| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
Immunex Corporation, Seattle, WA 98101
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
response. Our results link the required
CD40/CD40L interactions for healing with DC-derived IL-12p70 production
and provide a mechanism to explain the genesis of a protective T
cell-mediated response in the face of local immune evasion within the
macrophage at the site of Leishmania
delivery. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Skin-dwelling APC, specifically epidermal Langerhans cells (LC)3 and dermal dendritic cells (DC), are actively involved in the surveillance of their environment (2, 3). These cells use phagocytosis, macropinocytosis, micropinocytosis, and receptor-mediated endocytosis (4) to sample their environment and capture many types of exogenous molecules and microorganisms. Data conflict on the ability of DC to take up Leishmania parasites, in particular on whether or not the promastigote can enter DC. LC and DC within cutaneous lesions are reportedly parasitized by Leishmania major (Lm) in vivo in both human cutaneous leishmaniasis (5, 6, 7) and experimental murine cutaneous leishmaniasis (8, 9). In humans with cutaneous leishmaniasis, parasites (or their breakdown products) have been found in and around LC in biopsy specimens. Additionally, murine LC have been shown to take up Leishmania parasites and migrate to local draining lymph nodes for T cell activation (10). Another in vitro study reported that although primate and human epidermal LC do not take up stationary-phase leishmania promastigotes, dermal macrophages do under similar conditions (11). More recently, it has been shown that murine fetal skin-derived DC (FSDDC) are preferentially infected by Lm amastigotes compared with metacyclic promastigotes (12), whereas mouse CD11c+ splenic DC take up Lm promastigotes (13).
In this study, immature cytokine-derived myeloid DC (driven by GM-CSF and IL-4) cultured from human peripheral blood were used to assess early events occurring at the site of delivery of Leishmania and to examine their ability to activate/drive T cells along a type 1 pathway and initiate immunity. We also evaluated the ability of the infectious stage of Lm (metacyclic promastigotes) to be taken up by human mononuclear cell-derived DC and detailed the events pursuant to that process. The data show that as a consequence of the interaction between Lm metacyclic promastigotes and DC, IL-12p70 can be produced in a CD40L-dependent manner. Furthermore, these parasite-harboring DC can process Lm metacyclic promastigote-derived molecules and present their processed products to human T cells from the blood of patients with local cutaneous leishmaniasis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
DC were generated from buffy coats obtained from normal
volunteer blood donors at the National Institutes of Health (NIH)
Clinical Center, Department of Transfusion Medicine. The four patients
in this study with localized cutaneous leishmaniasis were seen at the
NIH Clinical Center for diagnosis and treatment. They had no previous
therapy and had the onset of ulceration 
2–3 mo before presentation.
The time of presentation and ulcerated appearance of the lesions were
typical for cutaneous leishmaniasis. All lesional biopsies were culture
positive for Leishmania. Subsequent species confirmation of
each isolate was performed, and all were typed as Lm. Informed consent
was obtained from each of the patients.
Parasites
Lm clone V1 (MHOM/IL/80/Friedlin) was cultured in 199 medium supplemented with 20% heat-inactivated FCS (HyClone Laboratories, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 40 mM HEPES, 0.1 mM adenine (in 50 mM HEPES), 5 µg/ml hemin (in 50% triethanoamine), and 1 µg/ml 6-biotin (M199/S). Infective-stage metacyclic promastigotes of Lm were isolated from stationary culture (4–5 days old) by negative selection using peanut agglutinin (Vector Laboratories, Burlingame, CA) as previously described (14). Amastigotes were isolated from footpad lesion homogenates of BALB/c mice by differential centrifugation and were cryopreserved. Parasites tested below the detection limits for endotoxin (<0.1 endotoxin units/ml, LAL assay; BioWhittaker, Walkersville, MD) and tested negative for mycoplasma (PCR detection method; American Type Culture Collection, Manassas, VA). Before infection of DC, parasites were opsonized with 5% normal human serum by incubation at 37°C for 30 min.
Reagents, cytokines, and Abs
Recombinant human GM-CSF (rhGM-CSF) (1 x
107 U/mg) and rhIL-4 (2 x
106 U/mg) were obtained from PeproTech (Rocky
Hill, NJ). rhIFN- (500 U/ml) was obtained from PharMingen (San
Diego, CA). mAbs to the following molecules were obtained commercially:
CD14 (63D3) and glycophorin (10F7) (American Type Culture Collection);
mouse IgG1 FITC and mouse IgG1 PE (Dako, Glostrup, Denmark); mouse
IgG2b FITC, mouse IgG2b PE, CD56-PE (B159), CD3-FITC, and CD86-PE
(B70/B7-2) (PharMingen); CD56-PE (Leu19), CD14-PE (Leu-M3), HLA-DR-PE,
and CD80-PE (BB1/B7) (Becton Dickinson, San Jose, CA); CD1a-PE (Caltag,
San Francisco, CA); CD1a-FITC (OKT6; Ortho Diagnostic Systems, Raritan,
NJ); CD54-FITC (ICAM-1; Amac, Westbrook, ME); and CD19-FITC
(Immunotech, Marseilles, France). mAbs to the following molecules were
obtained as generous gifts: CD40 ligand trimer (CD40LT) and
anti-CD40LT M90 (Immunex, Seattle, WA); CD19 (FMC63; H. Zola,
Woman’s & Children’s Hospital, Adelaide, Australia); CD16 (3G8; J.
Jaffe, Hahnemann University, Philadelphia, PA); CD3 (OKT3) (Orthoclone;
Dr. Fuccello, Ortho-Biotech, Raritan, NJ); and
anti-lipophosphoglycan-FITC (LPG WIC-79.3; D. L. Sacks,
National Institute of Allergy and Infectious Diseases).
Staphylococcus aureus Cowan 1 bacteria (SAC) (1/10,000
dilution, Sansorbin) was obtained from Calbiochem (La Jolla, CA).
Generation and characterization of immature DC
Cells were prepared exactly as previously reported (15), using the IL-4 and GM-CSF protocol for generation of large numbers of immature DC from peripheral blood-derived monocytes (16). All cells were cultured in RPMI 1640 (BioWhittaker) supplemented with 2 mM L-glutamine, 10 mM HEPES, 50 mM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FCS (Life Technologies, Gaithersburg, MD). All tissue culture media were found to be free of endotoxin (to the detection limits of the LAL assay). Briefly, PBMCs were isolated from normal blood bank donors or patients with cutaneous leishmaniasis using Ficoll-diatrizoate (Lymphocyte Separation Medium; ICN Biochemicals, Aurora, OH) centrifugation separation. The mononuclear cells were suspended at 5 x 106 cells/ml and incubated for 2 h at 37°C under 5% CO2. The nonadherent cells were removed with gentle rinsing. The full volume of culture medium was replaced, and rhGM-CSF and rhIL-4 (1000 U/ml) were added. These cells were then cultured for 7 days, adding half-volume fresh media and full-volume fresh cytokines every other day. The DC were purified using two rounds of negative selection to remove lineage marker positive cells (anti-CD3 at 1:150; anti-CD14, anti-CD16, and anti-CD56 at 1:200; and anti-CD19 at 1:1000) with either magnetic cell sorting technique, magnetic cell separation system microbeads (Miltenyi Biotec, Sunnyvale, CA), or Dynal (Lake Success, NY) goat anti-mouse IgG beads as follows. The cells were suspended in ice-cold HBSS/5% FCS at 107 cells/ml with negative-selection Ab and were rocked at 4°C for 30 min, washed twice in HBSS/5% FCS, and suspended at 2 x 107 cells/ml. The magnetic beads were added at 4:1 bead-to-cell ratio and incubated for 15 min (magnetic cell separation system) or for 30 min with rocking (Dynal) at 4°C. The cells were then placed onto a prepared column or into the magnetic field twice to enhance purity. The DC used in this study were routinely CD1a+ (>90%), HLA-DR+ (>95%), CD86+ (20%), CD40+ (>95%), and negative for CD3, CD14, CD19, and CD56 by flow cytometry (FACSCalibur; Becton Dickinson).
Monocytes were isolated using a human monocyte isolation kit (Miltenyi Biotec) according to the manufacturer’s guidelines after enriching for monocytes by cold aggregation of PBMCs (17) and were routinely >95% CD14+ using flow cytometry.
Coculture of DC/monocytes and Lm parasites
DC or monocytes were suspended at 106 cells/ml, and 0.3 ml of this cell suspension was placed into individual wells of 48-well plates (CoStar, Cambridge, MA). Opsonized parasites (ratio 4:1 per cell) were added to DC or monocyte cultures for 16–18 h at 37°C under 5% CO2. At the time of coculture, Polymixin B (10 µg/ml; Sigma, St. Louis, MO) was added to the culture media for use in all wells. In experiments using CD40LT, the trimer was used at 1 µg/ml. Cells and supernatants were pooled from triplicate wells to minimize well-to-well variability. Cell-free supernatants were collected after centrifugation and stored at -70°C until ELISA-based cytokine measurements were performed. Collected cells were washed four times with PBS and aliquoted for further analysis. Brefeldin A (10 µg/ml; Sigma) was added during the final 6 h of culture when performing intracellular cytokine staining.
Cytospin preparations
Cytospins were prepared using a Shandon II cytocentrifuge (Shandon Lipshaw, Pittsburgh, PA) set at 500 rpm for 5 min. After Wright-Giemsa staining, light microscopic analysis of the cells was performed in a blinded fashion, counting a minimum of 250 cells per slide to estimate both the infection rate and parasite number per cell. The cytospins were run in parallel with flow cytometry using anti-lipophosphoglycan (LPG)-FITC labeling.
Flow cytometry
Cells were incubated with normal mouse sera to block nonspecific binding before staining. Staining was done in 100 µl volumes with titrated amounts of directly conjugated mAb for optimal labeling. A minimum of 10,000 cells were acquired on a FACSCalibur and were analyzed using CellQuest Software (Becton Dickinson). For some experiments, the parasites were prelabeled with carboxyfluorescein diacetate (Molecular Probes, Eugene, OR) before coculture with DC, and then the DC were subsequently stained for surface molecules.
For intracellular cytokine staining, cells were fixed in 2% paraformaldehyde and washed with ice-cold PBS/0.1% BSA. The cells were blocked for 1 h or overnight in PBS/5% nonfat milk/0.1% saponin (Fischer Scientific, Pittsburgh, PA). The anti-IL-12-PE or mIgG1-PE isotype control Ab (PharMingen) was diluted (5 µg/ml) in this same blocking buffer and added to the cells in a final volume of 50 µl. Cells were stained for 30 min, washed twice with PBS/0.1% saponin, and then washed once with PBS alone. A minimum of 10,000 cells were analyzed as above. For specificity control, excess rhIL-12p70 (1 µg) was coincubated with the Ab in the staining process.
ELISA-based cytokine detection assays
All cytokines were detected as secreted protein products in
culture supernatants using cytokine-specific ELISA assays. Paired Abs
were from R&D Systems (Minneapolis, MN; IL-1ß, TNF-
, IFN-, and
IL-6) and PharMingen (IL-12p70 and IL-10). Assays were performed
according to the manufacturer’s guidelines. The lower limits of
detection for the assays were as follows: for IL-12p70, 31 pg/ml; for
IFN-, IL-1ß, IL-6, and IL-10, 39 pg/ml; and for TNF-, 48
pg/ml.
Autologous T cell proliferation assays
DC were generated from Lm-infected patients. Some of these DC were infected overnight with Lm metacyclic promastigotes as described above and were washed extensively to remove any external parasites. Other DC were simply plated out in parallel but were not exposed to Lm. All DC were irradiated with 2000 rad before use in assays. Autologous T cells (responder cells) were isolated (Lympho-Quik T; One Lambda, Canoga Park, CA) and cocultured at 105 cells/well with varying concentrations of DC. The plates were incubated at 37°C under 5% CO2 for 5 days, pulsed with 1 µCi/well of [3H]thymidine (DuPont, Boston, MA) for 16 h, and harvested onto filter mats for scintillation counting.
Statistical analysis
Statistical analysis was performed using the Wilcoxon signed-rank test, and the correlation coefficient was determined using simple linear regression. All statistics were performed using StatView 5 (SAS Institute, Cary, NC).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To compare infection of DC with the two developmental stages that
either initiate or maintain parasitism in the vertebrate host, DC were
cultured with metacyclic promastigotes or amastigotes of Lm opsonized
with human serum. After overnight incubation, supernatants were
collected and cells harvested for further analysis. The infection rate
with amastigotes was typically 1.5-fold greater than it was with the
metacyclic promastigotes (average, 58 vs 38%), but the number of
intracellular parasites was similar (3 parasites/cell). Because we
were interested in the initial events associated with the "natural
route" of infection (i.e., the delivery of metacyclics into the
dermis through the epidermis), the subsequent studies focused on DC
infected with metacyclic promastigotes (Fig. 1
). Most infected cells contain more than
one parasite, and many of these intracellular forms are located
peripherally within the cell. The inset in Fig. 1 shows a metacyclic
promastigote entering a DC. Select DC + Lm metacyclic promastigote
cocultures (n = 3) were kept for 72 h and examined
at 24 h intervals for parasite growth. Over the 3-day period,
there was an approximate doubling of both the number of DC infected
(from a mean of 38% at day 1 to 70% at day 3) and the number of
intracellular parasites (from a mean of 3.3 organisms per cell at day 1
to 6.2 organisms per cell at day 3) within each infected cell.
|
To assess the effect of intracellular Lm on DC surface molecule
expression, we labeled parasite-harboring DC (hereafter referred to as
infected DC) using a mAb, anti-LPG FITC, that is specific for the
LPG-rich surface of metacyclic-stage parasites. We compared unexposed
DC (media control) with parasite-exposed, uninfected DC
(LPG-) and infected DC
(LPG+). The distinction of infected and
uninfected cells by flow cytometry was consistent with the microscopic
analysis of stained cells (Fig. 2A). Fig. 2B shows
a representative dot plot of an ungated DC population stained with both
LPG-FITC and HLA-DR PE after overnight exposure to Lm metacyclic
promastigotes. In Fig. 2C, unexposed DC (upper
panels) and exposed populations (LPG-,
uninfected, middle panels; or LPC+, infected,
lower panels) with mean fluorescence intensities are shown.
Compared with cells cultured in media alone, Lm-infected DC showed a
2.8-fold increase in HLA-DR surface expression, a 2.2-fold increase in
CD86, and a 1.5-fold increase in CD40 in this representative experiment
(minimum of four experiments). Similar results were obtained when the
parasite was fluorescein-labeled before infection using
carboxyfluorescein diacetate (n = 6). In comparison to
cells cultured in media alone, infected DC showed significant
(p < 0.05) average increases in surface
expression of HLA-DR (3-fold), CD86 (2.4-fold), and CD40 (1.9-fold).
Similar increases in these surface costimulatory molecules were
observed when DC were infected with Lm amastigotes (data not
shown).
|
Although DC could be infected with Lm and, as a result,
up-regulate surface accessory molecules, there were no cytokines
detected in the overnight culture supernatants (IL-12p70, IL-1ß,
IL-6, IL-10, and TNF-; data not shown). Because CD40L is a known
inducer of cytokines (including IL-12p70) in DC, we examined the
effects of sequential additions of parasites and CD40LT on cytokine
production (Fig. 3). To mimic the events
that likely occur after entry of Lm into skin DC, including activation,
maturation, and migration to the draining lymph node where they could
receive the CD40L signal from primed T cells, CD40LT was added 8–10
h after the parasites. As shown in Fig. 3A, no IL-12p70 was
detected with media alone or with parasites alone (Lm). Although
IL-12p70 production could be seen with CD40LT alone (242 pg/ml ±
88), there was a 10-fold increase in IL-12p70 when both Lm plus CD40LT
were added (2108 pg/ml ± 489). In all donors tested
(n = 13), there was a significant increase in IL-12p70
production using both stimuli sequentially when compared with using
either stimulus alone (CD40LT vs CD40LT + Lm, p =
0.008; Lm vs CD40LT + Lm, p = 0.008). Similar results
were obtained with other cytokines including TNF-, IL-6, IL-10, and
IL-1ß (data not shown). SAC, another potent inducer of IL-12p70, was
tested in conjunction with Lm metacyclic promastigotes to determine
whether there was any modulatory effect on IL-12p70 production by Lm.
In seven donors tested, with sequential exposure to metacyclic
parasites and then to SAC, there was no significant alteration in
IL-12p70 production (p = 0.12; Fig. 3B).
|
Using intracellular cytokine flow cytometry, IL-12 could be
colocalized to LPG+ DC (Fig. 4). In the absence of costimulation with
CD40LT, infected DC did not produce IL-12. With CD40LT alone, 10.7% of
cells stained for IL-12. When both Lm and CD40LT were added, 22% of
all Lm-infected DC stained positively for intracellular IL-12, and 76%
of all IL-12+ cells were Lm-infected cells. There
was an augmentation of IL-12 production based not only on the cell
frequency (1.5-fold increase) but also on the mean fluorescence
intensity (1.6-fold increase) when comparing CD40LT alone to Lm plus
CD40LT (n = 8). This synergistic effect paralleled that
seen in the secreted protein assays (not shown). The intracellular
detection of IL-12 was specific with near complete blockade in the
presence of exogenous rhIL-12p70.
|
To determine whether DC can process live Lm metacyclic
promastigotes and present the Lm-derived peptides (Ag) to sensitized T
cells, DC were generated from monocytes recovered from Lm-infected
patients; these DC were subsequently infected in vitro, and autologous
T cells were added (n = 4) (Fig. 5). As the number of infected DC added to
a fixed number of T cells was increased, there was an increase in
[3H]thymidine incorporation and IFN-
production compared with those T cells cultured with uninfected DC
(Fig. 5). The IFN- response was markedly inhibited by the addition
of an anti-CD40L Ab (Fig. 5B), an effect seen less
dramatically when [3H]thymidine incorporation
was used as the readout (Fig. 5A).
|
To test whether the effect of CD40LT was restricted to the
induction of IL-12 in DC or if it could induce IL-12 in other APC
exposed to Lm, we compared both DC and monocytes derived from the same
normal donors (n = 4) and exposed them to Lm metacyclic
promastigotes (Fig. 6). With monocytes,
there was no IL-12p70 detected in overnight supernatants using media
alone, Lm metacyclic promastigotes alone, CD40LT alone, or the
combination of Lm metacyclic promastigotes plus CD40LT. In marked
contrast, DC from the same donors showed increased IL-12p70 in the
presence of CD40LT alone and a significant 3-fold increase with the
combination of CD40LT plus Lm under the same conditions. There were
comparable infection rates in the monocytes and DC (64 vs 48%,
respectively). The monocytes were capable of producing IL-12p70 when
stimulated with IFN- plus SAC, demonstrating that their capacity to
produce IL-12p70 in response to a different stimulus was intact.
Consistent with previous findings (18, 19, 40), when
IFN- plus SAC was used to stimulate Lm-infected monocytes (MO), no
IL-12 was detected (in two of two donors tested: MO + IFN- + SAC
936, 378 pg/ml; and Lm-MO + IFN- + SAC < 39, <39 pg/ml of
IL-12p70, respectively).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this study we used human DC for three reasons: 1) their proximity to the site of delivery of the parasite by the sandflies; 2) their potential ability to generate a protective IL-12-driven cell-mediated immune response, in part as a result of their ability to migrate to local draining lymph nodes; and 3) murine data suggesting alternative (nonmacrophage) cellular sources of IL-12. That DC may indeed be that IL-12 source is suggested by the well-described inhibition of IL-12 production within parasite-harboring macrophages in murine models (18, 19, 20), the observed in vivo production of IL-12 in response to Leishmania infection (21), and the absolute requirement for IL-12 to mediate healing even in a low-inocula model (22).
Using light microscopy and flow cytometry, we determined that metacyclic promastigotes were taken up by DC, the stage of the parasite that initiates parasitism in the vertebrate host. Our results are in agreement with murine studies using both Lm amastigotes and metacyclic promastigotes to infect FSDDC (12), splenic DC (13), or epidermal LC (9), although the present study suggests (for human DC at least) that infection with metacyclic promastigotes occurs more efficiently than has heretofore been appreciated. In contrast to these results, others have reported the inability to infect human or primate LC with stationary-phase promastigotes (11). Possible explanations for this discrepancy could be differences in the subpopulations of DC used, culture conditions, human serum opsonization, and the variability in the number of infectious-stage parasites (metacyclic promastigotes) when using stationary-phase vs purified metacyclic parasites.
The presence of Lm within DC resulted in an increase in HLA-DR, CD40,
and CD86 surface expression, findings that suggest the DC were
undergoing maturation (4). The up-regulation of these
molecules was a direct consequence of infection, as indicated by
two-color flow analysis of DC surface molecules and parasite Ag (Fig. 2
). Using murine FSDDC infected with amastigotes, a similar increase in
MHC expression, CD40, and CD86 has been described, although when
metacyclic promastigotes were used, no surface changes were appreciated
in bulk FSDDC populations (12). A similar absence of
surface changes with exposure to the metacyclic stage has been recently
reported using mouse spleen-derived DC (13). These
conflicting data point to the importance of specifically identifying
those cells infected with the parasite (Figs. 1 and 2) because when the
frequencies of infected cells are small (12), significant
changes are difficult to identify. Our phenotypic findings are
supported by data using recombinant Leishmania brasiliensis
Ag (LeIF)-treated human myeloid DC in which the expression of B7-1,
CD40, and CD54 is increased (23).
Interestingly, we found that although the DC were clearly harboring Lm
and had surface changes associated with activation/maturation, there
were no cytokines produced (in particular IL-12) in the absence of
appropriate costimulation. Because it has been reported that ligation
of CD40 on the surface of DC is a key step in the induction of IL-12
production (24, 25), the role of the CD40/CD40L
interaction in IL-12 production by Lm-infected DC was examined. Our
study demonstrates a second signal requirement for IL-12p70 production
by Lm-infected human DC and shows that CD40L meets this requirement.
Further, using two-color analysis of intracellular cytokines and
parasite LPG, the production of IL-12 by Lm metacyclic
promastigote-infected DC in the presence of CD40L could be easily
documented (Fig. 4). Therefore, we suggest that the DC is rendered
capable of producing IL-12, once it is infected, only after receiving
an additional signal provided by CD40L. DC infected with amastigotes
(rather than metacyclic promastigotes) had similar requirements for
IL-12 production. Indeed, in a number of parallel studies
(n = 4) comparing promastigotes to amastigotes,
IL-12p70 production after amastigote infection was also
CD40L-dependent. As with the promastigotes, DC surface CD86, CD40, and
HLA-DR was increased after amastigote infection (data not shown). The
idea that both stages have similar costimulatory requirements for IL-12
production may enhance the biological relevance of this finding because
as the infection develops, the amastigote stage (the stage that
maintains infection) is clearly more abundant and likely more
accessible to peripheral DC.
The critical role for CD40/CD40L interactions has been demonstrated in
the protective immune response in experimental leishmaniasis by using
mice with the targeted disruption of CD40/CD40L (26, 27, 28).
This protective response is related to IL-12 production (27, 29, 30, 31, 32). Recently, several murine studies detected DC-derived
IL-12p40 in response to Leishmania (Leishmania
donovani (33) and Lm (12, 13)), a
response that was not clearly CD40L-dependent; however, IL-12p70
production was detectable in only one of these studies. IL-12p70
secretion by human DC in response to a recombinant
Leishmania Ag or Toxoplasma infection has been
detected and, in each case, was in part CD40L-dependent (23, 34). The requirement for CD40L for IL-12p70 production in
Lm-infected human DC may be beneficial from the parasite standpoint in
that Leishmania is simply less likely to induce IL-12 unless
there is sufficient and appropriate costimulation, a process that would
most likely occur after DC migration to the draining lymph node. Other
important costimulatory molecules have been studied in the setting of
early leishmaniasis (35), although they were not directly
studied for this article. However, the consistent increase in
expression of CD86 (B7-2) and HLA-DR (MHC class II) molecules in DC
harboring Lm parasites likely reflects their involvement in the immune
response. In fact, the incomplete inhibition of the proliferative
response (compared with cytokine production; see Fig. 5) by CD40/CD40L
blockade speaks to additional, and more dominant, costimulatory
requirements for proliferative responses.
The particular type of DC used in this study is a less mature DC, more
equipped for capturing extracellular molecules/microorganisms for
further processing than for immunostimulation (4, 36).
This exact cell type has been shown to migrate to the T cell area of
draining LN when generated ex vivo and reinfused s.c. in a primate
model (37). Very clearly these DC, once infected, can
reactivate primed T cells. Further, the Lm-infected DC can reveal the
presence of IFN--secreting T cells, a process that is inhibited by
blocking CD40L with an antagonizing monoclonal Ab. This inhibition of
IFN- production suggests a role for CD40L-driven IL-12p70
in recall immune responses to Lm metacyclic promastigotes.
Our data also suggest that Lm metacyclic promastigotes may exert
different effects once inside DC vs monocytes. We show that although
CD40L costimulation of Lm-infected DC induces large amounts IL-12, no
IL-12 was induced in monocytes from the same donors under identical
stimulation (Fig. 6). Indeed, specific inhibition of mononuclear
phagocyte cytokine production has been well described in the setting of
infection with Leishmania (in particular with metacyclic
promastigotes) (18, 19, 20, 38, 39, 40). Alternatively, the
observed differential effects of CD40L on DC vs monocytes may reflect
the need for higher levels of costimulation or for additional stimuli
to induce IL-12 in monocytes (41). Unlike what has been
seen in murine macrophages (18, 19, 20) and in bulk human PBMC
culture systems (40), Lm infection of human DC does not
significantly inhibit SAC-driven IL-12 production (Fig. 3B).
Our data fit into the existing body of literature and may help to
explain an apparent discrepancy regarding in vitro and in vivo
findings. Although in vitro studies show that macrophage IL-12
production is clearly prevented by Leishmania metacyclic
infection at both the mRNA and protein levels (18, 19, 39), infection of mice with Lm metacyclic promastigotes in vivo
drives IL-12 production and a protective IFN- response
(21) and is an absolute requirement for healing. Our in
vitro results suggest that Lm-infected DC are capable of producing
IL-12p70, given an appropriate second signal delivered through
CD40.
The events described in this paper suggest that although the draining
lymph node may be a site for initiation of IL-12-driven Th1 immune
responses through Lm-infected DC, there may be concomitant IL-12
inhibition within the macrophage compartment operating at the site of
the lesion (i.e., the site of parasite delivery and development).
Additionally, overproduction of TNF- and TGF-ß at the site of the
lesion may contribute to the local pathology, inhibit healing
(42, 43), and provide a temporary refuge for parasite
growth.
Lending support to the importance of CD40-mediated induction of IL-12 is a recent study in a murine vaccine model in which a strong IL-12-dependent adjuvant effect of CD40LT DNA was seen when coadministered with soluble Leishmania Ag (44). This requirement for CD40L costimulation may give the parasite a temporal advantage, allowing peripheral amplification while the infected DC migrate to the draining lymph node, where they presumably engage in immunologic cross-talk with T cells (and perhaps NK cells). This DC-T cell interaction results in up-regulation of CD40L on clustered T cells and CD40 triggering on the DC (24).
The idea that the type of APC involved influences the nature of the ensuing immune response is one that has been previously addressed by others (45, 46) and is supportive of our data. In a BALB/c murine model, GM-CSF-treated bone-marrow-derived macrophages pulsed with Lm Ag have been shown to protect against a lethal challenge of L. major. This protection was contingent upon three factors: 1) specialized "activation" of the macrophages with GM-CSF (as M-CSF-treated and -untreated macrophages failed to protect), 2) IL-12 production, and 3) T cell contact. These GM-CSF-activated macrophages may resemble DC, as murine myeloid DC can be generated from GM-CSF-stimulated bone marrow-derived macrophages (47). Similarly, it seems likely that the requirement for contact with sensitized T cells involves CD40/CD40L interactions. Additionally, others have directly compared murine epidermal LC and macrophages that were pulsed with leishmania Ag and found that only the LC (not macrophages) could protect against challenge (48).
This in vitro model of parasite/human DC interactions can be readily
adapted to any leishmania species. We have used it to delineate
appropriate costimulatory requirements for IL-12p70 production, a
prerequisite for inducing naive T lymphocytes to produce IFN-.
Ongoing efforts in our laboratory suggest that other leishmanial
species (e.g., Leishmania tropica) differ from Lm once taken
up by human DC (unpublished data). In the case of Lm, usually a
self-healing cutaneous infection (49, 50), production of
IL-12p70 by DC costimulated in the lymph node may be the force that
drives a protective and healing immune response.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: HIV Research Program, Henry M. Jackson Foundation, 13 Taft Court, Suite 200, Rockville, MD 20850.
3 Abbreviations used in this paper: LC, Langerhans cell; DC, dendritic cell; Lm, Leishmania major; FSDDC, fetal skin-derived DC; rhGM-CSF, recombinant human GM-CSF; CD40LT, CD40 ligand trimer; SAC, Staphylococcus aureus Cowan 1 bacteria; LPG, lipophosphoglycan.
Received for publication December 21, 1999. Accepted for publication March 20, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J. Exp. Med. 179:1109. production and expression of B7-1 in human monocyte-derived antigen-presenting cells. Eur. J. Immunol. 27:2634.[Medline]
This article has been cited by other articles:
|
|
 |
|
  A. E. Field, S. Wagage, S. M. Conrad, and D. M. Mosser Reduced Pathology following Infection with Transgenic Leishmania major Expressing Murine CD40 Ligand Infect. Immun., June 1, 2007; 75(6): 3140 - 3149. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Hewitson, G. R. Jenkins, P. A. Hamblin, and A. P. Mountford CD40/CD154 Interactions Are Required for the Optimal Maturation of Skin-Derived APCs and the Induction of Helminth-Specific IFN-{gamma} but Not IL-4. J. Immunol., September 1, 2006; 177(5): 3209 - 3217. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Korner, V. Fuss, J. Steigerwald, and H. Moll Biogenesis of Leishmania major-Harboring Vacuoles in Murine Dendritic Cells Infect. Immun., February 1, 2006; 74(2): 1305 - 1312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yasumi, K. Katamura, T. Yoshioka, T.-a. Meguro, R. Nishikomori, T. Heike, M. Inobe, S. Kon, T. Uede, and T. Nakahata Differential Requirement for the CD40-CD154 Costimulatory Pathway during Th Cell Priming by CD8{alpha}+ and CD8{alpha}− Murine Dendritic Cell Subsets J. Immunol., April 15, 2004; 172(8): 4826 - 4833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Prina, S. Z. Abdi, M. Lebastard, E. Perret, N. Winter, and J.-C. Antoine Dendritic cells as host cells for the promastigote and amastigote stages of Leishmania amazonensis: the role of opsonins in parasite uptake and dendritic cell maturation J. Cell Sci., January 15, 2004; 117(2): 315 - 325. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Colmenares, A. L. Corbi, S. J. Turco, and L. Rivas The Dendritic Cell Receptor DC-SIGN Discriminates among Species and Life Cycle Forms of Leishmania J. Immunol., January 15, 2004; 172(2): 1186 - 1190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. M. Padigel, N. Kim, Y. Choi, and J. P. Farrell TRANCE-RANK Costimulation is Required for IL-12 Production and the Initiation of a Th1-Type Response to Leishmania major Infection in CD40L-Deficient Mice J. Immunol., November 15, 2003; 171(10): 5437 - 5441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Chaussabel, R. T. Semnani, M. A. McDowell, D. Sacks, A. Sher, and T. B. Nutman Unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites Blood, July 15, 2003; 102(2): 672 - 681. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D. Straw, A. S. MacDonald, E. Y. Denkers, and E. J. Pearce CD154 Plays a Central Role in Regulating Dendritic Cell Activation During Infections That Induce Th1 or Th2 Responses J. Immunol., January 15, 2003; 170(2): 727 - 734. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. McDowell, M. Marovich, R. Lira, M. Braun, and D. Sacks Leishmania Priming of Human Dendritic Cells for CD40 Ligand-Induced Interleukin-12p70 Secretion Is Strain and Species Dependent Infect. Immun., August 1, 2002; 70(8): 3994 - 4001. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Companjen, L. I. v. d. Wel, L. Boon, E. P. Prens, and J. D. Laman CD40 ligation-induced cytokine production in human skin explants is partly mediated via IL-1 Int. Immunol., June 1, 2002; 14(6): 669 - 676. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Belkaid, E. Von Stebut, S. Mendez, R. Lira, E. Caler, S. Bertholet, M. C. Udey, and D. Sacks CD8+ T Cells Are Required for Primary Immunity in C57BL/6 Mice Following Low-Dose, Intradermal Challenge with Leishmania major J. Immunol., April 15, 2002; 168(8): 3992 - 4000. [Abstract] [Full Text] [PDF]
|
|
|
|
) or absence (
) of Ab to CD40L (A).
B, IFN-