| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1
*
Immunobiology Program, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, Brazil;
Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and
Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, GIPL
ceramide induced marked apoptosis in J774 cells and macrophages,
independent of nitric oxide secretion. When cells were preincubated
with the GIPL-derived glycan chain, addition of intact GIPL induced
macrophage apoptosis in the presence of IFN-. Synthetic
C2-dihydroceramide also induced
apoptosis in the presence of IFN-. Induction of apoptosis in
T. cruzi-infected macrophages by GIPL ceramide plus
IFN- led to increased parasite release compared with IFN-
treatment alone. Viable parasites released comprised both infective
trypomastigote and spheromastigote forms. These results identify a
novel pathway by which T. cruzi
glycosylphosphatidylinositol family molecules affect host macrophages,
with implications for the infectious process. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The structural features of T. cruzi GIPLs enable exposure of
a bioactive ceramide moiety to the signal-transducing machinery of host
cells. Ceramides were characterized as important intracellular
mediators of cell signaling through a range of host cell surface
receptors, including TNF-
(9) and IL-1ß (10) receptors, as well as
immunoregulatory molecules such as Fas (CD95) (11) and CD28 (12).
Ceramides mediate activation of the JNK-1 kinase in response to
stress-related stimuli (13), as well as cell cycle arrest or apoptosis
caused by cytokines or irradiation (9, 14, 15). However, ceramides were
also implicated in macrophage (M
) activation (16, 17) and in
lymphocyte costimulatory responses to CD28 engagement (12), indicating
that distinct or even opposite cellular responses to
ceramide-mobilizing agents arise, depending on context and
differentiation state of the responding cell (18).
Since M infection by protozoan parasites characterizes an early and
recurrent target for host innate and acquired effector immune responses
(19), it was of interest to investigate whether T. cruzi
GIPL affects M function. In this report, we investigated whether
this glycophosphosphingolipid from a protozoan pathogen subverts host
cellular responses due to induction of ceramide-controlled processes.
We show that, among other effects, GIPL-derived ceramide from T.
cruzi synergizes with the host cytokine IFN- to induce intense
M apoptosis. Under these conditions, viable intracellular parasites
are released into the extracellular medium. The results suggest that
ceramide-containing glycolipids from T. cruzi could be
implicated in the spread of the infectious process.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Primary M were derived from peritoneal lavage cells from
female BALB/c mice 6 to 8 wk old (Fiocruz Animal Section, Rio de
Janeiro, Brazil). After overnight culture, nonadherent cells were
removed, leaving adherent M. The J774.G8 murine M cell line,
originally recloned from the J774 cell line (20), was maintained by
weekly passages in complete DMEM supplemented with 10% FCS (Life
Technologies, Grand Island, NY). Complete DMEM also contained 2 mM
L-glutamine, 1 mM sodium pyruvate, 10 µg/ml gentamicin,
MEM nonessential amino acids, 10 mM HEPES, and 50 µM 2-ME.
Isolation and purification of T. cruzi GIPL and GIPL glycan and lipid moieties
GIPLs were purified from epimastigotes of T. cruzi (Y strain) grown in brain-heart infusion medium supplemented with 10 mg/liter hemin and 5% v/v FCS (5 days at 26°C, with shaking), as previously described (2). Briefly, after cold water extraction, cell debris was extracted with 45% v/v aqueous phenol at 75°C, and the aqueous layer was dialyzed, freeze dried, reconstituted with water, and applied to a column of Bio-Gel P-100. The excluded material was lyophilized, and GIPLs were recovered by extraction with chloroform:methanol:water (10:10:3). Nuclear magnetic resonance and fast atom bombardment mass spectroscopy analyses were used to confirm the absence of contaminating peptidic material and LPS in purified GIPL preparations. For isolation of the phosphoinositol-oligosaccharide chain and the lipid moiety, intact GIPL was subjected to alkaline degradation (1 M KOH at 37°C for 48 h) (21). After neutralization with acetic acid, nonpolar material was recovered by chloroform extraction, purified by silica chromatography, and dried under an N2 stream to yield the GIPL-derived ceramide. This material was dissolved in serum-free culture medium containing 10% ethanol in a heated water bath (65 to 80°C) to a stock solution of 500 µg/ml, sterile filtered, and stored in the cold. The aqueous extract of alkaline hydrolyzed GIPL was mixed with Dowex 50W-X8 resin (25 to 50 mesh; H+ form) and filtered through glass wool. Phosphoinositol oligosaccharides were further desalted on a Bio-Gel P-2 column, lyophilized, dissolved in water, and sterile filtered. The structure of T. cruzi GIPL, Y strain, has been resolved by chemical and physical analyses (i.e., nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectroscopy) (1). The major molecular species found in the lipid moiety is the ceramide N-lignoceroyl-dihydrosphingosine (1).
M cultures
For treatment with parasite glycolipids, primary M or J774.G8
cells were cultured in complete DMEM supplemented with 1% Nutridoma-SR
(Boehringer Mannheim, Indianapolis, IN) instead of FCS. Primary M or
J774 cells (2.5 x 105) were incubated in 1 ml of
complete culture medium in 24-well vessels (Corning Glass Works,
Corning, NY) with culture medium alone or with intact T.
cruzi GIPL (40 µg/ml), isolated GIPL glycan chain
(phosphoinositol oligosaccharides) (30 µg/ml), isolated GIPL-derived
ceramide (10 µg/ml or lower), synthetic
C2-ceramide
(N-acetyl-D-sphingosine; ICN Pharmaceuticals,
Costa Mesa, CA), or C2-dihydroceramide
(N-acetylsphinganine; ICN) and bacterial LPS (10 ng/ml,
Escherichia coli O111:B4; Difco, Detroit, MI) for 20 h at
37°C. Synthetic C2-ceramides were dissolved in
DMSO at 2 mg/ml, serially diluted to DMEM-10% DMSO, and added to
cultures after brief heating (at 65°C). In addition, some
cultures received GIPL ceramide together with the indicated doses of
NG-monomethyl-L-arginine monoacetate
(L-NMMA; Calbiochem-Novabiochem, La Jolla, CA). After
20 h, half of the cultures received murine recombinant IFN- (40
U/ml; PharMingen, San Diego, CA) and were cultured for an additional
24 h. Cultures were then examined in an inverted phase contrast
microscope, supernatants were collected, and cells were either
recovered and counted by trypan blue exclusion or processed for
transmission electron microscopy. Some experiments employed
modifications of this basic protocol, as noted in the figure legends.
In additional experiments, M and J774 cells were also treated with
GIPL or GIPL ceramide and glycan moieties together with IFN-, added
at the start of culture. In some experiments, percentage cell loss,
instead of viable cell number, is presented and was calculated as 100
- [(viable cell number in experimental group)/(mean viable
cell number with solvent alone)]. Results are the means and SE of
triplicate cultures. For assessing induction of M fluid phase
endocytosis (22), the fluorescent dye Lucifer Yellow (LY; excitation
wavelength 430 nm, detection at 540 nm; Sigma Chemical, St. Louis, MO)
was added to cultures at 250 µg/ml in the absence or presence of GIPL
ceramide. Cells were incubated for 20 h at 37°C and examined
under fluorescence microscopy in an inverted microscope equipped with
an LY filter set.
Release of fragmented DNA into culture supernatants
After 24 h of IFN- addition, supernatants from M or
J774.G8 cell cultures subjected to several experimental treatments
were collected, depleted of contaminating cells by centrifugation, and
treated as previously described (23), to isolate released DNA. Briefly,
0.6 ml of each supernatant was treated with 0.2 ml of digestion buffer
consisting of 0.5% SDS, 200 µg/ml proteinase K (Sigma), and 50 mM
NaCl in TE buffer (10 mM Tris and 1 mM EDTA, pH 7.8) for 1 h at
42°C. The mixture was treated with 50% isopropanol/0.5 M NaCl at
-20°C overnight, for DNA precipitation. Precipitates were pelleted,
washed with 70% ethanol, allowed to dry in air, and reconstituted with
20 µl of TE buffer. Aliquots (10 µl) were applied to horizontal
agarose gels (1.5%) and subjected to a standard electrophoresis
procedure. Gels were stained with 5 µg/ml ethidium bromide (Sigma)
and photographed under UV light.
Nitric oxide (NO) and cytokine production assays
NO levels produced by primary M cultures (assayed in
quadruplicate) were estimated by measuring
NO-2 accumulated over 24 or 48 h,
as assayed by the Griess reaction (24). Levels of IL-6 secreted into
M culture supernatants were assayed by sandwich ELISA, using a
previously described IL-6 detection protocol (25). Recombinant murine
IL-6, plus capture and detection anti-IL-6 mAbs, were purchased
from PharMingen.
Electron microscopy
After culture with different treatments, J774.G8 cells, which are poorly adherent, were recovered by aspiration after tapping the bottles and washed and fixed with 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer, pH 7.2, for 1 h at 4°C. After washing in the same buffer, pellets were postfixed with 1% OsO4, dehydrated in acetone, and embedded in Epon. Thin sections were produced with a Reichert Ultramicrotome (Reichert Division Leica AG, Austria) and observed either unstained or stained with uranyl acetate in a Zeiss EM10C (Carl Zeiss, Oberkuchen, Germany) electron microscope.
Cytokines
Murine recombinant cytokines were used at the following final
concentrations: IFN-, 40 U/ml; IL-1ß, 20 ng/ml; IL-2, 50 U/ml;
IL-4, 10 ng/ml; TNF-, 40 ng/ml; and GM-CSF, 1 ng/ml. All cytokines
were purchased from PharMingen.
Infection of M monolayers with T. cruzi
Primary BALB/c peritoneal M (2 x 105
adherent cells/well in 24-well culture vessels) were infected overnight
with chemically induced metacyclic forms of T. cruzi clone
Dm28c, obtained as described (23), at a 10:1 parasite:cell ratio in 1
ml of complete culture medium containing 10% FCS at 37°C. The next
day (day 1), monolayers were extensively washed to remove extracellular
parasites and recultured with complete culture medium containing 1%
Nutridoma instead of FCS. At day 4, triplicate cultures were treated or
not with GIPL-derived ceramide (10 µg/ml) and with IFN- (40 U/ml),
to induce M apoptosis. Delaying massive apoptosis induction up to
day 4 was required to allow for sufficient intracellular parasite
replication and differentiation to take place. The next day (day 5),
cultures were gently aspirated, and extracellular parasites were
counted in a hemocytometer chamber. Typical motile trypomastigotes and
spheromastigotes with rudimentary flagellum were readily identified.
Statistical analysis was done by Student’s t test, using a
log transformation of released parasite number to normalize the data.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We investigated the ability of T. cruzi GIPL or its
isolated glycan and ceramide components to induce M secretion of
either NO or the cytokine IL-6. We used primary peritoneal M in
these assays, since we were unable to detect significant NO production
in the J774.G8 subtype cells we employed. After a 20-h incubation,
neither intact GIPL nor its isolated components induced measurable NO
production in M, while a combination of low doses of LPS plus
IFN- induced intense NO production. After 48-h incubation, GIPL, but
not its isolated components, induced NO production (6.28 ±
1.2 µM nitrites, compared with 0.56 ± 1.4 µM nitrites in
untreated M). Incubation of M with isolated GIPL, GIPL glycan, or
GIPL-derived ceramide for 20 h, followed by treatment with
IFN-, resulted in no detectable NO production after an additional
24 h, while the response to LPS plus IFN- was intense
(20.5 ± 0.4 µM nitrites) under these conditions. Similarly, no
detectable NO production was seen following the simultaneous addition
of GIPL or its components and IFN-, at the start of culture (data
not shown). Stimulation with LPS plus IFN- in the presence of the
inducible nitric oxide synthase (iNOS) inhibitor L-NMMA
(1000 µM) blocked 84% of NO production (5.26 ± 0.48 µM
nitrites) induced by the stimuli in the absence of L-NMMA
(33.51 ± 2.73 µM nitrites). Very limited secretion of IL-6 was
detected following M exposure to intact GIPL (263 ± 22 pg/ml,
compared with 141 ± 9 pg/ml in untreated M) or to its isolated
glycan (306 ± 76 pg/ml) and ceramide (192 ± 40 pg/ml)
components, after a 48-h incubation, and this response was not
appreciably enhanced by IFN- addition after a 24-h incubation. On
the other hand, IL-6 secretion in response to LPS alone was intense
(1875 ± 345 pg/ml) and could be further increased by IFN-
addition (4020 ± 295 pg/ml). Neither intact GIPL nor its isolated
components induced any measurable secretion of TNF- (not shown).
Induction of M endocytosis and cell death by GIPL-derived
ceramide
Despite a lack of NO secretion, treatment with T. cruzi
GIPL-derived ceramide induced marked morphologic changes both in the
J774.G8 M cell line (Fig. 1
) and in
primary M (not shown). Addition of GIPL ceramide (10 µg/ml) for 20
to 48 h resulted in intense vacuole formation (Figs. 1B
and 2, B and C),
compared with untreated cells (Fig. 2A). Large and numerous
cytoplasmic vesicles were induced within 2 to 4 h of GIPL ceramide
addition (Fig. 2B). After overnight culture, large vacuoles
were present (Fig. 2C). Intact GIPL at an equimolar ceramide
concentration (40 µg/ml) and a correspondent sugar dosage of the GIPL
glycan moiety (30 µg/ml) both failed to induce vesicle formation.
Treatment of J774.G8 cells with GIPL ceramide in the presence of the
fluid phase fluorescent tracer LY (molecular mass, 900 Da) resulted in
induction of LY-containing fluorescent vesicles, including large,
single fluorescent vacuoles (Fig. 1D). Staining of vacuoles
with LY was also seen in primary M treated with GIPL ceramide (not
shown). These results indicate that vesicle formation is due to fluid
phase endocytosis. We also investigated effects of exposure to T.
cruzi GIPL in the presence of IFN-. Addition of IFN- alone
had no effect on cell viability or morphology (Fig. 1A).
However, addition of IFN- to GIPL ceramide-treated cells resulted in
intense J774 cell shrinkage and death in culture (Fig. 1C).
Addition of IFN- to cells treated with intact GIPL or isolated
glycan did not result in changes in cell morphology or cell death. In
dose-response studies, maximal cell loss was induced in J774.G8 cells
with concentrations of GIPL ceramide as low as 1.0 µg/ml in the
presence of IFN- (Fig. 3A).
The small but significant cell loss induced by GIPL ceramide alone
(Fig. 3A) presumably has resulted from a block in the
J774.G8 cell cycle, since no morphologic evidence of cell death was
seen (also, see below). To investigate whether J774 cell death was
dependent on minimal NO secretion not detected by the Griess assay,
J774.G8 cells were treated with GIPL ceramide plus IFN- in the
presence of increasing doses of the iNOS inhibitor L-NMMA
(Fig. 3B). Doses of L-NMMA up to 1000 µM,
which blocked more than 80% of NO secretion in M, had no effect in
the cell death induced by GIPL ceramide plus IFN- (Fig. 3B), further suggesting that cell death was NO independent.
Both M and J774.G8 cell apoptosis could also be induced in 20
h, following simultaneous addition of GIPL ceramide and IFN- at the
start of culture (not shown). Again, under these conditions, no
measurable NO production could be detected in M.
|
|
|
induces host
M apoptosis
The morphologic appearance of the cell death response, with cell
shrinkage, formation of membrane blebs, and membranous vesicles,
together with electron microscopic evidence of nuclear chromatin
condensation into crescents (Fig. 2
D), were suggestive of
apoptosis. Therefore, the presence of released fragmented DNA in
culture supernatants was investigated by agarose gel electrophoresis
(Fig. 4). In the absence of IFN-, no
treatment induced DNA release in either J774.G8 cells or M (Fig. 4, lanes 2 through 5, top and
bottom). However, in the presence of IFN-, isolated
T. cruzi GIPL ceramide moiety induced fragmented DNA release
in both J774.G8 cells and M (Fig. 4, lane 9,
top and bottom). Treatment of either cell type
with IFN- alone (Fig. 4, lane 6) or with IFN- plus
either LPS (lane 7) or intact GIPL (lane
8) did not result in DNA fragmentation. Isolated GIPL glycan, with
or without IFN-, did not induce DNA release. Exposure to a mixture
of GIPL glycan chain and isolated ceramide in the presence of IFN-
did not affect the degree of DNA release induced by ceramide (not
shown). Ceramides found in T. cruzi GIPLs are
dihydroceramides, which by themselves are poor inducers of apoptosis in
cultured cells (9). We investigated the effects of synthetic permeable
C2-ceramides on J774.G8 apoptosis. Exposure to
C2-ceramide, but not to
C2-dihydroceramide, induced J774 cell DNA
fragmentation (Fig. 5A,
lanes 3 and 5). However, similar to T.
cruzi GIPL-derived ceramide, addition of
C2-dihydroceramide combined with IFN-
resulted in DNA release into supernatants (Fig. 5A,
lane 4). We also tested whether additional cytokines could
synergize with GIPL ceramide to induce M apoptosis. Besides IFN-,
addition of GM-CSF also induced DNA fragmentation in ceramide-treated
primary M (Fig. 5B, lane 9). On the other
hand, IL-1ß, IL-2, IL-4 (Fig. 5B), and TNF- (not shown)
were ineffective. The inability of any endogenous TNF- to induce
M apoptosis was confirmed by the finding that apoptosis could not be
blocked by an excess amount of neutralizing anti-TNF mAb (not
shown).
|
|
. After 20 h in culture, this treatment previously
resulted in vacuole formation in J774.G8 cells but not in cell death.
However, intense cell death (Fig. 6A) and release of fragmented
DNA were seen after 48 h in culture (Fig. 6B).
Incubation with isolated glycan, the presence of IFN-, and the
presence of ceramide-containing intact GIPL, were all required for this
delayed apoptosis induction (Fig. 6, A and B).
These results indicate that extracellular GIPL can also induce
apoptosis and suggest that a large capacity M receptor for the GIPL
glycan could prevent this effect.
|
apoptosis
We addressed whether induction of M apoptosis results in
increased destruction of intracellular forms of T. cruzi. We
previously found that a major increase in trypomastigote release from
infected M occurred between days 4 and 5 in culture. Monolayers of
primary M were infected with T. cruzi and treated or not
with GIPL-derived ceramide plus IFN- at day 4. Extracellular
parasite number was assessed at day 5 (Fig. 7). Addition of IFN- alone led to a
decrease in the number of released trypomastigotes (Fig. 7). On the
other hand, exposure to GIPL ceramide plus IFN- resulted in M
apoptosis and in markedly increased numbers of released parasites,
compared with cultures treated with IFN- alone (Fig. 7). Both motile
infective trypomastigotes and transition forms (spheromastigotes)
increased in the extracellular medium, suggestive of M rupture
before completion of parasite differentiation. These results were
reproduced in a repeat experiment. The data indicate that M
apoptosis through GIPL ceramide increases the release of viable
parasites.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
function is their ability to synergize with or antagonize effects of
IFN- on M. Based on our studies showing effects of T.
cruzi GIPLs on host T and B cell activation (3, 4), we
investigated whether this class of glycolipid interferes with M
activation and synergizes with IFN-. Direct effects on NO and IL-6
secretion were modest or absent after 24 h, although intact GIPL
induced limited NO secretion in primary peritoneal M after 48 h
in culture. On the other hand, exposure to T. cruzi
GIPL-derived ceramide had marked effects on both endocytosis and
viability of J774.G8 cells and M. Intense vesicle formation was
induced, leading to a single or few large vacuoles that accumulated LY
(22), indicative of fluid-phase endocytosis. A proteophosphoglycan
secreted by Leishmania mexicana also induces vacuole
formation in M (26), suggesting a common set of biologic activities
exerted by this related family of parasite molecules.
Strikingly, in the presence of IFN-, T. cruzi
GIPL-derived ceramide induced intense apoptosis in the majority of
J774.G8 cells or primary M. Interestingly, this apoptotic effect was
absent in primary cultures of beating murine neonate myocardiocytes
(not shown), suggesting cell type specificity for the ceramide effect.
M apoptosis induced by GIPL ceramide was most likely NO independent,
since 1) no NO secretion was detected in primary M or J774.G8 cells
after stimulation with GIPL ceramide plus IFN-; 2) LPS plus IFN-,
which induced measurable NO secretion in the serum-free medium and low
dosages we employed, failed to induce M or J774.G8 cell apoptosis
under the same conditions; and 3) apoptosis could not be blocked by
iNOS inhibitor L-NMMA at doses that markedly blocked NO
production in M. Neither GIPL glycan nor intact GIPL had effects
comparable with those of GIPL ceramide at concentrations up to 50
µg/ml. The simplest explanation for the lack of effect with GIPL
would be that M bear glycan receptors able to capture and
internalize GIPLs into a distinct intracellular compartment, while
isolated ceramide would directly insert into the plasma membrane. We
did not directly address this issue. However, consistent with this
possibility, intact GIPL synergized with IFN- for apoptosis
induction after prior M incubation with GIPL glycan. Since T.
cruzi is located in the cytosol in infected M, it is likely
that both GIPL and free ceramides derived from intracellular parasites
could bypass this putative glycan receptor, but this possibility needs
to be tested. Molecules similar to GIPLs were found in infective
T. cruzi trypomastigote forms (27). Moreover, free ceramides
were found in amastigotes (28), and ceramide-containing GPI anchors
were described in abundant glycoproteins of amastigotes (28) and
metacyclic trypomastigotes (29). Regarding synergism with IFN-,
intense and continued production of IFN- by cells of the host innate
and acquired immunity control T. cruzi numbers, but IFN-
is unable to eradicate infection (reviewed in 30 .
Downstream events involved in apoptosis induction by GIPL ceramide are
currently unknown. Ceramide-dependent pathways are implicated as
messengers for M activation by LPS (16, 17). However, it is unlikely
that GIPL dihydroceramide uses LPS signal-transducing pathways to
induce M apoptosis, since LPS lacked all GIPL ceramide effects,
including induction of endocytosis or apoptosis. The proapoptotic role
of IFN- has been described in different systems. In the HT-29
epithelial cell line, a synergism between IFN- and either TNF- or
anti-Fas Ab, each of which mobilizes endogenous ceramide (9, 11),
led to apoptosis (31). Moreover, pretreatment of Fas-expressing human
keratinocytes with IFN-, but not with TNF-, induced apoptosis by
anti-Fas mAb (32). These results suggest functional complementation
between IFN- signals and ceramide-mobilizing stimuli for apoptosis
induction. We showed that a synthetic permeable dihydroceramide
(C2-dihydroceramide) also synergized with
IFN- for M apoptosis induction, confirming the result with
T. cruzi-derived ceramides, which are dihydroceramides (1, 2). It is tempting to speculate that the results with the T.
cruzi glycolipid could have implications for infection. In
infected M, IFN- could complement a truncated apoptotic signal
provided by a parasite-derived dihydroceramide. Since IFN- also
mediates intracellular T. cruzi killing (33), induction of
apoptosis instead of NO secretion in some of the infected M could
provide an escape mechanism for the intracellular parasite, while
effective trypanosome killing would also occur at the same time in
other M. We found that apoptosis in infected M increased the
release of motile infective trypomastigotes and also spheromastigote
transition forms, suggestive of premature M rupture. However, this
possible mechanism for parasite escape during host cellular immune
responses involving IFN- remains to be demonstrated. We are
currently investigating whether M apoptosis naturally occurs during
infection with the virulent Colombiana strain of T. cruzi
and whether IFN- secretion could be involved.
Induction of apoptosis in host M and other cell types is a recently
recognized mechanism of virulence for several bacterial pathogens, such
as Salmonella (34), Shigella (35), and
Listeria (36) species. On the other hand,
mycobacterium-induced regulation of M apoptosis is rather complex.
Infection of M by mycobacteria down-regulates Bcl-2 protein (37) and
induces apoptosis (37, 38). Apoptosis induced by virulent mycobacteria
has been correlated with NO production in the mouse (38). However,
mycobacterial lipoarabinomannan and LPS protected M from apoptosis,
despite inducing NO secretion (38). Other studies suggested a
host-protective role for apoptosis of human M infected with
Mycobacterium avium, which resulted in bacterial killing by
fresh, noninfected M (39). Mycobacterium-infected M
are also targets for two sets of human cytolytic T cell clones that
mediate lysis through granule- or Fas-dependent mechanisms (40).
Different from granule-mediated cytotoxicity, Fas-mediated apoptosis of
infected M by CTLs does not result in death of the released
mycobacteria (40), a result similar to our model with T.
cruzi. Protozoan parasites of the genera Toxoplasma and
Leishmania were also investigated regarding induction of
host cell apoptosis. Infection with Toxoplasma gondii
inhibits target cell apoptosis in response to several stimuli,
including Fas-dependent and -independent pathways (41). However, a more
virulent strain of T. gondii has been reported to induce
M apoptosis, and protection against it has been associated with
IFN--mediated induction of stress protein HSP 65 that prevented M
apoptosis (42). Bone marrow-derived M infected with Leishmania
donovani are resistant to apoptosis induction by factor
deprivation (43). Increased M survival could also be induced by
leishmanial lipophosphoglycan and was attributed to autocrine secretion
of TNF and GM-CSF by infected M (43). Together, these results
suggest that distinct intracellular parasites control host cell
apoptosis both to survive within infected cells and also as a virulence
mechanism to spread infection. Moreover, the data suggest that
microbial surface glycolipids are involved in the control of host cell
apoptosis. Apoptosis of infected cells has not been previously studied
in T. cruzi infection, although one suggestive report
described massive in vivo destruction of parasitized splenic M in
infected mice, coincident with a drop in parasitemia, onset of host
mortality, and chronification of infection (44). It would be
important to investigate whether induction of host M apoptosis is a
virulence mechanism in T. cruzi infection.
|
Footnotes |
|---|
2 Address correspondence to Dr. George A. DosReis, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco G, Ilha do Fundão, Rio de Janeiro, RJ, 21944-970, Brazil. E-mail:
3 Abbreviations used in this paper: GIPL, glycoinositolphospholipid; GPI, glycosylphosphatidylinositol; M, macrophage; NMR, nuclear magnetic resonance; LY, Lucifer Yellow; NO, nitric oxide; L-NMMA, NG-monomethyl-L-arginine monoacetate; GM-CSF, granulocyte-macrophage colony-stimulating factor; iNOS, inducible nitric oxide synthase.
Received for publication February 10, 1998. Accepted for publication June 26, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-activated human monocytes: involvement of the nitric oxide synthase pathway. J. Biol. Chem. 269:9811. and ligation of Fas antigen. J. Immunol. 155:4147.[Abstract]
-treated cultured keratinocytes. Arch. Dermatol. Res. 287:315.[Medline]
-treated macrophages against Trypanosoma cruzi involves an L-arginine-dependent, nitrogen oxide-mediated mechanism inhibitable by interleukin-10 and transforming growth factor-ß. Eur. J. Immunol. 22:2501.[Medline]
T cells prevents apoptosis of macrophages and contributes to host defense in mice infected with Toxoplasma gondii. J. Immunol. 159:2375.This article has been cited by other articles:
|
|
 |
|
  M. Bergeron and M. Olivier Trypanosoma cruzi-Mediated IFN-{gamma}-Inducible Nitric Oxide Output in Macrophages Is Regulated by iNOS mRNA Stability J. Immunol., November 1, 2006; 177(9): 6271 - 6280. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Petersen, K. A. Krumholz, J. Carmen, A. P. Sinai, and B. A. Burleigh Trypanosoma cruzi Infection and Nuclear Factor Kappa B Activation Prevent Apoptosis in Cardiac Cells Infect. Immun., March 1, 2006; 74(3): 1580 - 1587. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Shi, G. Wei, W. Pan, and H. Tabel Impaired Kupffer Cells in Highly Susceptible Mice Infected with Trypanosoma congolense Infect. Immun., December 1, 2005; 73(12): 8393 - 8396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ibata-Ombetta, T. Idziorek, P.-A. Trinel, D. Poulain, and T. Jouault Candida albicans Phospholipomannan Promotes Survival of Phagocytosed Yeasts through Modulation of Bad Phosphorylation and Macrophage Apoptosis J. Biol. Chem., April 4, 2003; 278(15): 13086 - 13093. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Brodskyn, J. Patricio, R. Oliveira, L. Lobo, A. Arnholdt, L. Mendonca-Previato, A. Barral, and M. Barral-Netto Glycoinositolphospholipids from Trypanosoma cruzi Interfere with Macrophages and Dendritic Cell Responses Infect. Immun., July 1, 2002; 70(7): 3736 - 3743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Zuniga, A. Gruppi, J. Hirabayashi, K. I. Kasai, and G. A. Rabinovich Regulated Expression and Effect of Galectin-1 on Trypanosoma cruzi-Infected Macrophages: Modulation of Microbicidal Activity and Survival Infect. Immun., November 1, 2001; 69(11): 6804 - 6812. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. S. Campos, I. C. Almeida, O. Takeuchi, S. Akira, E. P. Valente, D. O. Procopio, L. R. Travassos, J. A. Smith, D. T. Golenbock, and R. T. Gazzinelli Activation of Toll-Like Receptor-2 by Glycosylphosphatidylinositol Anchors from a Protozoan Parasite J. Immunol., July 1, 2001; 167(1): 416 - 423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. B. de Arruda Hinds, L. M. Previato, J. O. Previato, Q. Vos, J. J. Mond, and L. M. T. Pecanha Modulation of B-Lymphocyte and NK Cell Activities by Glycoinositolphospholipid Purified from Trypanosoma cruzi Infect. Immun., November 1, 1999; 67(11): 6177 - 6180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. BELLIO, A.-C. S. C. O LIVEIRA, C. S. MERMELSTEIN, M. A. M. CAPELLA, J. P. B. VIOLA, J.-P. LEVRAUD, G. A. DOSREIS, J. O. PREVIATO, and L. MENDONÇA-PREVIATO Costimulatory action of glycoinositolphospholipids from Trypanosoma cruzi: increased interleukin 2 secretion and induction of nuclear translocation of the nuclear factor of activated T cells 1 FASEB J, September 1, 1999; 13(12): 1627 - 1636. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
