| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Laboratory of Cellular Immunology, Flanders Interuniversity Institute for Biotechnology, Free University of Brussels (Vrije Universiteit Brussel), and
Department of Molecular Biology, Free University of Brussels (Université Libre de Bruxelles), Brussels, Belgium; and
Department of Biochemistry, The University Dundee, Scotland, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-inducing capacity of different trypanosome components
was analyzed in vitro, using as indicator cells a macrophage cell line
(2C11/12) or peritoneal exudate cells from LPS-resistant
C3H/HeJ mice and LPS-sensitive C3H/HeN
mice. The variant-specific surface glycoprotein (VSG) was identified as
the major TNF--inducing component present in trypanosome-soluble
extracts. Both soluble (sVSG) and membrane-bound VSG (mfVSG) were shown
to manifest similar TNF--inducing capacities, indicating that the
dimyristoylglycerol (DMG) compound of the mfVSG anchor was not required
for TNF- triggering. Detailed analysis indicated that the
glycosyl-inositol-phosphate (GIP) moiety was responsible for the
TNF--inducing activity of VSG and that the presence of the
GIP-associated galactose side chain was essential for optimal TNF-
production. Furthermore, the results showed that the responsiveness of
macrophages toward the TNF--inducing activity of VSG was strictly
dependent on the activation state of the macrophages, since resident
macrophages required IFN-
preactivation to become responsive.
Comparative analysis of the ability of both forms of VSG to activate
macrophages revealed that mfVSG but not sVSG stimulates macrophages
toward IL-1 secretion and acquisition of LPS responsiveness. The
priming activity of mfVSG toward LPS responsiveness was also
demonstrated in vivo and may be relevant during trypanosome infections,
since Trypanosoma brucei-infected mice became gradually
LPS-hypersensitive during the course of infection. Collectively, the
VSG of trypanosomes encompasses two distinct macrophage-activating
components: while the GIP moiety of sVSG mediates TNF- induction,
the DMG compound of the mfVSG anchor contributes to IL-1 induction
and LPS sensitization. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
is a
pleiotropic cytokine, produced mainly by activated macrophages (1).
Although its name is derived from its capacity to cause necrosis of
certain parenchymal organs and tumors, the molecule was initially also
isolated as a factor named "cachectin," responsible for systemic
suppression of lipoprotein lipase activity in trypanosome-induced
cachexia (2, 3). As TNF- is a main mediator in inflammatory
responses, its influence in a number of infectious disease has been
studied. Among other effects, TNF- was found to be involved in the
pathology of several parasitic diseases, including trypanosomiasis (4),
Chagas’ disease (5), leishmaniasis (6), and malaria (7). In several of
these infections, TNF- plays a bidirectional role, influencing both
parasite and host (8). In the case of trypanosome infections, TNF-
seems to be involved in the neuropathology of sleeping sickness, and a
correlation has been described between the disease severity in human
African trypanosomiasis and high serum levels of the cytokine (9).
Furthermore, a possible association between TNF- production by
monocytes from trypanosome-infected cattle and the severity of
disease-associated anemia has been documented (10). Using mouse models,
the immunopathology of experimental African sleeping sickness has been
linked to TNF--mRNA detection in the brain of Trypanosoma
brucei-infected mice (11), and a role for TNF- involvement in
trypanosomiasis-associated immunosuppression has been put forward (12).
Finally, a direct involvement of TNF- in trypanosomiasis control and
parasite growth was documented in vivo (4, 13), and a trypanolytic
effect of TNF- on T. brucei was confirmed in vitro
(14, 15, 16). Information concerning the trypanosome-derived factor(s) that
may be responsible for the induction of TNF- production by
host cells is scarce. In analogy with plasmodia-associated TNF-
induction, trypanosomiasis-associated TNF- induction has been
suggested to be mediated by the glycosylphosphatidylinositol
(GPI)3 anchor on the
variant-specific surface glycoprotein (VSG) of the trypanosome (17).
VSG is the crucial molecule involved in the escape of the host immune
response, as 107 identical VSGs cover each trypanosome and
form a dense coat around each cell (18, 19). The mechanism of immune
escape is based on a regular switch in the expression of VSG variants,
jeopardizing the induction of an effective Ab response against the
parasite. Interestingly, in response to environmental stress,
trypanosomes are capable of liberating their VSG through a VSG lipase
(20). This enzyme cleaves the GPI anchor, leaving the
dimyristoylglycerol (DMG) compound of the GPI anchored in the membrane,
and releasing the glycosyl-inositol-phosphate (GIP)-VSG part (21). In
this report, we compare the macrophage-activating capacity, analyzed in
terms of cytokine secretion (TNF-/IL-1, IL-6, IL-10, and IL-12)
and induction of LPS hyper-responsiveness, of the released soluble VSG
(sVSG) and the membrane-bound form VSG (mfVSG). We show that both forms
of VSG possess distinct macrophage-activating components. |
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2C11/12 macrophages (22) were grown in RPMI 1640 medium
supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 0.1
mg/ml streptomycin, and 0.3 mg/ml L-glutamine, all
purchased from Life Technologies Laboratories, Grand Island, NY.
Adherent cells were selected by a repetitive 2-day transfer on plastic
cell culture plates (Costar, Cambridge, MA). For in vitro TNF-
induction assays, cells were left to adhere for 2 h at 37°C in
fresh medium on 96-well culture plates before stimulation with
trypanosome components or LPS. Cells were used at a concentration of
4 x 105 cells/ml, 100 µl/well. Peritoneal exudate
cells (PECs) were harvested from both LPS-sensitive
C3H/HeNHsd mice and LPS-resistant C3H/HeJolaHsd
mice (Harlan, Zeist, The Netherlands) by a peritoneal wash with 10 ml
of ice-cold sucrose/H2O solution (116 g/L). The collected
cells were washed in RPMI 1640 and left to adhere for 2 h at
37°C on 96-well culture plates. Cells were used for TNF- induction
assays at a concentration of 2 x 106 cells/ml, 100
µl/well. When indicated, 2C11/12 and PECs were stimulated with
recombinant murine IFN- (Life Technologies) at a final concentration
of 100 U/ml. All cell incubations were done at 37°C in a humid 5%
CO2 atmosphere incubator.
Trypanosomes
The AnTat 1.1E clone of the EATRO 1125 stock of the pleomorphic bloodstream form was kindly provided by Dr. N. Van Meirvenne (Institute of Tropical Medicine, Antwerp, Belgium). To follow the course of the parasitemia, 6- to 8-wk-old female C3H/HeN mice (Harlan) were injected i.p. with 5000 parasites. At intervals of 2 or 3 days, the number of parasites present in the blood was counted using a light microscope.
Trypanosome-soluble extract and VSG preparation
Trypanosomes were harvested from infected blood by DE52 chromatography (23), using sterile PBS (pH 8.0) supplemented with 1% glucose for equilibration and elution. After separation, parasites were washed and resuspended in RPMI 1640 medium at a concentration of 109 cells/ml. Crude parasite lysate was obtained by three freeze/thaw cycles in the presence of 1 mM Pefabloc protease inhibitor (Boehringer Mannheim, Mannheim, Germany) and 0.01 mM E64 (Sigma Chemical Co., St. Louis, MO). Soluble extract was obtained by removing nonsoluble components by 15-min centrifugation at 13,000 x g. sVSGs were prepared from DE52-purified parasites by osmotic lysis for 5 min at 37°C at 109 cells/ml in 10 mM sodium phosphate (pH 8.0) containing 0.1 mM TLCK and 0.1 mM PMSF (both from Boehringer Mannheim). The supernatant was passed through a column of DE52 equilibrated in 10 mM sodium phosphate (pH 8.0). sVSG was further purified on a column of Sephacryl-S200 (Pharmacia Biotech, Uppsala, Sweden), dialyzed against water overnight at 4°C, and freeze-dried. mfVSG was prepared according to the method of Jackson et al. (24). Freeze-dried VSG was resuspended in sterile RPMI 1640 medium just before use. All soluble extract and VSG samples were incubated under gentle shaking for 2 h at room temperature with Prosep-Remtox (Bioprocessing, Princeton, NJ.) glass beads to remove possible LPS contamination. Afterward, beads were separated by sample filtration over a 22-µm sterile Spin-X centrifuge tube filter (Costar).
Protein concentration of the soluble extract and VSG was estimated by a detergent-compatible protein assay kit (Bio-Rad Laboratories, Hercules, CA) using BSA as a standard.
Enzyme treatments
All enzymes used to digest trypanosome-soluble extract or VSG
and protease inhibitors were purchased from Boehringer Mannheim. These
include recombinant N-glycosidase F from
Flavobacterium meningosepticum, -galactosidase from green
coffee beans, Pronase from Streptomyces griseus, Proteinase
K from Tritirachium album, and the protease inhibitors
Pefabloc SC, PMSF, and TLCK. All enzymes and inhibitors were used
according to the manufacturer’s instructions.
N-Glycosidase F digestion was performed in PBS using 5 U/ml enzyme (24 h/37°C).
-Galactosidase digestion of VSG was performed in 0.1 M sodium
acetate buffer, pH 5.0, using 50 U/ml enzyme (2 x 18
h/37°C).
Proteinase K digestion was performed in PBS, pH 8.0, using 5 U/ml enzyme (24 h/37°C).
Pefabloc SC was used when indicated (1 mM final concentration) to prevent proteolysis during lysate preparation by freeze/thaw cycles. Furthermore, it was used to stop Proteinase K digestion of trypanosome-soluble extracts or VSG.
Preparation of the glycosyl-inositol-phosphate (GIP) fraction
The GIP fraction, otherwise known as the sVSG COOH-terminal
glycopeptide (sCt-gp), was purified from a Pronase digest of sVSG
(MITat 1.4) by ion exchange chromatography on Dowex AG50 and
QAE-Sephadex A25, as described earlier (25). This fraction was digested
with coffee bean -galactosidase and repurified on QAE-Sephadex A25.
A negative ion electrospray mass spectrum of final GIP fraction
revealed [M-2H]2- pseudomolecular ions at
m/z 571.5, 625.5, and 735.5 (in a ratio of 2:1:1)
corresponding to the structures Asp-ethanolamine-HPO4-6
Man1–2 Man1–6
Man1–4GlcN1–6myo-inositol-1-HPO4 and
(most likely, based on the intact GPI structure of MITat 1.4 VSG)
Asp-ethanolamine-HPO4-6 Man1–2
Man1–6(Gal1–3)Man1–4GlcN1–6myo-inositol-1-HPO4
and Asp-ethanolamine-HPO4-6 Man1–2
Man1–6(Gal1–6Gal1–3)Man1–4GlcN1–6myo-inositol-1-HPO4,
respectively (26). The concentration of the GIP fraction was determined
by measuring the myo-inositol content by selection ion
monitoring gas chromatography-mass spectrometry (27).
LPS treatment
For both in vitro and in vivo analyzes of LPS sensitization of
macrophages, LPS from Escherichia coli 055:B5 was used
(Difco Laboratories, Detroit, MI). For in vitro experiments, different
LPS dilutions were made in RPMI 1640 medium and added to 2C11/12 cells
or to freshly harvested PECs. After 3 h incubation, culture
supernatants were taken and tested for the presence of TNF- using a
TNF--specific ELISA. A minimal LPS dose used in sensitization
experiments was determined as one-half of the highest concentration
that could be added to the cells without observing a reproducible
induction of TNF- production. For in vivo experiments, different LPS
dilutions were made in PBS and injected i.p. into LPS-sensitive
C3H/HeN mice. Blood was taken from these mice, and serum
TNF- levels were determined by ELISA. A minimal LPS dose was
determined as the lowest dose that gave reproducibly measurable TNF-
levels without causing any signs of morbidity.
To test the involvement of TNF- in LPS-induced mortality of T.
brucei-infected mice, an i.p. injection of 50 µg of neutralizing
anti-TNF- mAb (PharMingen, San Diego, CA) was used.
Cytokine detection
The levels of TNF- and IL-1 present in culture
supernatants or serum were measured using, respectively, a
TNF--specific and an IL-1-specific ELISA (Innogenetics, Gent,
Belgium). IL-6, IL-10, and IL-12 present in culture supernatants or
serum were measured by ELISA using specific Abs purchased from
PharMingen. Cytokine levels present in all culture supernatants were
measured in triplicates, and mean values were calculated for
presentation of the results. SEs never exceeded 15% within a given
bioassay. To analyze the significance levels of observation, a Student
t test was used and p values were indicated in
the text. Serum cytokine levels were measured using three mice for
every measurement. Mean serum cytokine levels were calculated for
presentation of the results.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-inducing component.
We have reported earlier that T. brucei-soluble
extracts contain soluble components capable of inducing the secretion
of TNF- by a differentiated macrophage cell line (2C11/12) (13). In
a first attempt to identify the T. brucei components
involved in the induction of TNF- production, efforts were focused
on the VSG, since this glycoprotein represents approximately 10% of
the total protein content of trypanosome lysates. To analyze the
possible contribution of VSG in the induction of TNF- production by
2C11/12 cells, T. brucei-soluble extract was depleted of VSG
by immunoprecipitation, tested for TNF- induction, and compared with
the TNF--inducing activity of total trypanosome-soluble extract and
purified soluble as well as membrane-bound forms of VSG (sVSG and
mfVSG, respectively). As shown in Figure 1
, both forms of VSG efficiently trigger
TNF- production by 2C11/12 cells, while depletion of VSG from the
trypanosome-soluble extract reduces significantly its TNF- inducing
capacity (p < 0.001 up to a dilution of 1/32).
Soluble extracts of procyclic forms that lack the VSG failed to induce
detectable levels of TNF- secretion. Furthermore, at identical
protein concentrations, both sVSG and mfVSG are more potent TNF-
inducers than trypanosome-soluble extract. According to these results,
VSG is the main trypanosome TNF--inducing component, and since sVSG
and mfVSG are equally potent TNF- inducers, the DMG compound of the
mfVSG anchor (Fig. 2) is not required for
this activity.
|
|
induction, sVSG was digested with Proteinase K,
N-glycosidase F, and -galactosidase. As shown in Figure 3a, only treatment with
-galactosidase had a marked effect on the TNF--inducing capacity
of sVSG (p < 0.001 up to a dilution of 1/32).
Neither digestion of the protein moiety nor the removal of
N-linked carbohydrates from the VSG preparation resulted in
the abolishment of TNF- induction, indicating that the GIP moiety at
the base of the VSG carries the TNF--inducing capacity. To confirm
this finding, the GIP moiety was purified and checked for its capacity
to trigger TNF- induction before and after -galactosidase
treatment. Indeed, it was found that the GIP moiety of sVSG is fully
capable of triggering TNF- induction and that the -galactose side
chain of this moiety has an amplifying effect on the TNF--inducing
capacity of the GIP moiety (Fig. 3b).
|
induction, the TNF--inducing capacity of sVSGs that
differ in their galactose side chain composition was compared. AnTat1.1
and MITat 1.4 belong to the same VSG class (class I) and have the same
galactose side chains, while MITat 1.5 belongs to another class (class
III) and is unique, since it lacks the galactose side chains (28, 29).
From Figure 4a, it is clear
that both of the class I VSG are potent TNF- inducers, while the
class III VSG is significantly less capable of triggering TNF-
production by 2C11/12 cells (p < 0.001 up to a
dilution of 1/32). This results confirm the requirement for the
galactose side chain for optimal TNF--inducing capacity of the GIP
moiety of sVSG.
|
-inducing
activity of VSG requires IFN- priming
Thus far, the TNF--inducing potential of trypanosome-soluble
extracts and VSG has been evaluated on 2C11/12 macrophages that are
highly differentiated (22). It was therefore of importance to reanalyze
the TNF--inducing activity of sVSG on resident, nonactivated
macrophages. To this end, the TNF--inducing activity of different
VSGs was tested on PECs from LPS-resistant C3H/HeJ mice, and as shown
in Figure 4
b, these cells barely respond to TNF--inducing
class I sVSGs. The differences in the responses of 2C11/12 cells and
PECs toward sVSG might reside in the activation status of the two
macrophage cell types. Indeed, preactivation of PECs with IFN-
renders these macrophages fully responsive toward the TNF--inducing
activity of class I VSG, but not of class III VSG (Fig. 4d). In the case of 2C11/12 cells, pretreatment with
IFN- does not significantly increase their responsiveness toward
TNF--inducing VSGs (compare Fig. 4, a and
c), indicating that these cells are inherently
activated and do not require preactivation.
Since the responsiveness of normal macrophages toward the
TNF--inducing activity of sVSG requires IFN- priming, it was
important to reanalyze the involvement of the DMG compound of the VSG
anchor in TNF- induction by normal macrophages, either activated or
not. As shown in Figure 5, both mfVSG and
sVSG moderately trigger PECs from LPS-resistant C3H/HeJ (Fig. 5a) to produce TNF-. Although the levels of
TNF- induction are rather low, the TNF--inducing activity of
mfVSG is consistently higher than that of sVSG. Yet, following
activation with IFN-, both sVSG and mfVSG are equally capable of
triggering high levels of TNF- production by PECs of LPS-resistant
mice (Fig. 5b). In fact, the pattern of
responsiveness of IFN--primed PECs toward both types of VSGs is
similar to that recorded for 2C11/2 cells (Fig. 1). Collectively, these
results show that normal macrophages acquire, upon activation with
IFN-, a responsiveness toward the GIP moiety of sVSG, and
furthermore, that the galactose side chain of this moiety acts as an
enhancer for TNF- induction. Apparently, the DMG compound of mfVSG
is not of crucial importance in the triggering of TNF- production by
IFN--activated macrophages. It is important to mention here that the
same cytokine induction pattern, with equal levels of secretion, was
found when PECs from LPS-sensitive C3H/HeN mice were used
in these bioassays (results not shown).
|
To analyze whether trypanosome components, including sVSG and
mfVSG, are involved in a more general way in the induction of
inflammatory cytokine production by macrophages, both 2C11/12 cells and
C3H/HeJ PECs were incubated with trypanosome
components and tested for secretion of IL-1, IL-6, IL-10, and IL-12.
As shown in Figure 6a, when
2C11/12 cells were incubated with total soluble trypanosome extract
from T. brucei AnTat 1.1, high levels of IL-1 and IL-6
were detected in the culture supernatants, but secretion of both IL-10
and IL-12 remained below background levels (i.e., secretion by
unstimulated cells). When C3H/HeJ PECs were used, only
IL-1 secretion was recorded (Fig. 6b).
Preactivation of 2C11/12 cells or PECs with IFN- did not alter the
pattern of cytokine secretion. To analyze the role of VSG in the
induction of IL-1 and IL-6 secretion, 2C11/12 cells and
C3H/HeJ PECs were incubated with different concentrations
of sVSG and mfVSG. Induction of cytokine secretion was compared with
secretion induced by total trypanosome extract. While both forms of VSG
failed to induce IL-6 secretion by 2C11/12 cells as well as
C3H/HeJ PECs (data not shown), IL-1 induction could be
detected only when cells were stimulated with mfVSG or total
trypanosome extracts (Fig. 7,
a and b). sVSG did not induce detectable IL-1
secretion, and prestimulation of cells with IFN- did not alter these
induction patterns.
|
|
Although the VSGs are not potent inducers of TNF- production by
resting macrophages (PECs), such agents might prime macrophages to
respond to a secondary triggering agent such as LPS. To test this
possibility, 2C11/12 cells and C3H/HeN PECs were
preactivated with parasite-soluble extract, sVSG, and mfVSG, and
restimulated with 1 pg/ml LPS. This LPS concentration does not induce a
detectable TNF- production by itself (data not shown). According to
Figure 8a, both
trypanosome-soluble extract and mfVSG prime 2C11/12 cells to become
hypersensitive to LPS, resulting in high TNF- production. In
contrast, preactivation with sVSG does not sensitize the cells to
respond to LPS (compare Fig. 8a and Fig. 1).
|
inducers in the absence of LPS (Fig. 4b),
these components are capable of preactivating cells to become high
TNF- producers upon contact with LPS (Fig. 8b).
C3H/HeN PECs produce only low levels of TNF- upon
contact with sVSG, and furthermore, sVSG is not capable of sensitizing
macrophages to respond to LPS. Finally, when 2C11/12 macrophages or PECs were stimulated with 1 pg/ml LPS before stimulation with trypanosome-soluble extract, sVSG, or mfVSG, no prestimulation effects were observed (data not shown).
Collectively, these results indicate that the DMG compound of the mfVSG anchor is capable of priming macrophages to become hyper-responsive to LPS.
The DMG compound of the VSG anchor renders mice hypersensitive to
LPS : possible involvement in TNF- production during
trypanosomiasis
To analyze the in vivo relevance of the above-described
observations, C3H/HeN mice were treated i.p. with
sVSG, mfVSG, or parasite-soluble extract and challenged with LPS. The
inoculated protein content corresponded to approximately
108 parasites (i.e., 50 µg VSG and 500 µg total
trypanosome-soluble extract). Following injection of sVSG, mfVSG, and
parasite-soluble extract, serum TNF- levels remained below detection
limit 24 h postinoculation. However, when trypanosome-soluble
extract or mfVSG-treated animals were challenged with a minimal dose of
LPS (1 µg/mouse) 21 h postinoculation, a significant increase in
TNF- serum levels was recorded within 3 h (Fig. 9). As
expected, pretreatment with sVSG did not result into hypersensitivity
toward LPS.
|
might be relevant during infection with trypanosomes, since
serum levels of LPS were reported to increase during trypanosome
infection (30). Furthermore, TNF- levels were documented as
correlating with the severity of the disease in both humans and cattle
(9, 10). Therefore, LPS-induced TNF- levels were measured during the
course of the parasitemia (Fig. 10, a and
b). As shown in Figure 9b, when a minimal
dose of LPS (1 µg/mouse) was injected during the early phase of the
infection, a slight increase in the levels of serum TNF- was
measured after 3 h, and the treated mice manifested no signs of
morbidity. However, when the first peak of parasitemia was reached and
parasite numbers dropped, a dramatic increase in the serum levels of
TNF- was measured upon LPS challenge; 2 h after LPS injection,
severe signs of morbidity were recorded, resulting in 100% lethality
within 24 h after LPS administration.
|
, IL-6, IL-10, and IL-12.
These measurements indicated that only increased levels of IL-1 and
IL-6 could be detected (data not shown), yet these increased levels did
not correlate with the occurrence of LPS-induced mortality. Finally,
i.p. treatment with a neutralizing anti-TNF- mAb (50 µg/mouse)
24 h before LPS challenge rescued T. brucei-infected
mice from LPS-induced mortality, indicating that TNF- is a crucial
mediator in this event.
These results suggest that during infection the presence of the DMG
compound of the VSG anchor may activate macrophages, rendering these
cells competent for hyperproduction of TNF- in the presence of low
levels of LPS.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
is an inflammatory cytokine that was originally isolated
based on its capacity to induce trypanosomiasis-associated cachexia in
rabbits (2, 3). Today, it is known that TNF- is also involved in
trypanosomiasis-associated anemia and in the occurrence of
meningoencephalits signs during late stage infections (9, 10). Although
as such, TNF- can be considered crucially important in the general
immunopathology of African trypanosomiasis, it is still unclear which
trypanosome factors and which underlying mechanisms contribute to
TNF- induction in the host. Using a macrophage cell line-based
bioassay, we report here that sVSG, which is released from the parasite
surface through a GPI-specific phospholipase C (20), is the main
TNF--inducing component of trypanosome-soluble extracts.
Furthermore, we report that the GIP moiety at the base of the sVSG is
the key to TNF- induction and that the GIP-associated galactose side
chain is crucial for optimal TNF- induction mediated by
sVSG.
TNF- induction assays with PECs further corroborate the role of both
GIP and its galactose side chain in the TNF--inducing capacity of
sVSG. However, in the case of PECs, sVSG-mediated induction of TNF-
production requires preactivation of macrophages with IFN-. This
observation does not preclude a role for sVSG during trypanosome
infections, since there is ample evidence that African trypanosomes
sensitize host T cells, either directly or indirectly, to produce
IFN- (12, 31, 32, 33). Thus, during trypanosome infections, macrophages
may be primed with IFN- and as such may become responsive toward the
TNF--inducing capacity of sVSG. In testing the TNF--inducing
activity of mfVSG, which differs from sVSG solely by the presence of
the DMG moiety, no clear-cut differences were observed between the two
forms of VSG. mfVSG may be slightly more potent than sVSG in triggering
TNF- production by resident macrophages, yet when IFN--activated
PECs or differentiated 2C11/12 macrophages are used as responder cells,
both sVSG and mfVSG exert similar activities. The capacity of mfVSG to
stimulate TNF- production by thioglycolate-elicited macrophages via
its GPI anchor was reported earlier by Tachado and Schofield (17).
Furthermore, Schofield and Hacket (34) reported that Plasmodium
falciparum GPI, free or associated with protein, induces TNF-
production by thioglycolate-elicited macrophages and that removal of
the DMG moiety of GPI abolishes the cytokine production. Others,
however, have partially disagreed on the functional role of intact
malaria GPI, showing that it is the inositol monophosphate of the
malaria toxin that is crucial for TNF- induction (35). As a control,
a number of nonparasite-derived GPI-linked membrane proteins were
tested for their TNF--inducing capacity, and all were found to have
a negative score (36). Moreover, it was also shown that Abs against the
inositol monophosphate were capable of inhibiting plasmodium-triggered
TNF- induction (37). On the other hand, it was shown that the
GPI-anchored iM4 surface molecule of Leishmania mexicana was
incapable of inducing the secretion of TNF- although it was capable
of inducing intracellular activation of macrophages (38). With respect
to still another parasite, it was shown that in the case of
Toxoplasma gondii, the major component involved in TNF-
secretion by PECs was the carbohydrate moiety of a glycoprotein present
in soluble extracts of the parasite (39). Again, however, for
comparative studies of all of these results, it is important to stress
that in these experiments as well as in experiments performed with
Plasmodium and Leishmania components (17, 39),
thioglycolate-elicited PECs and not naive, nonstimulated cells were
used. Our results, obtained with naive PECs isolated without the use of
thioglycolate, are in keeping with those of Schofield and colleagues
concerning the TNF--inducing capacity of T. brucei mfVSG.
However, clear evidence is presented herein that the DMG compound of
the GPI of T. brucei is indeed not required for TNF-
induction per se, but is needed to obtain proper macrophages
stimulation in the absence of IFN-.
Although the DMG moiety of mfVSG is not important for TNF- induction
itself, our results indicate that it plays a crucial role in the
stimulation of other macrophage-mediated responses such as IL-1
production and hyper-responsiveness toward LPS. Indeed, both total
trypanosome-soluble extract and mfVSG trigger IL-1 secretion by
resting PECs and 2C11/12 cells, while sVSG is unable to do so, even
when the cells are preactivated with IFN-. Similarly, while total
trypanosome soluble extract and mfVSG efficiently prime macrophages to
respond to suboptimal concentrations of LPS, sVSG completely lacks this
priming activity. Hence, the DMG compound of the mfVSG anchor seems to
be crucial for its IL-1-inducing and -priming activity, and similar
findings were reported for the glycolipid toxin of P.falciparum (34). Interestingly, however, an early report by
Mathias et al. on the induction of IL-1 secretion by macrophages
showed that when the macrophage cell line P388D1 was pulsed with sVSG,
secretion of IL-1 could be measured (40). This corroborates our
results obtained with the 2C11/12 cell line, namely that appropriately
activated macrophage cell lines can respond toward sVSG without prior
stimulation. In summary, VSG encompasses distinct macrophage-activating
components: 1) The GIP moiety, and in particular its galactose side
chain, is responsible for TNF- production; and 2) the GPI moiety,
and in particular its DMG anchor, mediates macrophage priming and LPS
hyper-responsiveness.
The mechanisms underlying the macrophage-activating capacity of the GIP
and GPI components of sVSG and mfVSG are not yet clearly defined. This
holds true especially for the bioactivity of the GIP moiety. Although
it has been suggested that this moiety might function as a second
messenger in certain signal transduction pathways (17, 41), no formal
proof exists that this is the case for trypanosome-derived GIP. The GPI
derived from the mfVSG of T. brucei, on the other hand, was
reported to activate in macrophages an endogenous protein tyrosine
kinase pathway (17). In this context, it is appropriate to mention that
CD14, the main LPS receptor of macrophages, is a GPI-anchored receptor
that utilizes a tyrosine kinase-mediated signal transduction pathway
(42). Hence the CD14 receptor, which mediates the induction of TNF-
and IL-1 production (43), most probably requires an intracellular or
transmembrane signal-transducing partner (44). The mfVSG-mediated
hyper-responsiveness could be due to a recruitment of this CD14-GPI
partner on the macrophage membrane, facilitating CD14 signal
transduction.
Having demonstrated the macrophage-activating capacity of sVSG and
mfVSG, it remains to be determined which form of VSG is physiologically
relevant during trypanosomiasis. Taking into account that trypanosomes
utilize a potent phospholipase C enzyme to release sVSG in stress
situations (19) and that sVSG can be detected in the serum during
T. brucei infections (45), trypanosome-mediated induction of
TNF- release may be mediated by sVSG. Furthermore, as mentioned
above, trypanosome-elicited production of IFN- will render
macrophages very responsive toward sVSG-mediated TNF- production. It
should be mentioned that a similar situation could occur during natural
infections of cattle with Trypanosoma congolense. Indeed,
both the sVSG and mfVSG of T. congolense trigger similar
TNF- production by bovine monocytes, provided however that the cells
are preactivated with IFN- (Dr. M. Sileghem, unpublished
observations). The fact that trypanosome infections also sensitize
IL-1 production suggests an important role for the DMG compound of
the mfVSG anchor. Since IL-1 is described as a TNF- inducer (46),
its release may further fuel TNF- production. Furthermore, the
capacity of mfVSG to prime macrophages to become hyper-responsive
toward LPS may be physiologically relevant, since trypanosome
infections were reported to increase the serum levels of LPS
(30).
In conclusion, the VSG of African trypanosomes exert potent
macrophage-activating properties resulting in the production of
cytokines such as TNF- and IL-1. Given the potentially noxious
effects of these cytokines on the host, one might wonder whether their
production is beneficial for the parasite. As far as TNF- is
concerned, its parasite-mediated production could make sense because
TNF- has been shown to be involved in trypanosome-elicited
immunosuppression (12, 33) and parasite growth regulation (13, 14, 15, 16).
|
Acknowledgments |
|---|
1–6myo-inositol-1-phosphate, Prof. B. Fraser-Reid
for supplying Gal1–2Gal1–3
Man1-O-(CH2)4-CH3, and Prof. J.
van Boon for supplying GLcNa1–6myo-inositol-1,2 cyclic
phosphate. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Stefan Magez, Eenheid CIMM (IMOL 2), Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640 Sint Genesius Rode, Belgium.
3 Abbreviations used in this paper: GPI, glycosylphosphatidylinositol; DMG, dimyristoylglycerol; GIP, glycosyl-inositol-phosphate; VSG, variant-specific surface glycoprotein; sVSG, soluble VSG; mfVSG, membrane form VSG; PEC, peritoneal exudate cell; TLCK, N-(p-tosyl)lysine chloromethyl ketone.
Received for publication April 17, 1997. Accepted for publication October 31, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
during African trypanosomiasis: involvement in parasite control, immunosuppression and pathology. Res. Immunol. 144:370.[Medline]
, Il-1 and Il-6 in the immune response to protozoan parasites. Immunoparasitol. Today 12:13.
as a growth factor for Trypanosoma brucei. Eur. J. Immunol. 26:1359.[Medline]
is involved in growth control of Trypanosoma brucei. J. Cell Biol. 137:715. and TNF- expression in macrophages by protein tyrosine kinase mediated signal transduction. Biochem. Biophys. Res. Commun. 205:984.[Medline]
production that stimulates parasite growth. Eur. J. Immunol. 21:2447.[Medline]
after induction with the surface coat of Trypanosoma brucei rhodesiense or lipopolysaccharide. J. Immunol. 145:3450.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  N. Antoine-Moussiaux, P. Buscher, and D. Desmecht Host-Parasite Interactions in Trypanosomiasis: on the Way to an Antidisease Strategy Infect. Immun., April 1, 2009; 77(4): 1276 - 1284. [Full Text] [PDF]
|
|
|
|
 |
|
 R. Lopez, K. P. Demick, J. M. Mansfield, and D. M. Paulnock Type I IFNs Play a Role in Early Resistance, but Subsequent Susceptibility, to the African Trypanosomes J. Immunol., October 1, 2008; 181(7): 4908 - 4917. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Stijlemans, T. N. Baral, M. Guilliams, L. Brys, J. Korf, M. Drennan, J. Van Den Abbeele, P. De Baetselier, and S. Magez A Glycosylphosphatidylinositol-Based Treatment Alleviates Trypanosomiasis-Associated Immunopathology J. Immunol., September 15, 2007; 179(6): 4003 - 4014. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Leppert, J. M. Mansfield, and D. M. Paulnock The Soluble Variant Surface Glycoprotein of African Trypanosomes Is Recognized by a Macrophage Scavenger Receptor and Induces I{kappa}B{alpha} Degradation Independently of TRAF6-Mediated TLR Signaling J. Immunol., July 1, 2007; 179(1): 548 - 556. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Caljon, J. Van Den Abbeele, B. Stijlemans, M. Coosemans, P. De Baetselier, and S. Magez Tsetse Fly Saliva Accelerates the Onset of Trypanosoma brucei Infection in a Mouse Model Associated with a Reduced Host Inflammatory Response Infect. Immun., November 1, 2006; 74(11): 6324 - 6330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Drennan, B. Stijlemans, J. Van Den Abbeele, V. J. Quesniaux, M. Barkhuizen, F. Brombacher, P. De Baetselier, B. Ryffel, and S. Magez The Induction of a Type 1 Immune Response following a Trypanosoma brucei Infection Is MyD88 Dependent J. Immunol., August 15, 2005; 175(4): 2501 - 2509. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Shi, G. Wei, W. Pan, and H. Tabel Trypanosoma congolense infections: antibody-mediated phagocytosis by Kupffer cells J. Leukoc. Biol., August 1, 2004; 76(2): 399 - 405. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Maina, J. M. Ngotho, T. Were, J. K. Thuita, D. M. Mwangangi, J. M. Kagira, J. M. Ndung'u, and J. Sternberg Proinflammatory Cytokine Expression in the Early Phase of Trypanosoma brucei rhodesiense Infection in Vervet Monkeys (Cercopithecus aethiops) Infect. Immun., May 1, 2004; 72(5): 3063 - 3065. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Debierre-Grockiego, N. Azzouz, J. Schmidt, J.-F. Dubremetz, H. Geyer, R. Geyer, R. Weingart, R. R. Schmidt, and R. T. Schwarz Roles of Glycosylphosphatidylinositols of Toxoplasma gondii: INDUCTION OF TUMOR NECROSIS FACTOR-{alpha} PRODUCTION IN MACROPHAGES J. Biol. Chem., August 29, 2003; 278(35): 32987 - 32993. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Delorenzi, A. Sexton, H. Shams-Eldin, R. T. Schwarz, T. Speed, and L. Schofield Genes for Glycosylphosphatidylinositol Toxin Biosynthesis in Plasmodium falciparum Infect. Immun., August 1, 2002; 70(8): 4510 - 4522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. C. Almeida and R. T. Gazzinelli Proinflammatory activity of glycosylphosphatidylinositol anchors derived from Trypanosoma cruzi: structural and functional analyses J. Leukoc. Biol., October 1, 2001; 70(4): 467 - 477. [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]
|
|
|
|
|
|
D. M. Paulnock and S. P. Coller Analysis of macrophage activation in African trypanosomiasis J. Leukoc. Biol., May 1, 2001; 69(5): 685 - 690. [Abstract] [Full Text]
|
|
|
|
|
|
B. Namangala, P. De Baetselier, W. Noël, L. Brys, and A. Beschin Alternative versus classical macrophage activation during experimental African trypanosomosis J. Leukoc. Biol., March 1, 2001; 69(3): 387 - 396. [Abstract] [Full Text]
|
|
|
|
|
|
C. Ropert, I. C. Almeida, M. Closel, L. R. Travassos, M. A. J. Ferguson, P. Cohen, and R. T. Gazzinelli Requirement of Mitogen-Activated Protein Kinases and I{{kappa}}B Phosphorylation for Induction of Proinflammatory Cytokines Synthesis by Macrophages Indicates Functional Similarity of Receptors Triggered by Glycosylphosphatidylinositol Anchors from Parasitic Protozoa and Bacterial Lipopolysaccharide J. Immunol., March 1, 2001; 166(5): 3423 - 3431. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Magez, M. Radwanska, A. Beschin, K. Sekikawa, and P. De Baetselier Tumor Necrosis Factor Alpha Is a Key Mediator in the Regulation of Experimental Trypanosoma brucei Infections Infect. Immun., June 1, 1999; 67(6): 3128 - 3132. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Cardoso De Almeida, M Geuskens, and E Pays Cell lysis induces redistribution of the GPI-anchored variant surface glycoprotein on both faces of the plasma membrane of Trypanosoma brucei J. Cell Sci., January 12, 1999; 112(23): 4461 - 4473. [Abstract] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) from bloodstream form trypanosomes, total soluble
extract from procyclic form trypanosomes (x), sVSG (
), mfVSG
(
), or total trypanosome extract depleted from VSG by
immunoprecipitation (•). Cells were incubated for 24 h with
different dilutions of trypanosome-soluble extract or VSG, starting at
500 µg/ml. TNF-
