| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, Calcutta, India
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-activated macrophages. In a
45-day BALB/c mouse model of visceral leishmaniasis, complete
elimination of spleen parasite burden was achieved by cystatin in
synergistic activation with a suboptimal dose of IFN-. In contrast
to the case with promastigotes, cystatin and IFN- inhibited the
growth of amastigotes in macrophages. Although in vitro cystatin
treatment of macrophages did not induce any NO generation,
significantly enhanced amounts of NO were generated by macrophages of
cystatin-treated animals. Their splenocytes secreted soluble factors
required for the induction of NO biosynthesis, and the increased NO
production was paralleled by a concomitant increase in antileishmanial
activity. Moreover, splenocyte supernatants treated with
anti-IFN- or anti-TNF-
Abs suppressed inducible NO
generation, whereas i.v. administration of these anticytokine Abs along
with combined therapy reversed protection against infection. mRNA
expression and flow cytometric analysis of infected spleen cells
suggested that cystatin and IFN- treatment, in addition to greatly
reducing parasite numbers, resulted in reduced levels of IL-4 but
increased levels of IL-12 and inducible NO synthase. Not only was this
treatment curative when administered 15 days postinfection, but it also
imparted resistance to reinfection. These studies provide a promising
alternative for protection against leishmaniasis with a switch of
CD4+ differentiation from Th2 to Th1, indicative of
long-term resistance. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
An attractive target for new therapy is a family of cathepsin L-like
and cathepsin B-like cysteine proteases, found in all species of
Leishmania examined and required for parasite growth and
virulence (3, 4). Elimination of cathepsin L-like gene
families by homologous recombination resulted in loss of virulence in
highly susceptible BALB/c mice (3, 4), whereas deletion of
the cathepsin B-like gene led to reduced survival of parasites in
macrophages (3, 5). Therefore, it was thought worthwhile
to investigate the possible role of cystatin, a natural cysteine
protease inhibitor, in modulating L. donovani infection.
However, one major complicating factor in chemotherapeutic treatment is
the depressed immune functions exhibited by the victims of disseminated
leishmaniasis. Appropriate T cell-mediated responses are of primary
importance in an effective host defense in visceral leishmaniasis
(6). There is also a correlation between host control over
parasite replication and the activation of Th1-type effector cells
that produce the macrophage-activating cytokines IFN- and IL-2
(7). Both in humans (8, 9) and in
experimental animal models (10, 11), L.
donovani infection is accompanied by parasite-specific immune
depression mediated by T cells and macrophages, thereby preventing
spontaneous cure and the development of protective immunity. Moreover,
nonhealing infections in susceptible strains of mice such as BALB/c are
accompanied by the preferential expansion of IL-4-producing Th2-type
cells (12). Therefore, immunostimulation of the infected
host is an effective strategy for circumventing immunosuppression.
IFN- could be used as an immunopotentiator for augmenting the
capacity of macrophages to eliminate Leishmania
infection. Moreover, the safety of parenteral human rIFN- has been
demonstrated for various diseases, including leprosy, cancer, and AIDS
(13, 14, 15). However, IFN- treatment alone is not
sufficient to promote a Th1 response and/or suppress in vivo activation
of Th2 cells because even continued IFN- therapy has little effect
on the eventual outcome of a Leishmania major infection in
BALB/c mice (16).
Studies to date suggest that the parasite cysteine proteases may
themselves help to ensure a Th2-like response in BALB/c mice that leads
to parasite proliferation (17). Thus, inhibition of such
cysteine proteases might slow or even prevent parasite proliferation
and allow the host immune system to function effectively and confer
protective immunity by effecting a switch in CD4+
T cell differentiation from Th2 to Th1 (18). Tight binding
and reversible natural inhibitors that belong to the cystatin
superfamily regulate cysteine proteases. Experiments have revealed that
chicken cystatin, the best-characterized inhibitor of cysteine
proteases (19, 20), stimulated NO (3)
production by IFN--activated macrophages (21). Because
NO is the key effector molecule for antileishmanial activity, we tested
the capacity of cystatin to elicit a Th1-mediated adaptive response and
to prevent and treat infections with L. donovani, a lethal
Th2-mediated disease, in BALB/c mice. Our investigation was aimed
toward elucidating the dual role of cystatin in suppressing the
functional differentiation of Th2-type CD4+ T
cells and in turn augmenting Th1 response, together with the ability to
up-regulate NO, the latter property being totally unrelated to the
former. Here we present data demonstrating that cystatin can synergize
with subthreshold concentrations of IFN- in inducing Th2-Th1
conversion and generation of NO, resulting in abrogation of parasite
infection.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
L. donovani strain AG83 (MHOM/IN/1983/AG83) was isolated from an Indian patient with kala azar (22). The strain was maintained in BALB/c mice by i.v. passage every 6 wk. L. donovani promastigotes for use in experiments were obtained by allowing isolated splenic amastigotes to transform in parasite growth medium for 72 h at 22°C. The growth medium consisted of medium 199 (Life Technologies, Grand Island, NY) supplemented with 10% (v/v) FCS. Soluble leishmanial Ag (SLA)3 was prepared from promastigotes by freeze-thawing the cell suspension (5 x 109 cells/ml in 100 mM Tris-HCl, pH 8.0, containing 1 mM EDTA, 50 µg/ml leupeptin, and 1.6 mM PMSF) three to five times followed by sonication for 5 x 45 s at 20 kilocycles/min in an ice bath. The contents were centrifuged at 10,000 x g for 20 min, and the supernatant was dialyzed, filtered, and stored at -70°C. It was used at a concentration of 20 µg/ml.
Macrophages
Macrophages were collected by peritoneal lavage from mice (BALB/c; 20–25 g) given i.p. injection of 0.5 ml 4% thioglycolate broth 5 days before harvest and were used as described earlier (23). The culture medium consisted of RPMI 1640 supplemented with 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS. A total of >90% of the cell preparation was identified as macrophages by microscopic observation, and the macrophages were routinely found to be >95% viable by trypan blue exclusion.
Splenocyte culture
Spleens were aseptically removed and teased into single-cell suspensions in RPMI 1640 supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), 2-ME (50 µM), L-glutamine (2 µM), HEPES (10 µM), and 10% (v/v) FCS (24). RBCs were removed by lysis with 0.83% (w/v) NH4Cl. The remaining cells were washed twice with culture medium, and the viable mononuclear cell number was determined by counting trypan blue-unstained cells in a hemocytometer. Splenocyte suspensions (1 x 106 cells/ml) were dispensed into 35-mm tissue culture plates and incubated at 37°C in 5% CO2 for 48 h. Cells were harvested for RT-PCR and flow cytometric analysis. Culture supernatants were removed and frozen at -20°C until further use.
In vitro L. donovani proliferation assay
Promastigotes of L. donovani were cultured in medium
199 containing 10% (v/v) FCS with or without chicken cystatin (egg
white, E2801% = 8.7; Sigma, St.
Louis, MO) and IFN- for 72 h at 22°C. The proliferation of
promastigotes was evaluated by counting them every 24 h in a
hemocytometer.
In vitro assay of L. donovani growth in macrophages
Promastigotes were used to infect cultures of adherent
macrophages on glass cover slips (18 mm2; 5
x 105 macrophages/cover slip) in 0.5 ml of RPMI
1640/10% FCS at a ratio of 10 parasites/macrophage. Infection was
allowed to proceed for 4 h, unphagocytosed parasites were removed
by washing with medium, and cells were resuspended in RPMI 1640/10%
FCS with or without chicken cystatin and IFN-, along with each
component added alone, for 48 h at 37°C. Cells were then fixed
in methanol and stained with Giemsa stain for determination of
intracellular parasite numbers. The mean percentages of survival in
treated cultures were calculated on the basis of considering the number
of Leishmania in untreated cultures as 100%.
Determination of NO concentration
NO, quantified by the accumulation of nitrite in the culture medium, was measured according to the method of Ding et al. (25). Briefly, 100 µl of culture supernatants was mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 2.5% H3PO4) and incubated at room temperature for 10 min. Absorbance at 540 nm was then measured. Sodium nitrite (NaNO2) diluted in culture medium was used as a standard.
Establishment and assessment of infection
Mice were inoculated with L. donovani AG83 through
the tail vein. Initially, 107 promastigotes/mouse
were injected, and for reinfection the same number of promastigotes was
injected 60 days after the first infection. At 1 and 15 days after
inoculation of parasites, cystatin, either alone or in combination with
a suboptimal dose of IFN- (104 U), was
injected into the tail vein in various doses for 4 consecutive days.
Forty-five days after the start of infection, animals were sacrificed,
and their spleens were weighed. Multiple spleen impression smears were
prepared and stained with Giemsa stain. Spleen parasite burdens,
expressed as Leishman-Donovan units (LDU), were calculated as the
number of amastigotes per 1000 nucleated cells x spleen weight
(grams) (26).
RT-PCR analysis of cytokine mRNA
RT-PCR was performed to determine the cytokine profile of mRNA for IL-12 p40, IL-4, inducible NO synthase (iNOS), and hypoxanthine phosphoribosyltransferase (HPRT). Reverse transcription of 1 µg of RNA was performed according to the manufacturer’s protocol for the Superscript One-Step RT-PCR system (Life Technologies). The primers for all these genes have been published (27). After the appropriate number of PCR cycles, the amplified cDNA was separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining.
Flow cytometric analysis
Mice were infected with L. donovani
(107 parasites/mouse) and treated with cystatin
plus IFN- 15 days after infection. For intracellular IL-4 staining,
splenocytes were isolated 45 days postinfection, plated aseptically
(1 x 106 cells/ml), and stimulated with SLA
(20 µg/ml) for 48 h, whereas for IL-12 p40 staining, purified
splenic macrophages were stimulated likewise. Cells were incubated with
monensin (Sigma) (2 µM) for 4 h, washed in PBS containing 0.1%
NaN3/1% FCS at 4°C, and fixed with
paraformaldehyde. They were then permeabilized with saponin and treated
with PE-conjugated anti-mouse IL-12 p40 and IL-4 mAbs. Cells were
analyzed on a FACSCalibur cytofluorometer using the CellQuest software
(BD Biosciences, San Jose, CA). The area of positivity was determined
using an isotype-matched mAb.
Statistical analysis
The significance of the data was evaluated by the two-tailed t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To determine whether chicken cystatin, a natural inhibitor of
cysteine proteases, could modulate the infection of macrophages by
L. donovani and NO synthesis, macrophages were subjected to
treatment with various agents (Fig. 1
).
Neither cystatin (0.5 µM) nor IFN- (100 U/ml) when added alone
could induce marked production of nitrite after 48 h of incubation
(1.51 ± 0.23 nmol/106 cells and 3.32
± 0.35 nmol/106 cells, respectively). However,
the nitrite level was significantly increased (6-fold) when cystatin
was added simultaneously with IFN-, as compared with IFN- alone.
Moreover, although L. donovani infection caused a
suppression of NO production in IFN--activated macrophages
(1.12 ± 0.17 nmol/106 cells), combined
treatment of infected macrophages with cystatin and IFN- for 48
h produced 12.18 ± 1.24 nmol
NO2-/106
cells. The up-regulation of NO by cystatin in IFN--stimulated
macrophages was not due to the presence of LPS as a contaminant because
preincubation of cystatin with polymyxin B, an LPS inhibitor, did not
alter NO production. However, the NO-inducing effect in the control
experiment, consisting of LPS preincubated with polymyxin B, was
completely abolished.
|
on the progression of leishmaniasis
Because cystatin together with IFN- can up-regulate NO, the
effector molecule responsible for antileishmanial activity, it was
thought worthwhile to evaluate the efficacy of the combination
chemotherapy in a BALB/c mouse model of visceral leishmaniasis. BALB/c
mice (6 wk old, 
20 g) were infected i.v. with L. donovani
AG83 promastigotes as described in Materials and Methods.
The infection was allowed to proceed for 45 days, during which spleen
weight increased from 100.46 ± 11.17 mg to 1650.16 ± 139.92
mg. Two types of drug treatment schedule were chosen, one at the onset
of infection and the other at established infection. IFN- or
cystatin or a combination of both were administered i.v. daily for 4
consecutive days beginning 1 day after infection (onset of infection)
and 15 days after infection (established infection). Various cystatin
dosages were used (from 0.1 to 10 mg/kg/day), with a constant dose of
104 U of IFN- per mouse. Three i.p. injections
of >105 U IFN- alone, given every other day,
halted the visceral replication of L. donovani
(7); however, treatment with four injections of
104 U produced only modest inhibition and no
killing (7). Therefore, a dose of
104 U IFN- was selected to use in combination
with cystatin. All animals were sacrificed 45 days after inoculation,
and the degree of leishmanicidal potency of cystatin or IFN- or both
was assessed in terms of spleen weight and splenic amastigote burden.
The combination therapy with cystatin and IFN- was found to be much
more potent than either component in terms of 100% parasite
suppression. In the case of both onset of infection and established
infection, a dose of 5 mg/kg/day of cystatin together with
104 U IFN- per mouse given for 4 days greatly
reduced and possibly eliminated all parasites from the spleen, with
subsequent reduction in its weight to nearly normal levels (Fig. 2). Absence of parasites in the spleen
was further confirmed by culturing spleen specimens in transformation
medium at 22°C for 96 h. Cystatin or IFN- alone at the same
dose had little effect.
|
on the growth of promastigotes
To ascertain the possibility that cystatin along with IFN-
might exert a direct cytotoxic effect on L. donovani, the
influence of the combination regimen on the in vitro proliferation of
L. donovani promastigotes was assessed (Fig. 3). L. donovani proliferated
comparably regardless of the presence or absence of cystatin and
IFN- in the growth medium.
|
To assess the effects of combination treatment on L.
donovani amastigotes, the inhibition of amastigote multiplication
within macrophages by IFN- and cystatin was compared with that of
either component given alone. Leishmania-infected cultures
were treated with cystatin or IFN- or both for 48 h at 37°C
in macrophage culture medium. Controls were placed in medium alone.
After drug treatment, cells were washed and placed in drug-free medium
for an additional 20 h. They were then stained, and viable
parasites per macrophage were counted. It is difficult to differentiate
viable from nonviable amastigotes immediately after treatment.
Therefore, a 20-h interval between drug treatment and staining was
chosen to allow for the disposal of dead parasites. Combination IFN-
and cystatin treatment was most effective with an
IC50 of 4.3 µg/ml for cystatin, whereas
cystatin alone had no inhibitory effect (Fig. 4A). To test whether reactive
NO is involved in the growth inhibition of amastigotes, we used the
specific NO synthase inhibitor,
NG-monomethyl-L-arginine
(NMMA). Complete reversal of antileishmanial effect was observed upon
the addition of NMMA (Fig. 4A). Moreover,
NO2- release by macrophages
treated with a suboptimal dose of IFN- (100 U/ml) progressively
increased with increasing cystatin concentration until 1 µM, when it
reached a maximum level (Fig. 4B). In contrast, cystatin
when added to macrophage culture alone was unable to induce any NO
production. There was no toxic effect from cystatin on macrophages in
vitro at the highest concentration used for treatment. Therefore, these
results suggest that increased antileishmanial activity after
combination treatment with IFN- and cystatin may be correlated with
increased production of NO.
|
Because cystatin was unable to induce any NO production in
peritoneal macrophages, we set about to determine whether the
generation of NO by macrophages isolated from cystatin-treated mice
also corroborated their in vitro effect. Peritoneal macrophages
isolated from BALB/c mice given i.v. injections of cystatin produced
significantly higher levels of
NO2- (13.02 ± 1.56
nmol/106 cells) compared with control untreated
counterparts (1.20 ± 0.18 nmol/106 cells;
p < 0.001; Fig. 5A). These cells also
inhibited the replication of L. donovani (Fig. 5B). NMMA, the NO synthase inhibitor, was found to reverse
the stimulatory effect of cystatin. Moreover, macrophages isolated from
BALB/c mice given i.v. administration of 5 mg/kg/day cystatin and
104 U IFN- produced much higher levels of
NO2- (27.50 ± 2.47
nmol/106 cells) compared with their in vitro
counterparts (12.18 ± 1.24 nmol/106 cells;
p < 0.001; Fig. 5A). The level of nitrite
produced by various regimens was reflected in the expression of iNOS
mRNA, which, after isolation of total RNA, was subjected to RT-PCR
analysis as described in Materials and Methods (Fig. 5C).
|
To look into the mechanism of regulation of NO in the in vivo
situation, spleen cells were isolated from mice given i.v. injections
of cystatin. It was found that the generation of NO by peritoneal
macrophages from control untreated mice was increased after culturing
them in supernatants of splenocytes obtained from cystatin-administered
animals (12.10 ± 1.10 compared with 1.20 ± 0.18
nmol/106 cells; Fig. 6). The increase in NO production was
paralleled by an increase in the antileishmanial activity of these
cells (Fig. 6). Splenocyte supernatant from mice treated with both
cystatin and IFN- had a considerably stronger effect on NO
production by virgin macrophages (data not shown). In an effort to
determine the involvement of specific cytokines in the in vivo
activation of macrophages by cystatin, peritoneal macrophages from
untreated mice were cultured in splenocyte supernatants obtained from
either cystatin-treated or PBS-treated mice in the presence or absence
of mAbs against various cytokines. It was found that the addition of
either anti-IFN- or anti-TNF- greatly reduced the ex vivo
NO2- production as well as the
antileishmanial activity (Fig. 6). No detectable levels of nitrite were
found in splenocyte supernatants. To further ascertain the roles of
IFN- and TNF- in vivo, mice were i.v. administered 5 mg/kg/day of
cystatin alone or in combination with 200 µg of anti-IFN-,
anti-TNF-, or control hamster IgG at the onset of infection.
Spleen parasite burden was then determined 45 days after infection
(Table I). Anticytokine mAbs reactive
against either IFN- or TNF- greatly reduced cystatin-mediated
protection, whereas the parasite load in mice treated with cystatin
plus control Abs was similar to that in mice treated with cystatin
alone. As an additional control, neutralizing mAbs to other cytokines,
IL-1, IL-1
, and IL-6, were also examined. However, these
cytokines were ineffective in stimulating NO production by macrophages
in culture (Fig. 6), and administration of mAbs against these cytokines
had little effect on cystatin protection.
|
|
To gain an insight into the levels of various cytokines and iNOS
after combination chemotherapy, we examined the flow cytometric pattern
as well as the mRNA expression for a Th1 cytokine (e.g., IL-12), a Th2
cytokine (e.g., IL-4), and iNOS, which catalyzes the generation of NO
from L-arginine and mediates the leishmanicidal activity of
treated macrophages. Because both the treatment protocols (onset of
infection as well as established infection) for combined therapy
resulted in almost complete suppression of spleen parasite burden, we
selected the therapeutic treatment of IFN- and cystatin (15 days
after injection) for the measurement of cytokines. RT-PCR analysis of
cytokine mRNA levels confirmed that susceptible BALB/c mice treated
with a combined dose of IFN- and cystatin could reverse an
established Th2 response into a dominant Th1 response (Fig. 7A). Thus,
Leishmania-infected cells from mice treated with IFN- and
cystatin contained more IL-12 p40 mRNA and less IL-4 mRNA than those
from infected untreated controls. Mice treated with either component
alone at that dose exhibited little amounts of IL-12 p40 mRNA, although
administration of cystatin alone showed some increase over untreated
ones. Similarly, mice treated with both IFN- and cystatin also had
low levels of parasite-specific IgG1, a Th2-associated isotype, but
higher levels of parasite-specific IgG2a (data not shown). In addition,
the iNOS mRNA expression, which was very low in the spleen cells of
L. donovani-infected mice, was significantly induced by the
combined therapy of IFN- and cystatin.
|
. For this, splenocytes and purified macrophages
were separately stimulated in vitro with SLA and permeabilized, and a
one-color flow cytometry for IL-12 p40 and IL-4, respectively, was
performed. Results reveal that a higher frequency of cells from mice
subjected to combination chemotherapy (9.13%; Fig. 7
Be)
produced IL-12 p40 compared with untreated ones, in which the
intracellular IL-4 status in CD4+ T cells was
14.24% (Fig. 7Bh). Moreover, mice treated with cystatin
produced more IL-12 p40 and less IL-4 than those treated with a
suboptimal dose of IFN-. These results suggest that Th2 functions
have been effectively suppressed in BALB/c mice by combination
chemotherapy so that Th1 function could be activated sufficiently to
resolve infection with L. donovani.
Reinfection with L. donovani in BALB/c mice treated
with cystatin and IFN-
To further ascertain that the combination regimen has conferred
long-standing immunity, infected BALB/c mice treated with cystatin and
IFN- were later reinfected i.v. 60 days after primary infection.
Spleen parasite burden in the reinfected animals progressed prominently
in PBS-treated BALB/c mice, whereas cystatin- and IFN--treated mice
were largely resistant, as observed up to 7 wk (Fig. 8). Thus, infected BALB/c mice subjected
to a combination chemotherapy with cystatin (5 mg/kg/day) and a
suboptimal dose of IFN- (104 U/mouse) acquired
protective immunity.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. The impetus for this combination
chemotherapy was the earlier observation that cystatin could
up-regulate NO release from IFN--activated macrophages
(21). NO produced by cytokine-activated macrophages during
parasite infections is known to play a central role in the control of
parasite killing (29). That the mice treated with cystatin
and a suboptimal dose of IFN- were indeed cured was shown by the
reversion of spleen size to near-normal levels and the complete
suppression of spleen parasite burden. Moreover, this therapy was
effective in mice with ongoing infections in which a nonprotective Th2
response had been established. After treatment and the resulting
resolution of parasitism, the cytokine profile in these mice indicated
a switch to a protective Th1 pattern. Treatment with either component
alone had very little effect. The superior efficacy of the combination
treatment in eliminating intracellular amastigotes of L.
donovani in both an in vitro macrophage model and an in vivo mouse
model of visceral leishmaniasis demonstrated the effectiveness of this
approach. Neither the components nor the dosage used proved toxic to
macrophages, as evidenced by their viability (trypan blue exclusion)
and the release of lactate dehydrogenase from cells (data not shown).
During the experimental period, all the animals remained healthy,
without any apparent weight loss.
Increased microbicidal activity of cystatin- and IFN--activated
macrophages is achieved by a nitrogen-dependent mechanism. Enhanced NO
generation resulted upon incubating cystatin with IFN--activated
mouse peritoneal macrophages in vitro, and the leishmanicidal activity
acquired correlated with the induction of NO production. It may be
mentioned that Engel et al. (30) showed a parasiticidal
effect of synthetic cysteine protease inhibitors on intracellular
Trypanosoma cruzi by inactivation of cruzain, a major
protease of the parasite. The reason cystatin may induce an increase in
NO synthesis from activated macrophages remains unclear. However, it is
known that the biological effect of cystatin as a synergic NO inducer
is not related to the inhibition of cysteine protease activity because
the irreversible and structurally unrelated cysteine protease inhibitor
E64 did not induce any increase in nitrite level (21).
Also, from the structural standpoint, the observation that saturation
of cystatin-inhibitory sites by reduced-alkylated papain did not
interfere with cystatin-induced NO release from activated macrophages
suggests the noninvolvement of inhibitory sites in the process
(21). Although our in vitro studies demonstrated the
inability of cystatin alone to induce NO synthesis in macrophages, our
in vivo studies argue against this. Thus, cystatin given in vivo
induced NO synthesis in peritoneal macrophages, along with an
enhancement of inhibition of parasite growth. This suggests that NO
generation by macrophages may be an indirect effect of cystatin
activation requiring the cooperation of macrophages and other cells of
the immune system. The ability of cystatin to induce NO in synergy with
IFN- in vitro is suggestive of the compensatory role of IFN- for
the effector molecules of immune system cells. Therefore, protection
against infections with L. donovani induced by i.v.
administration of cystatin and IFN- correlated with the development
of activated macrophages secreting NO. Moreover, strong evidence for
the participation of both IFN- and TNF- in cystatin regulation of
NO production and protection against leishmaniasis in vivo was
suggested by the observation that anti-IFN- or anti-TNF-
mAbs could effectively block the significant increase in NO production
by normal untreated macrophages when activated with the splenocyte
supernatant from cystatin-treated mice. Further, the in vivo
administration of anti-IFN- or anti-TNF- mAbs could block
the induction of cystatin-mediated protection against L.
donovani infection. The immunologic stimulus for the production of
cytotoxic levels of NO in vitro by murine macrophages is the
synergistic effect of IFN- and exogenous TNF or microbes and
microbial products to stimulate endogenous release of TNF- by
macrophages (31, 32, 33, 34). Exogenous agent-stimulated TNF acts
in an autocrine fashion to amplify the actual synthesis and release of
NO by IFN--primed cells (33, 35). The data obtained
with anti-IFN- and anti-TNF- mAbs in cystatin-treated
mice suggest that a similar synergism exists in vivo. It is likely that
the in vivo administration of cystatin stimulates TNF synthesis, which
in turn triggers IFN- production by spleen lymphocytes. Therefore,
IFN- and TNF- are the principal agents in macrophage activation
by cystatin in vivo, and the protection afforded by combined treatment
against experimental visceral leishmaniasis ultimately depended on the
physiologic generation of NO. Indeed, cystatin has been shown very
recently to cause production of increased amounts of TNF- in
IFN--primed macrophages (36). Because LPS alone can
induce NO production, we examined the effect of the LPS inhibitor
polymyxin B and confirmed that the LPS contamination of cystatin used
was insufficient to induce NO production from macrophages.
Several studies have emphasized the importance of Th1 cytokines in host
defense mechanisms against infection caused by various microbial
pathogens (37). Some insight as to how therapy with
IFN- and cystatin influences the production of various cytokines and
macrophage NO was gained by examination of mRNA levels and flow
cytometric analysis of spleen cells 45 days after infection. Transcript
levels of IL-4 were reduced in mice given combined therapy, whereas
those for iNOS and IL-12 p40 were significantly elevated. Flow
cytometric analysis of cells secreting cytokines also corroborated the
mRNA transcript results. Because IL-4 can suppress both NO and IL-12
p40 production, it is possible that a reduction in the IL-4 level after
combined treatment may assist IFN- in promoting both macrophage NO
and IL-12 production. In contrast, IL-12 induces IFN- production and
cytotoxic activity by NK and T cells (38) and can initiate
the differentiation of Th1 cells from naive T cells
(39, 40, 41). IL-12 enhances IFN- production by Th1 clones
(42) and promotes the proliferation of Th1 but not Th2
cells (39). However, because IFN- can prime macrophages
to produce IL-12 p40 (43), it is possible that the
administration of exogenous IFN- as done in this study acts as a
positive stimulus for enhanced IL-12 production, which in turn may
promote both higher IFN- production and Th1 cell development.
Taken together, the findings in this report support the view that a complex series of cytokines and cell-mediated interactions contributes to the host’s innate response to visceral leishmaniasis. The stimulatory capacity of cystatin may also confound studies involving site-specific targeting for enhanced macrophage activation. The absence of any host cell or animal toxicity at therapeutic doses suggests that the parasites are more susceptible to inhibitor, perhaps because of the redundancy of cysteine proteases in mammalian cells vs the parasite. It may be mentioned that administration of a cathepsin B-specific inhibitor to highly susceptible BALB/c mice resulted in a switch from the usual inefficient Th2 cytokine response to a Th1 response that cleared the L. major infection (18). In contrast, the administration of vinyl sulfone inhibitor, an irreversible cysteine protease inhibitor, to a mouse model of cutaneous leishmaniasis did not result in a switch from Th2 to Th1 cytokines. It exerted its antileishmanial effect by inhibiting parasite replication (44). In the present study, cure as well as resistance acquired by susceptible BALB/c mice due to combination chemotherapy were attributed to two mechanisms: 1) the direct action of cystatin for the induction of the NO that kills the parasite; and 2) the switch of CD4+ T cell-mediated immune responses from the disease-promoting Th2 to the protective Th1 type. The switching of an established inappropriate Th response to an appropriate one has implications not only for the treatment of nonhealing leishmaniasis but also for the treatment of other chronic infectious diseases.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Pijush K. Das, Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700032, India.
3 Abbreviations used in this paper: SLA, soluble leishmanial Ag; iNOS, inducible NO synthase; HPRT, hypoxanthine phosphoribosyl transferase; NMMA, NG-monomethyl-L-arginine; LDU, Leishman-Donovan units.
Received for publication September 7, 2000. Accepted for publication December 29, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, tissue immune reaction, and response to treatment with IL-2 and IFN-. J. Immunol. 138:2290.[Abstract]
or interleukin 4 during the resolution or progression of murine leishmaniasis: evidence for expansion of distinct helper T cell subsets. J. Exp. Med. 169:59. differentially regulates interleukin-12 and interleukin-10 production in leprosy. J. Clin. Invest. 99:336.[Medline]
and lipopolysaccharide. Cancer Immunol. Immunother. 45:250.[Medline]
. Ann. Intern. Med. 108:36.
-independent mechanism. J. Exp. Med. 171:115.-activated mouse peritoneal macrophages. J. Biol. Chem. 271:28077. synergizes with IFN- in mediating killing of Leishmania major through the induction of nitric oxide. J. Immunol. 145:4306.[Abstract]
B and inducible isoform of nitric oxide synthase induction by interferon- and tumor necrosis factor- in INS-1 cells. J. Cell. Physiol. 184:46.[Medline]
-stimulated macrophages by induction of TNF-. J. Immunol. 145:4290.[Abstract]
triggers antitoxoplasmal activity of IFN--primed macrophages. J. Immunol. 147:2340.[Abstract]
induced L-arginine dependent toxoplasmastatic activity in murine peritoneal macrophages is mediated by endogenous tumor necrosis factor-. J. Immunol. 148:568.[Abstract]
-activated mouse peritoneal macrophages via cytokine synthesis. Eur. J. Biochem. 266:1111.[Medline]
(IFN-) production during differentiation of human T helper (Th) cells and transient IFN- production in established Th2 cell clones. J. Exp. Med. 179:1273. production by mouse T helper clones that are unresponsive to B7 costimulation. J. Exp. Med. 180:223. in monocytic cells. J. Exp. Med. 183:147.This article has been cited by other articles:
|
|
 |
|
  J. Covert, A. J. Mathison, L. Eskra, M. Banai, and G. Splitter Brucella melitensis, B. neotomae and B. ovis Elicit Common and Distinctive Macrophage Defense Transcriptional Responses Experimental Biology and Medicine, December 1, 2009; 234(12): 1450 - 1467. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Bryson, S. Besteiro, H. A. McGachy, G. H. Coombs, J. C. Mottram, and J. Alexander Overexpression of the Natural Inhibitor of Cysteine Peptidases in Leishmania mexicana Leads to Reduced Virulence and a Th1 Response Infect. Immun., July 1, 2009; 77(7): 2971 - 2978. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Bhattacharjee, G. Gupta, P. Bhattacharya, A. Mukherjee, S. B. Mujumdar, A. Pal, and S. Majumdar Quassin alters the immunological patterns of murine macrophages through generation of nitric oxide to exert antileishmanial activity J. Antimicrob. Chemother., February 1, 2009; 63(2): 317 - 324. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mukherjee, A. Ukil, and P. K. Das Immunomodulatory Peptide from Cystatin, a Natural Cysteine Protease Inhibitor, against Leishmaniasis as a Model Macrophage Disease Antimicrob. Agents Chemother., May 1, 2007; 51(5): 1700 - 1707. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ponte-Sucre, R. Vicik, M. Schultheis, T. Schirmeister, and H. Moll Aziridine-2,3-dicarboxylates, peptidomimetic cysteine protease inhibitors with antileishmanial activity. Antimicrob. Agents Chemother., July 1, 2006; 50(7): 2439 - 2447. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Holzmuller, D. Sereno, and J.-L. Lemesre Lower Nitric Oxide Susceptibility of Trivalent Antimony-Resistant Amastigotes of Leishmania infantum Antimicrob. Agents Chemother., October 1, 2005; 49(10): 4406 - 4409. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ukil, A. Biswas, T. Das, and P. K. Das 18{beta}-Glycyrrhetinic Acid Triggers Curative Th1 Response and Nitric Oxide Up-Regulation in Experimental Visceral Leishmaniasis Associated with the Activation of NF-{kappa}B J. Immunol., July 15, 2005; 175(2): 1161 - 1169. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. DE SOUZA DIAS, P. H. DA COSTA PINHEIRO, S. KATZ, M. R. M. D. SANTOS, and C. L. BARBIERI A RECOMBINANT CYSTEINE PROTEINASE FROM LEISHMANIA (LEISHMANIA) CHAGASI SUITABLE FOR SERODIAGNOSIS OF AMERICAN VISCERAL LEISHMANIASIS Am J Trop Med Hyg, February 1, 2005; 72(2): 126 - 132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Schierack, R. Lucius, B. Sonnenburg, K. Schilling, and S. Hartmann Parasite-Specific Immunomodulatory Functions of Filarial Cystatin Infect. Immun., May 1, 2003; 71(5): 2422 - 2429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
K. G. J. Pollock, K. S. McNeil, J. C. Mottram, R. E. Lyons, J. M. Brewer, P. Scott, G. H. Coombs, and J. Alexander The Leishmania mexicana Cysteine Protease, CPB2.8, Induces Potent Th2 Responses J. Immunol., February 15, 2003; 170(4): 1746 - 1753. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Paolucci, S. E. Burastero, P. Rovere-Querini, C. De Palma, S. Falcone, C. Perrotta, A. Capobianco, A. A. Manfredi, and E. Clementi Synergism of nitric oxide and maturation signals on human dendritic cells occurs through a cyclic GMP-dependent pathway J. Leukoc. Biol., February 1, 2003; 73(2): 253 - 262. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D.P. Dickinson SALIVARY (SD-TYPE) CYSTATINS: OVER ONE BILLION YEARS IN THE MAKING--BUT TO WHAT PURPOSE? Critical Reviews in Oral Biology & Medicine, November 1, 2002; 13(6): 485 - 508. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Holzmuller, D. Sereno, M. Cavaleyra, I. Mangot, S. Daulouede, P. Vincendeau, and J.-L. Lemesre Nitric Oxide-Mediated Proteasome-Dependent Oligonucleosomal DNA Fragmentation in Leishmania amazonensis Amastigotes Infect. Immun., July 1, 2002; 70(7): 3727 - 3735. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and the amount of NO2- in
the medium (
) were determined. The number of amastigotes per
macrophage and the amount of NO2- generated in
infected controls cultured in splenocyte supernatants of PBS-treated
mice were 4.53 ± 0.10 and 1.26 ± 0.18 nmol/106
cells, respectively. Values are means of three experiments; error bars
indicate SDs.
