependent immune responses as well. First, lesions of MC-deficient mice contained fewer CD8+ T cells at early time points. Second, the percentage of Leishmania-primed CD8+ T cells was significantly lower in KitW/KitW-v than in Kit+/+ mice at early time points. Earlier studies have revealed that MHC class I-dependent antigen presentation in Leishmania infections is promoted by infected DCs only (and not by infected M{Phi}) (33) . Thus, our data suggest that impaired MHC class I-dependent CD8 priming in MC-deficient mice is the result of decreased recruitment of DCs to skin lesions of KitW/KitW-v mice.

In addition to defects in T cell priming, we also found qualitative differences in Th/Tc education in MC-deficient and -competent mice. The ratio between Th1 (IFN{gamma}) and Th2 cytokines (IL-4, IL-10) was significantly altered toward Th2 soon after infection of KitW/KitW-v mice with L. major. In parallel, we detected decreased levels of IL-12 in lesions of L. major-infected KitW/KitW-v mice compared with Kit+/+ mice. IL-12 is the most important/critical cytokine for inducing protective Th1 immunity in murine experimental leishmaniasis (17) . It is well known that L. major infected DCs, and not M{Phi}, release IL-12 (18 , 29 , 34 35 36) . Therefore, MC-dependent recruitment of DCs to sites of infection is likely to be responsible for the release of bioactive IL-12 in L. major infections. Thus, impaired Th1/Tc1 priming is observed in MC-deficient KitW/KitW-v mice.

Our data suggest that MCs strongly influence the development of protective T cell immunity against L. major. In support of this conclusion, we demonstrate that skin MC populations at sites of infection are required for normal systemic T cell responses against L. major: body sites in MC-deficient KitW/KitW-v mice distant to those reconstituted locally with MCs raised normal host defense responses after L. major infections. Thus, our data suggest that MC-mediated control of L. major infections is not limited to the induction of local inflammation. MC-dependent recruitment of proinflammatory cells to sites of L. major inoculation (e.g., DCs) also contributes to the development of protective, long-lasting memory responses against L. major.

In summary, MCs appear to provide protection against L. major infection by inducing and orchestrating local host defense reactions as well as systemic acquired immune responses. MCs have been shown to regulate T cell-dependent immune responses both directly (MHC class II expression, production of chemotactic factors, release of Th1- or Th2-cytokines) and indirectly (e.g., via regulation of DC migration, maturation and function) (37) . Our findings extend the recently introduced role of MCs as sentinels in host defense reactions against pathogens. Our results demonstrate that skin MC-dependent protection from the pathological consequences of parasitic infection involves their influence on both innate and adaptive immune responses.


   ACKNOWLEDGMENTS
 
We thank Drs. Robert Sabat, Kerstin Steinbrink, Cord Sunderkötter, Mark C. Udey, and Peter Walden for helpful discussions and Jodie Urcioli for critical reading of the manuscript. This work was supported in part by grants from the Jürgen-Manchot-Stiftung and the DFG to M.M. (SFB 548, SPP1110) and E.v.S. (SFB 490, SFB 548).

Received for publication February 18, 2006. Accepted for publication July 11, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Galli, S. J., Maurer, M., Lantz, C. S. (1999) Mast cells as sentinels of innate immunity. Curr. Opin. Immunol. 11,53-59[CrossRef][Medline]
  2. Henz, B. M., Maurer, M., Lippert, U., Worm, M., Babina, M. (2001) Mast cells as initiators of immunity and host defense. Exp. Dermatol. 10,1-10[CrossRef][Medline]
  3. Echtenacher, B., Mannel, D. N., Hultner, L. (1996) Critical protective role of mast cells in a model of acute septic peritonitis. Nature 381,75-77[CrossRef][Medline]
  4. Maurer, M., Echtenacher, B., Hultner, L., Kollias, G., Mannel, D. N., Langley, K. E., Galli, S. J. (1998) The c-kit ligand, stem cell factor, can enhance innate immunity through effects on mast cells. J. Exp. Med. 188,2343-2348[Abstract/Free Full Text]
  5. Li, E., Zhou, P., Petrin, Z., Singer, S. M. (2004) Mast cell-dependent control of Giardia lamblia infections in mice. Infect. Immun. 72,6642-6649[Abstract/Free Full Text]
  6. Artis, D., Villarino, A., Silverman, M., He, W., Thornton, E. M., Mu, S., Summer, S., Covey, T. M., Huang, E., Yoshida, H., et al (2004) The IL-27 receptor (WSX-1) is an inhibitor of innate and adaptive elements of type 2 immunity. J. Immunol. 173,5626-5634[Abstract/Free Full Text]
  7. Knight, P. A., Pemberton, A. D., Robertson, K. A., Roy, D. J., Wright, S. H., Miller, H. R. (2004) Expression profiling reveals novel innate and inflammatory responses in the jejunal epithelial compartment during infection with Trichinella spiralis. Infect. Immun. 72,6076-6086[Abstract/Free Full Text]
  8. Onah, D. N., Nawa, Y. (2004) Mucosal mast cell-derived chondroitin sulphate levels in and worm expulsion from FcRgamma-knockout mice following oral challenge with Strongyloides venezuelensis. J. Vet. Sci. 5,221-226[Medline]
  9. Finkelman, F. D., Shea-Donohue, T., Morris, S. C., Gildea, L., Strait, R., Madden, K. B., Schopf, L., Urban, J. F., Jr (2004) Interleukin-4- and interleukin-13-mediated host protection against intestinal nematode parasites. Immunol. Rev. 201,139-155[CrossRef][Medline]
  10. Wershil, B. K., Theodos, C. M., Galli, S. J., Titus, R. G. (1994) Mast cells augment lesion size and persistence during experimental Leishmania major infection in the mouse. J. Immunol. 152,4563-4571[Abstract]
  11. Katakura, K., Saito, S., Hamada, A., Matsuda, H., Watanabe, N. (1993) Cutaneous leishmaniasis in mast cell-deficient W/Wv mice. Infect. Immun. 61,2242-2244[Abstract/Free Full Text]
  12. Saha, B., Tonkal, A. M., Croft, S., Roy, S. (2004) Mast cells at the host-pathogen interface: host-protection versus immune evasion in leishmaniasis. Clin. Exp. Immunol. 137,19-23[CrossRef][Medline]
  13. Reiner, S. L., Locksley, R. M. (1995) The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13,151-177[CrossRef][Medline]
  14. Weber, A., Knop, J., Maurer, M. (2003) Pattern analysis of human cutaneous mast cell populations by total body surface mapping. Br. J. Dermatol. 148,224-228[CrossRef][Medline]
  15. Bidri, M., Vouldoukis, I., Mossalayi, M. D., Debre, P., Guillosson, J. J., Mazier, D., Arock, M. (1997) Evidence for direct interaction between mast cells and Leishmania parasites. Parasite Immunol. 19,475-483[CrossRef][Medline]
  16. Von Stebut, E., Metz, M., Milon, G., Knop, J., Maurer, M. (2003) Early macrophage influx to sites of cutaneous granuloma formation is dependent on MIP-1alpha/beta released from neutrophils recruited by mast cell-derived TNFalpha. Blood 101,210-215[Abstract/Free Full Text]
  17. Sacks, D., Noben-Trauth, N. (2002) The immunology of susceptibility and resistance to Leishmania major in mice. Nat. Rev. Immunol. 2,845-858[CrossRef][Medline]
  18. Von Stebut, E., Belkaid, Y., Jakob, T., Sacks, D. L., Udey, M. C. (1998) Uptake of Leishmania major amastigotes results in activation and interleukin 12 release from murine skin-derived dendritic cells: implications for the initiation of anti-Leishmania immunity. J. Exp. Med. 188,1547-1552[Abstract/Free Full Text]
  19. Von Stebut, E., Ehrchen, J. M., Belkaid, Y., Lopez Kostka, S., Molle, K., Knop, J., Sunderkotter, C., Udey, M. C. (2003) Interleukin 1alpha promotes Th1 differentiation and inhibits disease progression in Leishmania major-susceptible BALB/c mice. J. Exp. Med. 198,191-199[Abstract/Free Full Text]
  20. Wershil, B. K., Murakami, T., Galli, S. J. (1988) Mast cell-dependent amplification of an immunologically nonspecific inflammatory response. Mast cells are required for the full expression of cutaneous acute inflammation induced by phorbol 12-myristate 13-acetate. J. Immunol. 140,2356-2360[Abstract]
  21. Galli, S. J., Kitamura, Y. (1987) Animal model of human disease. Genetically mast cell-deficient W/Wv and Sl/Sld mice: their value for the analysis of the roles of mast cells in biological responses in vivo. Am. J. Pathol. 127,191-198[Medline]
  22. Belkaid, Y., Mendez, S., Lira, R., Kadambi, N., Milon, G., Sacks, D. L. (2000) A natural model of Leishmania major infection reveals a prolonged "silent" phase of parasite amplification in the skin before the onset of lesion formation and immunity. J. Immunol. 165,969-977[Abstract/Free Full Text]
  23. Chen, J. C., Chang, M. L., Muench, M. O. (2003) A kinetic study of the murine mixed lymphocyte reaction by 5,6-carboxyfluorescein diacetate succinimidyl ester labeling. J. Immunol. Methods. 279,123-133[CrossRef][Medline]
  24. Adkins, B., Williamson, T., Guevara, P., Bu, Y. (2003) Murine neonatal lymphocytes show rapid early cell cycle entry and cell division. J. Immunol. 170,4548-4556[Abstract/Free Full Text]
  25. Ritter, U., Meissner, A., Scheidig, C., Korner, H. (2004) CD8 alpha- and Langerin-negative dendritic cells, but not Langerhans cells, act as principal antigen-presenting cells in leishmaniasis. Eur. J. Immunol. 34,1542-1550[CrossRef][Medline]
  26. Grimaldi, G., Jr, Soares, M. J., Moriearty, P. L. (1984) Tissue eosinophilia and Leishmania mexicana mexicana eosinophil interactions in murine cutaneous leishmaniasis. Parasite Immunol. 6,397-408[Medline]
  27. De Oliveira, M. P., Lima, M. C., Calheiros, A. S., Martins, M. A., Antas, P. R., De Luca, P. M., Pirmez, C. (2005) Leishmania (Viannia) braziliensis: human mast cell line activation induced by logarithmic and stationary promastigote derived-lysates. Exp. Parasitol. 109,72-79[CrossRef][Medline]
  28. Wershil, B. K., Mekori, Y. A., Murakami, T., Galli, S. J. (1987) 125I-fibrin deposition in IgE-dependent immediate hypersensitivity reactions in mouse skin. Demonstration of the role of mast cells using genetically mast cell-deficient mice locally reconstituted with cultured mast cells. J. Immunol. 139,2605-2614[Abstract]
  29. Flohe, S. B., Bauer, C., Flohe, S., Moll, H. (1998) Antigen-pulsed epidermal Langerhans cells protect susceptible mice from infection with the intracellular parasite Leishmania major. Eur. J. Immunol. 28,3800-3811[CrossRef][Medline]
  30. Ahuja, S. S., Reddick, R. L., Sato, N., Montalbo, E., Kostecki, V., Zhao, W., Dolan, M. J., Melby, P. C., Ahuja, S. K. (1999) Dendritic cell (DC)-based anti-infective strategies: DCs engineered to secrete IL-12 are a potent vaccine in a murine model of an intracellular infection. J. Immunol. 163,3890-3897[Abstract/Free Full Text]
  31. Nakae, S., Suto, H., Kakurai, M., Sedgwick, J. D., Tsai, M., Galli, S. J. (2005) Mast cells enhance T cell activation: importance of mast cell-derived TNF. Proc. Natl. Acad. Sci. U. S. A. 102,6467-6472[Abstract/Free Full Text]
  32. 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]
  33. Belkaid, Y., von Stebut, E., Mendez, S., Lira, R., Caler, E., Bertholet, S., Udey, M. C., Sacks, D. L. (2002) CD8+ T cells are required for primary immunity in C57BL/6 mice following low-dose, intradermal challenge with Leishmania major. J. Immunol. 168,3992-4000[Abstract/Free Full Text]
  34. Carrera, L., Gazzinelli, R. T., Badolato, R., Hieny, S., Muller, W., Kuhn, R., Sacks, D. L. (1996) Leishmania promastigotes selectively inhibit interleukin 12 induction in bone marrow-derived macrophages from susceptible and resistant mice. J. Exp. Med. 183,515-526[Abstract/Free Full Text]
  35. Belkaid, Y., Butcher, B., Sacks, D. L. (1998) Analysis of cytokine production by inflammatory mouse macrophages at the single-cell level: selective impairment of IL-12 induction in Leishmania-infected cells. Eur. J. Immunol. 28,1389-1400[CrossRef][Medline]
  36. Von Stebut, E., Belkaid, Y., Nguyen, B. V., Cushing, M., Sacks, D. L., Udey, M. C. (2000) Leishmania major-infected murine Langerhans cell-like dendritic cells from susceptible mice release IL-12 after infection and vaccinate against experimental cutaneous leishmaniasis. Eur. J. Immunol. 30,3498-3506[CrossRef][Medline]
  37. Galli, S. J., Nakae, S., Tsai, M. (2005) Mast cells in the development of adaptive immune responses. Nat. Immunol. 6,135-142[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. J. Jenkins, G. Perona-Wright, and A. S. MacDonald
Full Development of Th2 Immunity Requires Both Innate and Adaptive Sources of CD154
J. Immunol., June 15, 2008; 180(12): 8083 - 8092.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
T. Kambayashi, J. D. Baranski, R. G. Baker, T. Zou, E. J. Allenspach, J. E. Shoag, P. L. Jones, and G. A. Koretzky
Indirect involvement of allergen-captured mast cells in antigen presentation
Blood, February 1, 2008; 111(3): 1489 - 1496.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
F. Siebenhaar, W. Syska, K. Weller, M. Magerl, T. Zuberbier, M. Metz, and M. Maurer
Control of Pseudomonas aeruginosa Skin Infections in Mice Is Mast Cell-Dependent
Am. J. Pathol., June 1, 2007; 170(6): 1910 - 1916.
[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
Services
Right arrow Email this article to a friend
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 Maurer, M.
Right arrow Articles by von Stebut, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maurer, M.
Right arrow Articles by von Stebut, E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS