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*
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
Unité d’Immunophysiologie Cellulaire, Institut Pasteur, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in the epidermal compartment, and an expansion
of T cells capable of producing IFN- in the draining lymph node.
Parasite growth was not enhanced over the first 4.5 wk in
anti-CD4-treated mice, SCID mice, or C57BL/6 mice deficient in
IL-12p40, IFN-, CD40 ligand, or inducible NO synthase. These mice
all failed to ultimately control infection in the site, but in some
cases (anti-CD4 treated, IL-12p40-/-,
CD40 ligand-/-, and SCID) high dermal
parasite loads were associated with little or no pathology. These
results extend to a natural infection model a role for Th1 cells in
both acquired resistance and lesion formation, and document the
remarkable avoidance of this response during a prolonged phase of
parasite amplification in the skin. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which mediates
NO-dependent killing by infected macrophages (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). The extent to which the high dose, s.c. inoculation model accurately reflects dermal leishmaniasis that develops following natural transmission of L. major by sand fly bite has not been carefully addressed. We previously established a model of natural infection that focused on the exacerbating effects of vector saliva on lesion development in a dermal site (11). In the present studies the coinoculation of sand fly saliva has been avoided because of recent findings that the salivary gland sonicate used to mimic natural transmission elicits powerful effects that are not seen following actual transmission by bite (S. Kamhawi et al., manuscript in preparation). The model retains two main features of natural transmission: low dose (100 metacyclic promastigotes) and inoculation into a dermal site (the mouse ear dermis). The model has been used to re-examine the basic relationship among parasite growth, lesion formation, and immunity. The studies have revealed two distinct phases in the pathogenesis of cutaneous leishmaniasis that have not been previously discerned: a remarkably silent phase, lasting 4–5 wk, favoring the amplification of parasites in the dermis without the formation of a lesion, followed by the development of a cutaneous lesion that is coincident with the killing of the parasite in the site.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) mice were purchased from the Division of Cancer
Treatment, National Cancer Institute (Frederick, MD). C57BL/6 SCID mice
and knockout (KO) mice, deficient in IL-12 p40, iNOS, or IFN-, all
generated on a C57BL/6 genetic background, were purchased from Taconic
Farms (Germantown, NY). C57BL/6
CD40L-/- mice were
provided by Dr. R. Seder. The IL-4-deficient mice were generated from
an C57BL/6 embryonic stem cell line and were provided by Dr. N.
Noben-Trauth. All mice were maintained in the National Institute of
Allergy and Infectious Diseases animal care facility under
pathogen-free conditions.
Parasite preparation and intradermal inoculation
L. major clone V1 (MHOM/IL/80/Friedlin) was cultured in medium 199 with 20% HI-FCS (HyClone, 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 mg/ml hemin (in 50% triethanolamine), and 1 mg/ml 6-biotin (M199/S). Infective stage metacyclic promastigotes of L. major were isolated from stationary culture (5–6 days old) by negative selection using peanut agglutinin (Vector, Burlingame, CA). One hundred metacyclic promastigotes were inoculated intradermally into the ear dermis using a 27.5-gauge needle in a volume of 10 µl. The evolution of the lesion was monitored by measuring the diameter of the induration of the ear lesion with a direct reading Vernier caliper (Thomas, Swedesboro, NJ).
Analysis of the parasite-loaded dermis
The leukocytes in the inflammatory ear dermis were recovered as previously described (12). Briefly, at different time points after intradermal inoculation of L. major, the ears were collected, and the ventral and dorsal dermal sheets were separated and immediately processed; the two leaflets were transferred, dermal side down, on culture medium into a six-well plate (nontissue culture treated, Greiner Labortechnik, Osterreich, Austria) for 6 h. Each well contained 4 ml of RPMI 1640, NaHCO3 with 25 mM HEPES, 10% HI-FCS, and penicillin/streptomycin. The cell populations spontaneously emigrating from the dermis were recovered, filtered through a 70-µm pore size nylon cell strainer (Becton Dickinson, Mountain View, CA) and washed twice before being cytospun and stained with Diff-Quik (DADE, Miami, FL) or stained for flow cytometric analysis. For histologic studies, the complete ears were fixed in 10% phosphate-buffered formalin and embedded in paraffin, and 5-µm sections were stained using hematoxylin and eosin.
Immunolabeling and flow cytometry
Before staining, the cells emigrating from the ear dermis were
incubated with an anti-FcIII/II (PharMingen, San Diego, CA)
receptor and 10% normal mouse serum in PBS containing 0.1% BSA and
0.01% NaN3. The double or triple stainings were
performed using directly conjugated Abs incubated simultaneously. Each
incubation was conducted for 30 min on ice. The dermal cells were
identified by characteristic size (forward scatter) and granulosity
(side scatter) combined with two-color analysis, as previously
described (11). The dendritic cells (DC) were identified
as large cells, MHC class II (25-9-17, PharMingen) bright, and F4/80
(A3-1, Caltag, Burlingame, CA) and NLDC 145 (DEC-205) positive. The
mononuclear phagocytes were identified as F4/80 positive, and MHC class
II low or negative. The neutrophils were identified as small cells,
Ly-6G bright (RB6-8C5, PharMingen), and negative for F4/80 (or MHC
class II); the eosinophils were identified by their granulosity
associated with F4/80 staining and the absence of MHC class II
staining. The lymphocytes were identified by their small size, along
with their TCR ß-chain (H57-597) and CD3 (145-2C11, PharMingen)
expression. The isotype controls used were rat IgG2b (A95-1,
PharMingen) and rat IgG2a (R35-95, PharMingen). For each sample, 10,000
cells were analyzed. The data were collected and analyzed using
CellQuest software and a FACSCalibur flow cytometer (Becton Dickinson,
San Jose, CA).
Estimation of parasite load
The two sheets of the infected ears were separated; deposited dermal side down in DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mg/ml collagenase A (Sigma, St. Louis, MO); and incubated for 2 h at 37°C. The sheets were cut into small pieces and homogenized using a Teflon-coated microtissue grinder in a microfuge tube containing 100 µl of M199/S. The tissue homogenates were filtered using a 70-µm pore size cell strainer (Falcon Products, St. Louis, MO) and serially diluted in a 96-well flat-bottom microtiter plate containing biphasic medium, prepared using 50 µl of NNN medium containing 30% of defibrinated rabbit blood and overlaid with 50 µl of M199/S. The number of viable parasites in each tissue was determined from the highest dilution at which promastigotes could be grown after 7 days of incubation at 26°C .
Lymph node cell preparation and culture
The retromaxillar draining lymph nodes were recovered and
mechanically dissociated using a pellet pestle. Cell viability was
assessed by trypan blue exclusion. For measurement of in vitro cytokine
production, single-cell suspensions of lymph nodes were pooled from
five animals, diluted to 4 x 106 cells/ml,
and dispensed into 96-well plates without Ag or with soluble L.
major Ag (SLA; 25 µg/ml) or Con A (2 µg/ml) in 100 µl of
complete RPMI containing 2-ME. Cultures were incubated at 37°C in 5%
CO2. Supernatant fluids were harvested at 24 and
48 h and were assayed for IL-4 and IFN-, respectively, by ELISA
as previously described (11).
Epidermal cell preparation
The epidermal cells were recovered as previously described
(11) with modifications; briefly, the ventral and dorsal
ear sheets were separated and transferred dermal side down on
DMEM-penicillin/streptomycin with 0.5% trypsin (U.S. Biochemical,
Cleveland, OH) for the dorsal face and 1% trypsin for the ventral
face. The sheets were incubated for 30 min at 37°C. The epidermis was
separated from the dermis and deposited on a 70-µm nylon cell
strainer (Becton Dickinson, Mountain View, CA), which was placed in a
petri dish containing DMEM plus 20% FBS, 100 U/ml penicillin, 100
µg/ml streptomycin, and 0.05% DNase (Sigma). The filter was gently
shaken for 2 min, and the cells passing through the filter were washed,
plated, and incubated 6 h at 37 C in the presence of 10 µg/ml
brefeldin A (Sigma). The cells were collected, fixed with 4%
paraformaldehyde in PBS for 5 min, and washed with cold PBS containing
0.1% BSA before staining. The epidermal cells were incubated with
anti-FcR Ab in PBS containing 10% normal mouse serum, 0.1%
saponin (Fisher, Pittsburgh, PA), 0.1% BSA, 1 mM CaCl, 1 mM
MgSO4, and 40 mM HEPES (permeabilization buffer).
After washing, the cells were incubated with the cytokine-reactive Abs
or their corresponding isotype control. All Abs were obtained from
PharMingen and were directly conjugated to PE: anti-IL-12p40
(C15.6), anti IFN- (XMG1.2), anti-IL-4 (BVD4-1D11), and isotype
control, rat IgG1 or rat IgG2b. After staining, the cells were fixed
again with 1% paraformaldehyde and washed. For each sample, 40,000
cells were analyzed.
CD4+ cell depletion or IL-12 treatment of mice
For CD4 depletion the mice were inoculated weekly i.p. with 1 mg of anti-CD4 (GK1.5) or a rat isotype control (GL113). The IL-12 p40 KO mice were given 0.5 µg of recombinant murine IL-12 (provided by Genetics Institute, Cambridge, MA) i.p. during the first 3 days of infection.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One hundred L. major metacyclic promastigotes were
inoculated into the ear dermis (both ears) of C57BL/6 mice. The number
of parasites was monitored every 2 wk, and the evolution of the lesion
(measured by nodule diameter) was examined at least once a week over a
period of 10.5 wk (Fig. 1
A).
During the first 2 wk, the number of parasites increased only slightly,
followed by a dramatic increase during the third and fourth weeks,
reaching a peak load of 
3 x 105
parasites/ear. Surprisingly, this initial wave of parasite expansion
was not accompanied by the appearance of a lesion in any of the mice.
Two approaches were used to more carefully analyze the inflammatory
response in the parasite-loaded dermis. The first was to analyze the
cellular traffic through the dermis using a method based on the ability
of leukocytes that are nonadherent or weakly adherent to the matrix
(cells newly arrived or on their way to the draining node) to
spontaneously sediment out of the dermal sheets (12).
Second, histologic sections were prepared to observe the organization
of both the trafficking and matrix-bound cells within the involved
dermis. Following a transient cellular infiltrate due to the tissue
damage associated with needle inoculation (<48 h), the types and the
number of cells sedimenting out of the dermis remained unchanged from
the steady state dermis during the first 4 wk (Fig. 1B). The
tissue sections also failed to reveal any change in the cellularity or
architecture of the parasite-loaded dermis during the first 4 wk of
infection (Fig. 2).
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A). Between the fifth and ninth weeks, the
parasite load was reduced by >95%. The lesion size peaked at 1 mm
6 wk following the injection and resolved before the 12th week. Lesion
development was characterized by a wave of cells invading the dermis
beginning around the fifth week, comprised primarily of neutrophils,
macrophages, and eosinophils (Fig. 1B). The number of T
cells migrating through the site increased slightly during this time,
as did the number of DC. The number of DC increased substantially
during the latter stage of healing (8–10 wk). The tissue sections
obtained between the fifth and seventh weeks revealed a large influx of
cells into the dermal compartment (Fig. 2). The infiltrate was poorly
organized, and by the ninth week had largely resolved.
The final phase of the infection is characterized by the persistence of
100–10,000 parasites in the site for up to 1 year following resolution
of the cutaneous lesion (data not shown). The cells sedimenting from
the ear dermis during the chronic stage of infection were primarily
macrophages. Sections obtained after the 12th week showed an abundance
of lymphatic and blood capillaries and a persistent mononuclear cell
infiltrate embedded in a dense matrix (Fig. 2). High power
magnification revealed heavily infected macrophages surrounded by
uninfected mononuclear cells (not shown). Throughout the chronic phase,
neither the parasite nor the cellular infiltrate induced any additional
tissue damage, and this equilibrium between the parasite and the host
persisted until the natural death of the animal.
Immune responses in the epidermis and draining lymph node
The epidermis is in direct contact with the dermis, and the cells
that constitute this site in the mouse, essentially keratinocytes,
Langerhans cells, and dendritic epidermal T cells, are all
characterized by their capacity to rapidly produce cytokines in
response to tissue injury or microbial exposure (13). The
epidermal cells were collected during the course of the infection and
incubated for 12 h in the presence of Brefeldin A. The flow
cytometric analysis of the cells staining intracellularly for IL-4,
IL-12p40, or IFN- is shown in Fig. 3.
The first stage of the infection, corresponding to the sustained period
of parasite amplification in the underlying dermis, showed a consistent
production of IL-4 that decreased after the peak of the lesion (sixth
week). In contrast, IL-12p40 production in the epidermis was delayed
until 4 wk and peaked at 6 wk with an impressive number of cells
(500,000) staining for IL-12p40. The kinetics of the IFN-
response in the epidermal compartment were identical with those of
IL-12p40, with a delay until wk 4 and a peak response at 6–8 wk
involving up-regulated IFN- production by 900,000 cells/ear.
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and IL-4 in response to SLA was measured by ELISA (Fig. 4). Increased levels of IL-4 were
detected as early as the first week, and small amounts of IFN- were
detectable by wk 4. After the fourth week the production of both
cytokines by lymph node cells increased along with development of the
cutaneous lesion, with a peak at 6 wk (2.3 x
104 and 97 pg/ml for IFN- and IL-4,
respectively). As the dermal lesion resolved, the levels of IL-4 and
especially IFN- fell sharply, although a residual production of both
cytokines remained detectable during the chronic phase.
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A panel of immunodeficient mice, all on the C57BL/6 background,
were challenged in the ear dermis using 100 metacyclic promastigotes,
and the parasite loads, lesion development, and inflammatory infiltrate
in the inoculation site were compared with those of wild-type mice
throughout the first 10 wk postinfection. Surprisingly, the number of
parasites did not differ significantly between the untreated, wild-type
mice (5 x 105) and any of the
immunodeficient mice (0.4–8 x 105) over
the first 4.5 wk, with the exception of the CD40L KO mice, which had a
10-fold reduction in dermal parasite loads at 4.5 wk (Fig. 5). At each of the subsequent time points
examined (6.5 and 8.5 wk), the anti-CD4-treated mice, SCID mice,
and knockout mice deficient in IFN-, IL-12-p40, iNOS, or CD40L, all
failed to reduce the number of parasites in the site (Fig. 5). Of these
groups, the IFN--deficient and the anti-CD4-treated mice
appeared to be the most susceptible, yielding >100 million
parasites/ear at 8.5 wk. The peak parasite loads observed in the
IL-4-deficient mice were not significantly lower than those in the
wild-type mice, and the number of dermal parasites persisting
throughout the chronic phase was actually higher (data not shown).
Although the overall results extend to a natural infection model the
requirement for IL-12-driven Th1 responses in acquired immunity to
L. major, the more important findings relate to the fact
that for the first 4.5 wk the number of parasites in the ears of the
immunodeficient mice was no greater than that in control mice,
providing strong evidence that the activation or expression of
cell-mediated immunity is effectively absent even in the
immunocompetent mice throughout this period of parasite amplification
in the skin.
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Each of the groups of immunodeficient mice was also comparable to the
untreated, wild-type mice in their absence of lesion formation during
the first 4–5 wk (Fig. 5). Thereafter, three patterns of lesion
evolution were associated with the inability to reduce the parasite
burden in the site. In the anti-CD4-treated mice, CD40L KO mice,
and IL-12p40 KO mice, small nodules began to appear at the same time as
in the wild-type mice, and while the lesions failed to resolve, the
rate of lesion progression (Fig. 5) and the extent of tissue
destruction (Fig. 6) remained relatively
benign given the numbers of parasites that were accumulating in the
ear. In contrast, the infections in the IFN- KO and iNOS KO mice
were associated with rapidly progressing dermal lesions (Fig. 5) that
became ulcerative and necrotic by 8.5 wk (Fig. 6). At the other
extreme, in SCID mice no clinical pathology was ever detected until wk
9 despite the high parasite numbers in the site (Figs. 5 and 6).
Thereafter, nonulcerative nodular lesions progressed slowly in
these mice. These outcomes confirm a role for
CD4+ T cells in lesion formation and development.
Finally, the dermal lesions in the IL-4 KO mice, while resolved by wk
10, were not moderated in size or duration compared with those in
wild-type mice (Fig. 5).
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) or were fixed on a slide and stained
for microscopic evaluation of the cells harboring amastigotes (Table I,
expressed by the numbers in parentheses as a percentage
of the total population of infected cells). Mice depleted of
CD4+ cells showed an increased cellular
infiltrate compared with untreated, wild-type mice (6.25 x
105 vs 4.3 x 104
cells/ear), but there was little change in cellular composition. The
infiltrate was hardly altered in the IL-4 KO mice, apart from a much
lower number of eosinophils, presumably reflecting an impaired
regulation of IL-5. The types of inflammatory cells recovered from the
other immunodeficient mice could be divided into three groups that
correlated precisely with the three patterns of lesion development
described above. The first group displayed an increased cellular
infiltrate with a preponderance of neutrophils (IFN- KO and iNOS
KO). In the second group the infiltrating cells were primarily
eosinophils, with fewer numbers of neutrophils and macrophages
(IL-12p40 KO and CD40L KO). The accumulation of neutrophils led to the
rapid development of necrotic lesions in the iNOS and IFN- KO mice
(by wk 7 and 9, respectively), whereas the necrotic process was delayed
by 3–4 wk in the IL-12p40 and CD40L KO mice. The third group
corresponds to the mice with little or no dermal pathology up to 9 wk
postinfection (SCID), in which there was little increase in the total
number of leukocytes in the site, and the sedimenting cells were
predominantly macrophages and dendritic cells, with few eosinophils or
neutrophils.
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KO mice had a significant
number of infected eosinophils (29% and 7% of infected cells). The
infected neutrophils and eosinophils had a mean of six amastigotes per
cell, suggesting that the parasites persisted and even replicated
within these cells. Infected DC made up a small proportion (1–2%) of
the infected leukocytes recovered from the anti-CD4, IFN- KO,
and CD40L KO mice and a high proportion of the infected cells from SCID
mice (17% and 50%). The average number of amastigotes per infected DC
was low (1.6/DC). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of a challenge inoculum as low as 100 L. major promastigotes to initiate the development of small, healing footpad lesions in genetically resistant mice has been described previously (15, 16). In these studies, however, the number of parasites present in the inoculation site was not monitored over time, so that the relationship between parasite growth and lesion development could not be evaluated. In the few studies that have carefully monitored the parasite load in the footpad following high dose challenge, the early stage of lesion formation was associated with an increasing number of viable parasites in the site (17, 18, 19). In the present studies, by quantifying the parasite load at multiple time points, especially during the subclinical stage of infection, it was revealed that the maximum number of parasites in the site occurs before the onset of lesion formation. The quiescence of the parasite-loaded site was supported by the finding that apart from a transient increase in neutrophils immediately after needle injection, the number and types of leukocytes sedimenting out of the dermis remained unchanged during the first 4 wk postinfection. Presumably macrophages were recruited from the tissue or from blood to support the growth of amastigotes in the skin. These cells may have been sufficiently adherent to the matrix that they were missed in the restricted analysis of sedimenting cells. Nonetheless, analysis of tissue sections through the intact ear also failed to reveal any overt inflammatory process associated with the peak load of parasitized macrophages in the skin.
The dermal lesions that began to appear around wk 5 were associated with an acute infiltration of uninfected leukocytes, primarily neutrophils, macrophages, and eosinophils. The cell types in the inflammatory dermis are consistent with the few histological descriptions of human dermal lesions (20, 21, 22). The novel finding presented here is that the onset of this inflammatory process coincides with a reduction in the number of parasites in the site. Prior studies have noted the concurrence of pathology and immune response (23, 24), and others have determined that the number of parasites in the site does not necessarily correlate with the size of the lesion, particularly during the healing stage of disease (17, 19). So far as we are aware, however, there has not been any prior demonstration that lesion formation is so clearly timed with the onset of parasite clearance from the site.
The data suggest that induction of host responses that promote
immunity/pathology is avoided during a relatively sustained period of
intracellular growth in the skin. Parasite growth during the first 4–5
wk correlated with the absence of IL-12 and IFN- production by
potentially responsive cells in the epidermal compartment and with the
absence of Ag-reactive cells producing IFN- in the lymph node
draining the site. Because immune responses below the detection limits
of these assays might still be expected to moderate the early growth of
the parasite in the site, the finding that there was no enhancement of
parasite growth over the first 4.5 wk in any of the immunodeficient
mice, including the IL-12p40 KO and IFN- KO mice, is perhaps the
strongest evidence that in the intact mice the induction and expression
of host control mechanisms are effectively absent during this time. The
presence of IL-12 would appear to be a sufficient condition to initiate
a host protective response during the normally silent phase of
infection, because provision of exogenous IL-12 to IL-12p40 KO mice
during the first 3 days following inoculation resulted in a 10-fold
reduction in the numbers of parasites present in the site at 3 wk.
These data establish the in vivo relevance of a large series of in
vitro observations indicating that Leishmania avoid or
actively inhibit the immune initiation functions of their host
macrophages, including Ag presentation (25, 26, 27),
expression of MHC class II and costimulatory molecules
(28, 29, 30), and especially production of IL-12 in response
to infection (31, 32, 33, 34, 35). It is important to note that the
significance of these in vitro studies has remained controversial, in
so far as L. major-driven immune processes in vivo,
including IL-12 and IFN- production, have in many instances been
rapidly and efficiently induced following parasite delivery
(36, 37, 38, 39) .
The ability of the natural infection model to establish such a
discreet silent phase of infection is not dependent on intradermal
challenge, since we have observed that low dose challenge in the
footpad produces up to 6 wk of L. major growth without
footpad swelling, followed by lesion formation that is associated with
a reduced parasite burden in the site (R. Lira et al., unpublished
observations). The critical feature of the model seems to be dose. We
suggest that high dose inocula will undermine the ability of the
parasite to initiate infection in a quiescent manner, which may depend
on selective targeting of infectious stage parasites to macrophages in
the inoculation site. The excessive parasites and released Ags present
in high dose inocula, particularly inocula containing a high proportion
of noninfective forms, will probably be taken up by other cell types,
including dendritic leukocytes, which, in contrast to macrophages, have
been shown to produce IL-12 in response to Leishmania
infection (40, 41, 42) and to provide a potent source of APC
for T cell activation (42, 43, 44). In the present analysis,
low dose challenge in the dermis appeared to avoid activation of
epidermal cells for IL-12 production until wk 4, at which time the
activation of these cells, requiring perhaps a threshold level of
infected macrophages or released parasites in the dermis, correlated
with the strong up-regulation of IFN- production by T cells in the
draining node and the onset of both parasite killing and lesion
formation. The identity of the epidermal cells that stained positively
for IL-12p40 could not be confirmed due to defective expression of
surface markers following the trypsin treatment. Nonetheless, it is
likely that they are Langerhans cells based on their forward scatter
and the IL-12-producing capacity of these cells following L.
major infection in vitro (40).
Of interest was the increased frequency of IL-4-producing cells
observed in both the epidermis and the draining node as early as 2 wk
postinfection. Transient IL-4 responses following L. major
infection in resistant mouse strains have been described previously
(35, 45), although this is the first report that
IL-4-producing cells are detectable with such high frequency in the
epidermal compartment. The role that the early IL-4 response plays in
promoting the sustained growth of Leishmania in the dermis
is not clear, however, because the infections became just as well
established following low dose inoculation in IL-4-deficient mice.
Nonetheless, a role for additional type 2 cytokines that might be
induced early on needs to be considered in the light of recent findings
that IL-13 and IL-10 can also promote L. major infections in
conventional mouse footpad models (46, 47). The inability
of the IL-4-deficient mice to as efficiently control infection in the
chronic stage as wild-type mice was a consistent finding. Similar
findings were reported for IL-4-deficient mice infected with
Candida albicans (48) or with Toxoplasma
gondii (49) along with the surprising result that
IL-4 was required to sustain IFN- production by activated
CD4+ T cells (48).
Although the initial silent phase of infection was unaltered in
the immunodeficient mice, these mice ultimately confirmed that the
immune mechanisms responsible for control of L. major
infection in the conventional model continue to operate in a natural
infection model, i.e., a requirement for IL-12-driven,
CD40/CD40L-dependent CD4+ T cell activation,
IFN- production, and NO-mediated killing. The immunodeficient mice
also confirmed a role for T cells in pathology, with some interesting
comparisons. Lesion development, especially necrosis, was related
primarily to an acute infiltration of neutrophils, and secondarily
macrophages and eosinophils, into the parasite-loaded dermis, and this
response was at least in part CD4+ T cell
dependent, because each of the mice with more global defects in
CD4+ T cell activation and function (i.e.,
anti-CD4, CD40L KO, and IL-12p40 KO) had far less severe pathology
than the mice for which the defect was confined to effector molecules
involved in macrophage activation and intracellular killing (i.e.,
IFN- KO and iNOS KO mice). The complete absence of lesions in SCID
mice up to 9 wk postinfection despite the high parasite burden suggests
a role for other T cell subsets and/or B cells in dermal pathology.
Delayed development of footpad swelling in SCID mice (50)
and in MHC class II-deficient mice (7, 8, 51) has been
observed previously. The absence of pathology in these studies was
thought to be due to the lack of T cells that are required for the
recruitment of macrophages to the inoculation site (i.e., a lack of
host cells available for infection). It should be emphasized that in
our analysis the reduced pathology in the ears of the T cell-deficient
mice was in no case associated with a deficit in parasite growth or
host cells available for infection in the site. Instead, the prolonged
absence of lesions in these mice seems to reflect a maintenance of the
conditions associated with the initial silent phase of infection that
occurs in intact mice, in which the accumulation of infected
macrophages in the site, in the absence of an accompanying acute
inflammatory process, appears to have few pathological consequences.
The chronic phase also reflects the absence of an active
inflammatory process despite the persistence of infected macrophages in
the site. The persistence of L. major in the footpad and
draining node has been noted previously following the resolution of
footpad lesions in resistant mice (19, 52, 53, 54). The
natural infection model indicates that L. major persistence
in resistant mice is not an artifact of an inappropriate inoculation
site or high dose challenge and may prove a useful model to study the
mechanisms underlying low level, chronic infection and the events
associated with reactivation disease.
Another surprising aspect of the dermal pathology in the immunodeficient mice was the heterogeneity of the cells harboring parasites that were recovered from the infected dermis. In contrast to the wild-type mice, for which amastigotes were found only in macrophages, the intracellular amastigotes in the immunodeficient animals were variably associated with neutrophils, eosinophils, and dendritic leukocytes, which in the case of neutrophils and eosinophils harbored large numbers of parasites. Dendritic cells recovered from lymphoid tissue have been found to harbor persistent L. major (55), and transient or low multiplicity infections in neutrophils and eosinophils have been noted within cutaneous lesions (56, 57). Our results in the immunodeficient mice extend these findings by suggesting that neutrophils and eosinophils are under some circumstances capable of supporting sustained, productive intracellular infections. This would suggest that the cells are themselves long lived, due perhaps to the absence of cytokines or chemokines involved in their cell death signaling pathways. The data imply that infection outcomes in animals with specific immune defects need to be interpreted with care, since in addition to a compromised ability to activate infected macrophages for killing, the number and types of leukocytes permissive to infection might be significantly expanded.
In conclusion, the present studies have revealed an underlying feature of L. major infection in vivo that has been obscured by experimental approaches designed primarily to study parasite-driven Th1 and Th2 subset development. Our analysis of a natural infection model promotes the concept, raised repeatedly in the context of in vitro studies, that sustained intracellular growth in the absence of a parasite-driven host response is a hallmark of leishmanial infection and one that we believe is central to the maintenance of its transmission cycle in nature.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: CD40L, CD40 ligand; KO, knockout; iNOS, inducible NO synthase; DC, dendritic cells; SLA, soluble L. major Ag.
Received for publication January 14, 2000. Accepted for publication April 28, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major. J. Immunol. 143:266.[Abstract]
/ß) and type 2 nitric oxide synthase regulate the innate immune response to a protozoan parasite. Immunity 8:77.[Medline]
secreting CD4+ cells correlate with resolution of lesions in murine cutaneous leishmaniasis. J. Immunol. 149:2715.[Abstract]
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) following intradermal inoculation of 100 L.
major metacyclics. Values represent the mean induration
(millimeters) ± 1 SD (4–10 mice/group) and the geometric mean
parasite number per ear ± 1 SD (3 mice and 6 ears/group).
B, Leukocytes trafficking in the dermal compartment
during the course of the infection. Cells sedimenting out of the dermis
(pooled from 5 mice and 10 ears at each time point) were analyzed by
flow cytometry and monitored as the total number of leukocytes per ear
(
), eosinophils (
),
neutrophils (
), T lymphocytes (
), or dendritic cells (•) per
ear. The data shown are from a single experiment representative of two
separate experiments.
