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,
Departments of
*
Internal Medicine and
Microbiology, University of Iowa, and
Veterans Affairs Medical Center, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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is critical for the cure of leishmaniasis in humans and
mice. BALB/c mice are genetically susceptible to infection with the
visceralizing species of Leishmania, L.
chagasi. We have evidence that a soluble factor(s) inhibits
IFN- production by cultured liver granuloma cells from BALB/c mice
during L. chagasi infection. In contrast, liver granulomas
from C3H.HeJ mice, which are genetically resistant to L.
chagasi infection, produce abundant IFN-. According to ELISAs
and neutralization studies, there was not evidence that the Th2-type
cytokines IL-10 or IL-4 contributed to IFN- suppression. However,
both Ab neutralization and immunohistochemistry showed that
granuloma-derived TGF-ß was, at least in part, responsible for
inhibiting IFN- release by CD4+ cells in BALB/c liver
granuloma cultures. Consistently, TGF-ß levels were high in liver
granulomas from susceptible BALB/c mice but low in resistant C3H mice
or in BALB/c mice that were immunized against L. chagasi
disease. Administration of recombinant adenovirus expressing TGF-ß
(AdV-TGFß) but not IL-10 (AdV-IL10) caused genetically resistant C3H
mice to become significantly more susceptible to L. chagasi
infection. In contrast, either AdV-TGFß or AdV-IL10 could abrogate
the protective immune response achieved by immunization of BALB/c mice.
We conclude that locally secreted TGF-ß inhibits Th1-associated cure
of murine visceral leishmaniasis caused by L. chagasi,
independently of Th2-type cytokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Similar to human disease, there are differences in murine immune
responses to Leishmania sp. that cause human cutaneous
leishmaniasis (e.g., Leishmania major, Leishmani
amazonensis) vs organisms leading to human visceral leishmaniasis
(e.g., Leishmania chagasi, Leishmania donovani,
Leishmania infantum) (3, 4, 5, 6, 7). The outcome of
murine infection with L. major is largely determined by
expansion of different subsets of CD4+ T cells: Th1 cells
making IFN- provide protection, whereas Th2 cells making IL-4 and
IL-10 lead to progressive disease (8). In contrast,
studies of mice infected with either L. donovani or L.
chagasi indicate that Th2-type cytokine IL-4 is not required for
the susceptible phenotype to be manifest (7, 9).
Nonetheless, there is evidence in both humans and mice that the
Th1-type cytokine IFN- is critical for either innate or
immunization-induced protection against disease (10, 11, 12).
There is also evidence that an experimentally induced Th2-type
anti-parasite response can exacerbate murine L. donovani
disease, even though a Th2 response is not prominent during the usual
course of infection (13).
This ability of visceralizing leishmania to multiply in the absence of
an apparent Th2 response led us to investigate mechanisms whereby a
curative Th1 response is suppressed in this model. Our previous studies
provided evidence that a soluble factor inhibits IFN- production by
cultured liver granuloma cells from L. chagasi-infected mice
and that this inhibitor is distinct from either IL-4 or IL-10. The
inhibitor is derived from non-T cells in liver granulomas. IFN-
inhibition is not a prominent component of the systemic immune response
as reflected in splenocyte cultures. This local inhibitor of IFN-
correlates with, and probably facilitates, replication of L.
chagasi in the livers of susceptible mice (7).
Several macrophage-deactivating cytokines (e.g., IL-4, IL-10) have been
found to enhance the progression of murine cutaneous leishmaniasis
(14, 15). The macrophage-deactivating cytokine IL-10
inhibits IFN- and contributes to the progression of human visceral
leishmaniasis (16, 17), and neutralizing TGF-ß1 augments
IFN- and diminishes IL-4 mRNA levels in cutaneous leishmaniasis
(18). During the current study, we sought to identify the
soluble inhibitor of IFN- in the livers of mice infected with
L. chagasi. Our findings supported the fact that the
TGF-ßs inhibit IFN- production in liver granulomas, whereas there
was no evidence for an inhibitory role of IL-10. As such, the TGF-ßs
are likely to be important determinants of L. chagasi
disease outcome in susceptible BALB/c mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A strain of L. chagasi (MHOM/BR/00/1669) from a patient with visceral leishmaniasis in northeast Brazil was maintained by serial intracardiac injection in hamsters. Parasites were isolated from infected hamster spleens and cultured as promastigotes in liquid hemoflagellate-modified MEM (HOMEM) medium (19) and were used within 3 wk.
Mice were immunized either i.v. or s.c. Intravenously immunized mice
were inoculated with 1 x 107 -irradiated
promastigotes (150,000 rad, Cs irradiator) twice at 2-wk intervals and
challenged with viable promastigotes after an additional 2 wk.
Preliminary study showed -irradiated promastigotes remained motile
but did not multiply in culture, and they died after 7–10 days.
-Irradiated promastigotes were phagocytosed by human
monocyte-derived macrophages and transformed to amastigotes, but they
did not multiply intracellularly. Subcutaneously immunized mice
received 50 µg of soluble promastigote lysate, without or with
recombinant adenovirus, at the back of the neck. Promastigote lysate
was prepared by subjecting promastigotes in 10 mM Tris, pH 7.4, to
three rounds of freeze-thaw, then removing particulate material at
6000 x g. Proteins were quantified using the
bicinchoninic acid system (Bio-Rad, Hercules, CA).
BALB/c or C3H.HeJ mice were obtained from Charles River Laboratories (Bloomington, MA) or The Jackson Laboratory (Bar Harbor, ME), respectively. Mice were infected with 1 x 107 stationary phase promastigotes i.v. through a tail vein. Four weeks later, the mice were sacrificed, livers and spleens were weighed, and organ impression smears were examined microscopically. The parasite load in each organ was calculated as: [(ratio of amastigotes to mononuclear cells) x organ weight (mg)] x 2 x 105 parasites (20).
Splenocyte and liver granuloma cell cultures
Unless otherwise stated, granuloma cells and splenocytes were isolated 4 wk after challenge with L. chagasi promastigotes. Splenocytes or liver granuloma cells were suspended at 2 x 106 cells/ml in 200 µl Click’s medium as we have previously described (7, 21). Triplicate wells contained either no stimulus; 100 µg anti-CD3/ml (2C11, American Type Culture Collection, Manassas, VA); or 3 x 106 promastigotes/ml as a source of parasite Ag. After 3 days at 37°C (the time of maximal antiparasite response), culture supernatants were collected for cytokine assay. Some wells contained the following neutralizing Abs: 10 µg/ml panspecific Ab to TGF-ß (R&D Systems, Minneapolis, MN); 10 µg/ml 11B11 rat anti-mouse IL-4 (PharMingen, San Diego, CA); 10 µg/ml SXC-1 rat anti-IL-10 (PharMingen). To test for soluble inhibitor activity, splenocytes or liver granuloma cells from 4-wk infected mice were incubated for 3 days in tissue culture flasks in the absence of Ag. Supernatants were filtered to remove cells and stored at -20°C.
IFN-, IL-10, and IL-4 in triplicate supernatants were measured by
two-sandwich ELISAs as previously described (7). Levels of
TGF-ß were measured by its inhibition of mink lung epithelial cell
growth (22). Total TGF-ß was measured after
acidification to activate latent TGF-ß, followed by neutralization.
Some wells included anti-TGF-ß, which caused a 98.2% suppression
of cytokine activity. A standard curve was prepared with 0–1500 pg/ml
of TGF-ß1 (R&D Systems) in Click’s medium. Data show the mean
± SE for the indicated number of replicate experiments.
CD4+ or CD8+ cells were depleted from granuloma cell cultures by incubation with anti-CD4 (RL.172) or anti-CD8 (3.168) (American Tissue Collection, Manassas, VA) at 4°C for 45 min followed by Low-Tox guinea pig complement (Cedarlane, Westbury, NY) at 37°C for 1 h. After two rounds of lysis, cultures were depleted of 98.3% of CD4+ and 99.6% of CD8+ cells according to FACS analysis.
Immunohistochemistry
After 4 wk of infection, BALB/c mice were anesthetized and perfused systemically with PBS followed by 1% paramaformaldehyde. Livers and spleens were harvested and embedded in paraffin. Two-micrometer sections were deparaffinized, treated with hyaluronidase, blocked, and stained with rabbit polyclonal Ab to TGF-ß1 (provided by Dr. Leslie Gold, New York University, NY) as described (23). Controls were incubated with nonspecific rabbit IgG. All sections were then incubated in biotinylated anti-rabbit IgG followed by avidin-gold, followed by silver enhancement, and counterstained with hematoxylin (24).
Recombinant adenoviruses
Adenoviruses expressing IL-10 (AdV-IL-10),3 TGF-ß (AdV-TGF-ß), or Escherichia coli ß-galactosidase (AdV-lacZ) were generated by homologous recombination into Ad5 strain dl309 as previously described (25). Briefly, shuttle plasmids containing the insert sequences expressed from the Rous sarcoma virus long terminal repeat and flanked by adenoviral sequences were transfected into HEK 293 cells along with restriction enzyme-digested dl309 DNA. Recombinant plaques were identified by ß-galactosidase staining or IL-10 ELISA along with DNA slot blots of recombinant sequences. Plaques were amplified and replaqued three times (26). Titers were determined on HEK 293 cells and wild-type contamination assessed by an A549 plaque assay. All cells had a wild-type titer of <104 in 1010 recombinant pfu. Viruses were purified by CsCl2 gradient ultracentrifugation (26). Groups of mice were inoculated either i.v. or s.c. with 109 pfu of recombinant adenovirus in 100 µl of 3% sucrose/PBS. Control mice received 3% sucrose lacking adenovirus.
ß-galactosidase activity was measured using the Galacto-Light reporter system (Tropix, Bedford, MA). Five days after the introduction of adenovirus, tissues at the inoculation site or the draining lymph nodes were excised and frozen in liquid nitrogen. After homogenization and two rounds of freeze-thaw, ß-galactosidase activity was measured in supernatants according to the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred strains of mice are genetically either susceptible or
resistant to infection with L. donovani or L.
chagasi (5). We used susceptible BALB/c and resistant
C3H.HeJ mice as models of L. chagasi disease (3, 27). When L. chagasi promastigotes are introduced
i.v. into either of these mouse strains, the parasite load reaches a
maximum level in the liver after 4 wk, whereas the parasite load in the
spleen remains lower and peaks later. The course of infection is
illustrated in Fig. 1
A.
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in cultured liver
granuloma cells from infected BALB/c mice
We previously reported that IFN- levels are low or absent and
that there are few IFN--producing cells in liver granuloma cell
cultures of L. chagasi-infected BALB/c mice. In contrast,
high levels of IFN- are produced by splenocytes from the same mice
(7). Supernatants of unstimulated liver granuloma cell
cultures from infected BALB/c mice inhibited Ag-induced IFN-
production in splenocytes, whereas splenocyte culture supernatants did
not (Fig. 1
B). This result suggests that a soluble factor
(or factors) in liver cell culture supernatants is responsible, at
least in part, for attenuating IFN- levels at the site of parasite
growth.
Although all mouse strains acquire a measurable liver parasite load
after infection with L. chagasi, the magnitude of parasite
load differentiates susceptible from resistant phenotypes. The livers
of infected C3H.HeJ mice harbor four- to five-fold fewer granulomas
than susceptible BALB/c mice (see Fig. 1A), but by using
larger numbers of mice, amastigote-laden granulomas can be isolated and
studied. Liver granuloma cultures are normalized for equal numbers of
granuloma-derived immune cells. In contrast to BALB/c mice,
supernatants from infected C3H mouse liver granuloma cultures did not
inhibit IFN- production by syngeneic splenocytes (Fig. 1C). This finding may reflect relatively lower levels of
inhibitory factors in these cultures.
Kinetics of cytokine secretion
The above-described studies utilized livers from mice infected for
4 wk with L. chagasi. Studies conducted at different time
points showed that BALB/c liver granulomas secreted undetectable
amounts of IFN- during the first 4 wk of infection when parasites
were actively growing, but after spontaneous local resolution of liver
infection, the amount of IFN- increased from undetectable to >1
ng/ml (Figs. 1A and
2A). Splenocyte IFN-
remained high (
4 ng/ml) throughout infection, correlating with low
parasite numbers in this organ.
The amounts of two inhibitory cytokines, IL-10 (Fig. 2B) and
TGF-ß (Fig. 2C), were relatively high early in infected
BALB/c liver granuloma cell cultures when parasite loads were high.
Levels fell after subsequent spontaneous resolution of liver infection.
TGF-ß concentrations were statistically higher in liver than in
spleen cell cultures at all time points. The amount of IL-4 remained
low in both splenocyte and granuloma cell culture supernatants
throughout the course of infection (not shown). In contrast, the
amounts of both IL-10 and TGF-ß in splenocyte culture supernatants
were relatively low throughout the infection. These data raise the
possibility that IL-10, TGF-ß, or both are responsible for inhibiting
IFN- in BALB/c livers during the early stages of L.
chagasi disease.
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(Fig. 2D). At 4
wk after infection, the time of peak parasite load for both mouse
strains, C3H liver granuloma supernatants contained significantly lower
amounts of TGF-ß than BALB/c mice (4.24 ± 1.60 vs 574 ±
11 pg/ml, respectively; p < 0.01, n = 4), raising
the possibility that TGF-ß contributes to the susceptible phenotype
of BALB/c mice. At the same time point (4 wk), IL-10 levels were not
statistically different between liver granuloma culture supernatants
from C3H vs BALB/c mice (0.75 ± 0.20 and 1.87 ± 0.56 ng/ml,
respectively) or between splenocyte cultures from C3H vs BALB/c mice
(0.95 ± 0.42 vs 0.64 ± 0.17 ng/ml, respectively), although
there was a trend toward higher levels of IL-10 in BALB/c liver
granuloma cultures. IL-4 was not detected in spleen or liver cell
culture supernatants from either mouse strain during L.
chagasi infection, although it was detected during L.
major infection (not shown).
Immunohistochemistry showed silver-enhanced particles corresponding to
immunoreactive TGF-ß1 in liver granulomas from infected BALB/c mice
(Fig. 3B). Some particles were
visualized overlying granuloma macrophages and others apparently free
from cells, suggesting that the Ab detected both secreted and
cell-associated cytokine. Amastigotes could be seen within some
granuloma macrophages (Fig. 3A).
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i.v. immunization of BALB/c mice with -irradiated promastigotes
prior to challenge infection significantly lowered parasite loads in
immunized compared with control animals (Fig. 4A). Immunization caused a
significant increase in Ag-induced IFN- production by liver
granuloma cells but not splenocytes (Fig. 4B, Liver +
PM bars). Immunization caused a significant decrease in both splenocyte
and liver granuloma culture TGF-ß (Fig. 4D). There was a
paradoxical increase in IL-10, particularly pronounced in splenocyte
cultures, following immunization (Fig. 4C). This could
represent a compensatory increase due to augmented IFN- levels.
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inhibition
Splenocytes or liver granuloma cells from 4-wk-infected mice were
cultured without or with Abs that neutralize the activities of
potentially inhibitory cytokines (Fig. 5A). Neutralizing TGF-ß
caused a significant increase in IFN- in liver granuloma cell and
splenocyte culture supernatants; this increase was most dramatic in
liver granulomas. In contrast, Ab neutralization of either IL-10 or
IL-4 did not significantly augment IFN- in culture supernatants.
Combinations of neutralizing Abs did not further augment IFN- over
Ab to TGF-ß alone (not shown). We conclude that TGF-ß is a major
contributor to IFN- inhibition in BALB/c liver granuloma cell
cultures. The TGF-ß in cultured splenocytes of 4-wk infected BALB/c
mice may also inhibit IFN- to some extent, but the level is
apparently not high enough to abrogate Ag-induced IFN- production
(see Fig. 2, A and C).
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from 0.65–3.37 µg/ml. After CD8+
cell depletion, cells produced 1.80 µg IFN-/ml in the presence of
TGF-ß, whereas after CD4+ cell depletion, IFN- was
undetectable without or with anti-TGF-ß. We conclude that
CD4+ cells in liver granuloma cell cultures are a source of
IFN- once the inhibitory effects of TGF-ß are removed. Supplementing cytokines with recombinant adenovirus expressing IL-10 or TGF-ß
We used recombinant viruses expressing inhibitory cytokines to modulate the course of L. chagasi disease in genetically resistant C3H.HeJ mice, since adenovirus express recombinant cytokines in vivo over days to weeks. Preliminary studies showed adenovirus did not significantly alter the course of L. chagasi infection (17.27 ± 1.45 x 107 vs 15.96 ± 3.03 x 107 liver amastigotes in controls or mice infected with AdV-lacZ, respectively). The activity of recombinant viruses was verified in HEK 293 cell lysates (25,110.6 ± 95.5 vs 22.0 ± 3.2 pg of TGF-ß/ml in cultures infected with AdV-TGF-ß or AdV-lacZ, respectively), or in mouse sera (18.58 ± 8.85 vs 0.00 ng of IL-10/ml on day 1, and 8.91 ± 3.96 vs 0.00 ng of IL-10/ml on day 10 after inoculation of AdV-IL-10 vs AdV-lacZ, respectively).
Groups of resistant C3H.HeJ mice were pretreated with control
AdV-lacZ or with AdV-IL-10 or AdV-TGF-ß delivered i.v., a
route that causes recombinant viral protein expression in mouse livers.
Four days later, they were challenged i.v. with 107 live
L. chagasi promastigotes. An additional dose of recombinant
AdV was given 2 wk later and 4 wk after parasite challenge mice were
sacrificed. Measures of liver parasite loads showed that AdV-TGFß
caused a significant increase in L. chagasi infection (Fig. 5B). The modest increase in mice given AdV-IL10 did not
reach significant levels.
Susceptible BALB/c mice already express TGF-ß and IL-10 during
L. chagasi infection, and prior immunization caused a
significant decrease in TGF-ß (Fig. 4). We used recombinant AdV to
increase the local concentrations of IL-10 and TGFß at the site of
s.c. immunization. Local expression of recombinant adenoviral proteins
via this route was documented by inoculating mice s.c. with
AdV-lacZ or buffer. Skin homogenates contained 802,462 vs
2,181 units, and draining lymph nodes contained 23,609 vs 680 units of
ß-galactosidase activity, in mice receiving AdV-lacZ or
buffer, respectively.
BALB/c mice were subsequently "immunized" s.c. by infection with
buffer or recombinant AdV-lacZ, AdV-IL-10, or AdV-TGF-ß,
each delivered without or with 50 µg of soluble L. chagasi
promastigote lysate. Mice were challenged with live promastigotes 4 wk
after immunization. Mice that had received soluble promastigote lysate
plus either buffer alone (p < 0.03; not shown) or plus
AdV-lacZ (Fig. 5C) were significantly protected
against challenge infection with L. chagasi. However,
administration of either AdV-IL-10 or AdV-TGF-ß during immunization
prevented the development of resistance. Thus, either of these
inhibitory cytokines was able to prevent the development of an
immunization-induced protective immune response in BALB/c mice, even
though TGF-ß appears to be more prominent in facilitating parasite
growth during primary infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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plays a central role in limiting the growth of
Leishmania in murine and human macrophages and in limiting
the progression of leishmaniasis (10, 11, 28). Thus, the
modulation of either systemic or localized IFN- levels could be a
critical determinant of disease outcome. During our earlier studies, we
found evidence for a soluble factor or factors inhibiting the local
production of IFN- in liver granulomas of L.
chagasi-infected BALB/c mice. Similar inhibition was not detected
in splenocyte cultures, possibly reflecting the fact that high levels
of inhibitory factors and/or inhibitor-producing (e.g., Kupffer,
macrophage, stellate, dendritic) cells are present in the liver
(29). The liver-derived inhibitor(s) diminished Ag-induced
IFN- production by splenocyte cultures. The fact that splenocyte
cultures already contained high levels of both IL-10 and IFN-
suggested that IL-10 was not solely responsible for IFN- inhibition.
It is notable that L. chagasi replicate well in the livers
but poorly in the spleens of murine hosts. As such, cytokines produced
locally in the liver may be responsible for the susceptible vs
resistant phenotype of mice, and important interactions may not be
reflected in the systemic immune compartment as represented in
splenocyte cultures.
We found that TGF-ß levels were high in livers of BALB/c mice during
L. chagasi infection. Ab neutralization suggested that
TGF-ßs may be responsible, at least in part, for inhibiting IFN-
production by CD4+ cells locally in the liver. In contrast,
neutralization of the Th2-type cytokines IL-10 and IL-4 did not affect
the amounts of IFN- in these cultures. BALB/c mice that were
immunized and therefore partially protected against L.
chagasi infection had significantly lower levels of TGF-ß than
sham-immunized controls. Furthermore, TGF-ß levels were low in
genetically resistant C3H.HeJ mice, correlating with higher amounts of
IFN- produced by their cultured liver granuloma cells. These data
suggest the TGF-ßs may play a role in limiting IFN- production
during primary infection of susceptible mice, and that lowering TGF-ß
levels might be important for the development of immunity after
immunization. We previously observed that a soluble factor inhibited
IFN- release from granuloma T cells, even though granuloma T cells
released IFN- when cultured alone (7). The current
findings suggest TGF-ß may be at least partially responsible for
IFN- inhibition. TGF-ß, which could originate from liver Kupffer,
stellate, or dendritic cells or from liver macrophages, was localized
in granulomas by immunohistochemistry, suggesting that it is a product
of granuloma macrophages.
The cadence of murine visceral leishmaniasis is slow, and disease severity must be measured weeks after infection. Therefore, we chose to administer cytokines expressed by recombinant adenovirus that produced cytokines over days to weeks. Advantages of this viral delivery system include the relative stability of the virion so that deletion or recombination events are infrequent, and the fact that there is a precedent for the use of adenovirus in humans and mice (30, 31). Adenovirus delivered i.v. is ideal for this model, because recombinant virus, for the most part, is found in the liver where parasite growth is maximal. Disadvantages of adenovirus include the fact that repeated administration elicits an immune response to the adenovirus itself, limiting recombinant gene expression (30). The latter is not problematic during cytokine delivery because repeated administration is not necessary. Delivery of AdV-TGF-ß1 immediately before L. chagasi infection caused resistant mice to become more susceptible to infection, whereas adenovirus expressing the Th2-type cytokine IL-10 did not have a significant effect. These data suggest a role for TGF-ß1 in promoting susceptibility to L. chagasi disease in vivo.
Our findings could help explain the differences between murine immune
responses to parasites causing human cutaneous vs human visceral
leishmaniasis. Expansion of Th2-type CD4+ cells is the key
to progression of L. major (cutaneous) disease in BALB/c
mice, whereas Th1 expansion is important for disease control in
resistant mice (8). Initiators of Th1 development may
include macrophage-derived IL-12-stimulating NK cell IFN- or IL-18.
IL-4 is important in initiating the Th2 response, although the cell of
origin is under debate (14, 32, 33, 34). In contrast,
expansion of cells producing Th2-type cytokines is not required for
mice to manifest susceptibility to the visceralizing
Leishmania species (L. donovani (9, 35), L. chagasi (7)). Nonetheless,
recent evidence clearly shows that an experimentally induced Th2-type
antiparasite response is able to cause an exacerbation of visceral
leishmaniasis due to L. donovani in BALB/c mice
(13). According to our data, susceptibility to L.
chagasi infection in BALB/c mice correlates with the localized
production of TGF-ßs at the site of maximal parasite growth in the
liver. TGF-ß1 is a potent inhibitor of NK-cell derived IFN-
(32), and in this model it can substitute for Th2
cytokines as an inhibitor of Th1-type responses. The fact that either
TGF-ß1 or IL-10 could prevent antileishmanial protective immunity
from developing after s.c. immunization was not surprising, confirming
that both cytokines can prevent the development of protective Th1-type
responses in susceptible mice.
TGF-ßs constitute a family of related cytokines (TGF-ß1, TGF-ß2,
and TGF-ß3) of which TGF-ß1 is the major molecule produced by
immune cells (T cells, monocytes, macrophages) (36).
TGF-ß1 is released as a latent homodimer of 100 kDa, which must
undergo proteolytic activation to assume biological activity
(22). It inhibits T cell proliferation, CTL and LAK cell
generation, NK cell cytotoxicity, and MLR responses, and it
down-modulates IFN-, IL-2, and IL-12 levels (37, 38).
TGF-ß1 impairs IFN--induced macrophage activation and generation
of reactive oxygen intermediates, and it prevents formation of
reactive nitrogen intermediates (39, 40). Recent data
indicates that TGF-ß2 (which has overlapping biological effects with
TGF-ß1 (41)) impairs the ability of murine peritoneal
exudate cells to produce IL-12 and express CD40 and that the cytokine
promotes CD4+ cell development toward a Th2 phenotype
(42). IFN- has been found to antagonize, and IL-4 and
TGF-ß itself are found to promote TGF-ß expression
(43).
TGF-ßs contribute to the progression of infections due to Mycobacterium sp., Staphylococcus aureus, Trypanosoma cruzi, and Toxoplasma gondii (44, 45, 46, 47). Retention of an intact TGF-ß receptor signaling pathway is critical to T. cruzi invasion of epithelial cells (48), and the cytokine prevents vaccine induced immunity to Schistosoma mansoni (49). Paradoxically, TGF-ß has been found to contribute to host resistance to some pathogens (Candida albicans and Listeria monocytogenes) (50, 51).
Some species of Leishmania have been found to induce the production of TGF-ß by macrophages (18). Thus, the presence of the parasite could initiate and promote TGF-ß expression, and TGF-ß can also enhance its own expression (43). The fact that the cytokine influences disease-associated parameters was shown in vivo by administration of AdV-TGFß to C3H mice, and in vitro by neutralizing TGF-ß activity. It is possible that TGF-ß expression is initiated by the presence of parasites in BALB/c liver macrophages and that this TGF-ß, in turn, promotes both parasite growth and additional TGF-ß expression in a type of positive feedback loop resulting after parasite infection.
It is remarkable that both TGF-ß and IL-10 levels paralleled the
parasite load at different times of infection. Although neutralizing
IL-10 did not augment IFN- released into BALB/c liver cell
supernatants, this fact does not preclude a role for IL-10 in promoting
disease progression in concert with TGF-ßs in vivo. The exact
"trigger" leading to increased IFN- and decreased TGF-ß and
IL-10 in liver granuloma cell supernatants, and the actual cause of
disease resolution must be the subject of speculation at present. It is
possible that a late increase in Th1-promoting factors such as IL-12 or
IL-18 augments IFN-. Whether the small increase in IFN- shown in
Fig. 2A is sufficient to initiate disease resolution cannot
be determined from the present data. However, IFN- has been found
important for the cure of L. chagasi or L.
donovani disease in all murine models studied to date, and we must
consider this hypothesis as a distinct possibility.
A role for TGF-ß in promoting Th2-mediated progression of murine
cutaneous leishmaniasis was previously established using a model of
L. amazonensis disease. In this model, rTGF-ß augmented
lesion development, coincident with diminished Th1-type and increased
expression of Th2-type cytokines (18). In a hamster model
of L. donovani infection, TGF-ß was produced by adherent
cells causing impaired lymphocyte proliferation to parasite Ags
(52). TGF-ßs facilitate the growth of L.
braziliensis (a cause of mucosal leishmaniasis) and decrease
IFN- mRNA levels in BALB/c mice (18, 53). Our findings
suggest that TGF-ß1 may additionally present a means of suppressing
Th1-type responses in murine visceral leishmaniasis that is independent
of Th2-type cytokine expression. Thus, TGF-ß1 represents an
additional means by which Th1-type responses are down-modulated in
murine leishmaniasis, causing susceptibility to disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Mary E. Wilson, Dept. of Internal Medicine, University of Iowa, Iowa City, IA 52242. E-mail address:
3 Abbreviations used in this paper: AdV, adenovirus; pfu, plaque-forming unit.
Received for publication February 10, 1998. Accepted for publication July 13, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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have opposing effects, while TGF-ß positively regulates its own production. J. Immunol. 160:5719. production. Proc. Natl. Acad. Sci. USA 93:3193.This article has been cited by other articles:
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  S. Banerjee, J. Ghosh, S. Sen, R. Guha, R. Dhar, M. Ghosh, S. Datta, B. Raychaudhury, K. Naskar, A. K. Haldar, et al. Designing Therapies against Experimental Visceral Leishmaniasis by Modulating the Membrane Fluidity of Antigen-Presenting Cells Infect. Immun., June 1, 2009; 77(6): 2330 - 2342. [Abstract] [Full Text] [PDF]
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 C. F. Anderson, R. Lira, S. Kamhawi, Y. Belkaid, T. A. Wynn, and D. Sacks IL-10 and TGF-{beta} Control the Establishment of Persistent and Transmissible Infections Produced by Leishmania tropica in C57BL/6 Mice J. Immunol., March 15, 2008; 180(6): 4090 - 4097. [Abstract] [Full Text] [PDF]
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A. Maroof and P. M. Kaye Temporal Regulation of Interleukin-12p70 (IL-12p70) and IL-12-Related Cytokines in Splenic Dendritic Cell Subsets during Leishmania donovani Infection Infect. Immun., January 1, 2008; 76(1): 239 - 249. [Abstract] [Full Text] [PDF]
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U. Gaur, S. C. Roberts, R. P. Dalvi, I. Corraliza, B. Ullman, and M. E. Wilson An Effect of Parasite-Encoded Arginase on the Outcome of Murine Cutaneous Leishmaniasis J. Immunol., December 15, 2007; 179(12): 8446 - 8453. [Abstract] [Full Text] [PDF]
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H. K. Chang, C. Thalhofer, B. A. Duerkop, J. S. Mehling, S. Verma, K. J. Gollob, R. Almeida, and M. E. Wilson Oxidant Generation by Single Infected Monocytes after Short-Term Fluorescence Labeling of a Protozoan Parasite Infect. Immun., February 1, 2007; 75(2): 1017 - 1024. [Abstract] [Full Text] [PDF]
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 S. Rama Iniguez, M. A. Dea-Ayuela, J. A. Sanchez-Brunete, J. J. Torrado, J. M. Alunda, and F. Bolas-Fernandez Real-Time Reverse Transcription-PCR Quantification of Cytokine mRNA Expression in Golden Syrian Hamster Infected with Leishmania infantum and Treated with a New Amphotericin B Formulation. Antimicrob. Agents Chemother., April 1, 2006; 50(4): 1195 - 1201. [Abstract] [Full Text] [PDF]
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X. Shao, J. Rivera, R. Niang, A. Casadevall, and D. L. Goldman A Dual Role For TGF-{beta}1 in the Control and Persistence of Fungal Pneumonia J. Immunol., November 15, 2005; 175(10): 6757 - 6763. [Abstract] [Full Text] [PDF]
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H. W. Murray, K. C. Flanders, D. D. Donaldson, J. P. Sypek, P. J. Gotwals, J. Liu, and X. Ma Antagonizing Deactivating Cytokines To Enhance Host Defense and Chemotherapy in Experimental Visceral Leishmaniasis Infect. Immun., July 1, 2005; 73(7): 3903 - 3911. [Abstract] [Full Text] [PDF]
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 W. Z. Mehal, S. Z. Sheikh, L. Gorelik, and R. A. Flavell TGF-{beta} signaling regulates CD8+ T cell responses to high- and low-affinity TCR interactions Int. Immunol., May 1, 2005; 17(5): 531 - 538. [Abstract] [Full Text] [PDF]
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 S. M. Wahl, J. Swisher, N. McCartney-Francis, and W. Chen TGF-{beta}: the perpetrator of immune suppression by regulatory T cells and suicidal T cells J. Leukoc. Biol., July 1, 2004; 76(1): 15 - 24. [Abstract] [Full Text] [PDF]
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N. E. Rodriguez, H. K. Chang, and M. E. Wilson Novel Program of Macrophage Gene Expression Induced by Phagocytosis of Leishmania chagasi Infect. Immun., April 1, 2004; 72(4): 2111 - 2122. [Abstract] [Full Text] [PDF]
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S. Stager, J. Alexander, K. C. Carter, F. Brombacher, and P. M. Kaye Both Interleukin-4 (IL-4) and IL-4 Receptor {alpha} Signaling Contribute to the Development of Hepatic Granulomas with Optimal Antileishmanial Activity Infect. Immun., August 1, 2003; 71(8): 4804 - 4807. [Abstract] [Full Text] [PDF]
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A. Mookerjee, P. C. Sen, and A. C. Ghose Immunosuppression in Hamsters with Progressive Visceral Leishmaniasis Is Associated with an Impairment of Protein Kinase C Activity in Their Lymphocytes That Can Be Partially Reversed by Okadaic Acid or Anti-Transforming Growth Factor {beta} Antibody Infect. Immun., May 1, 2003; 71(5): 2439 - 2446. [Abstract] [Full Text] [PDF]
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K. R. Gantt, S. Schultz-Cherry, N. Rodriguez, S. M. B. Jeronimo, E. T. Nascimento, T. L. Goldman, T. J. Recker, M. A. Miller, and M. E. Wilson Activation of TGF-{beta} by Leishmania chagasi: Importance for Parasite Survival in Macrophages J. Immunol., March 1, 2003; 170(5): 2613 - 2620. [Abstract] [Full Text] [PDF]
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S. Ahmed, M. Colmenares, L. Soong, K. Goldsmith-Pestana, L. Munstermann, R. Molina, and D. McMahon-Pratt Intradermal Infection Model for Pathogenesis and Vaccine Studies of Murine Visceral Leishmaniasis Infect. Immun., January 1, 2003; 71(1): 401 - 410. [Abstract] [Full Text] [PDF]
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H. W. Murray, C. M. Lu, S. Mauze, S. Freeman, A. L. Moreira, G. Kaplan, and R. L. Coffman Interleukin-10 (IL-10) in Experimental Visceral Leishmaniasis and IL-10 Receptor Blockade as Immunotherapy Infect. Immun., November 1, 2002; 70(11): 6284 - 6293. [Abstract] [Full Text] [PDF]
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 A. Somanna, V. Mundodi, and L. Gedamu Functional Analysis of Cathepsin B-like Cysteine Proteases from Leishmania donovani Complex. EVIDENCE FOR THE ACTIVATION OF LATENT TRANSFORMING GROWTH FACTOR beta J. Biol. Chem., July 5, 2002; 277(28): 25305 - 25312. [Abstract] [Full Text] [PDF]
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B. Finkel-Jimenez, M. Wuthrich, and B. S. Klein BAD1, an Essential Virulence Factor of Blastomyces dermatitidis, Suppresses Host TNF-{alpha} Production Through TGF-{beta}-Dependent and -Independent Mechanisms J. Immunol., June 1, 2002; 168(11): 5746 - 5755. [Abstract] [Full Text] [PDF]
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H. W. Murray Clinical and Experimental Advances in Treatment of Visceral Leishmaniasis Antimicrob. Agents Chemother., August 1, 2001; 45(8): 2185 - 2197. [Full Text] [PDF]
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J. A. Streit, T. J. Recker, F. G. Filho, S. M. Beverley, and M. E. Wilson Protective Immunity Against the Protozoan Leishmania chagasi Is Induced by Subclinical Cutaneous Infection with Virulent But Not Avirulent Organisms J. Immunol., February 1, 2001; 166(3): 1921 - 1929. [Abstract] [Full Text] [PDF]
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N. Gomez-Escobar, W. F. Gregory, and R. M. Maizels Identification of tgh-2, a Filarial Nematode Homolog of Caenorhabditis elegans daf-7 and Human Transforming Growth Factor beta , Expressed in Microfilarial and Adult Stages of Brugia malayi Infect. Immun., November 1, 2000; 68(11): 6402 - 6410. [Abstract] [Full Text] [PDF]
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 C. H. Little, G. M. Georgiou, G. Fey, B. Ravindran, J. Levine, H. Ogedegbe, H. Yamase, and R. E. Cone Detection of Antigen-Specific Human Serum Proteins Related to the T-Cell Receptor in Infectious Disease and in an Immune Response to Milk Proteins or Chemicals Experimental Biology and Medicine, September 1, 2000; 224(4): 264 - 272. [Abstract] [Full Text]
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C. H. Little, G. M. Georgiou, A. Marceglia, H. Ogedgebe, R. E. Cone, and D. Mazza Measurement of T-Cell-Derived Antigen Binding Molecules and Immunoglobulin G Specific to Candida albicans Mannan in Sera of Patients with Recurrent Vulvovaginal Candidiasis Infect. Immun., July 1, 2000; 68(7): 3840 - 3847. [Abstract] [Full Text] [PDF]
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D. E. Jones, L. U. Buxbaum, and P. Scott IL-4-Independent Inhibition of IL-12 Responsiveness During Leishmania amazonensis Infection J. Immunol., July 1, 2000; 165(1): 364 - 372. [Abstract] [Full Text] [PDF]
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C. D. Mills, K. Kincaid, J. M. Alt, M. J. Heilman, and A. M. Hill M-1/M-2 Macrophages and the Th1/Th2 Paradigm J. Immunol., June 15, 2000; 164(12): 6166 - 6173. [Abstract] [Full Text] [PDF]
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S. C. Smelt, S. E. J. Cotterell, C. R. Engwerda, and P. M. Kaye B Cell-Deficient Mice Are Highly Resistant to Leishmania donovani Infection, but Develop Neutrophil-Mediated Tissue Pathology J. Immunol., April 1, 2000; 164(7): 3681 - 3688. [Abstract] [Full Text] [PDF]
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N. A. Gomes, C. R. Gattass, V. Barreto-de-Souza, M. E. Wilson, and G. A. DosReis TGF-{beta} Mediates CTLA-4 Suppression of Cellular Immunity in Murine Kalaazar J. Immunol., February 15, 2000; 164(4): 2001 - 2008. [Abstract] [Full Text] [PDF]
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B. Jiang, M. Jordana, Z. Xing, F. Smaill, D. P. Snider, R. Borojevic, D. Steele-Norwood, R. H. Hunt, and K. Croitoru Replication-Defective Adenovirus Infection Reduces Helicobacter felis Colonization in the Mouse in a Gamma Interferon- and Interleukin-12-Dependent Manner Infect. Immun., September 1, 1999; 67(9): 4539 - 4544. [Abstract] [Full Text] [PDF]
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G. Szalay, C. H. Ladel, C. Blum, L. Brossay, M. Kronenberg, and S. H. E. Kaufmann Cutting Edge: Anti-CD1 Monoclonal Antibody Treatment Reverses the Production Patterns of TGF-{beta}2 and Th1 Cytokines and Ameliorates Listeriosis in Mice J. Immunol., June 15, 1999; 162(12): 6955 - 6958. [Abstract] [Full Text] [PDF]
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