Department of Immunology, Parasitology and Ultrastructure, Flemish Interuniversity Institute for Biotechnology, Free University Brussels (VUB), Paardenstraat 65, B-1640 St-Genesius-Rode, Belgium
Correspondence: Dr. Alain Beschin, Cellular Immunology Unit, Flemish Interuniversity Institute for Biotechnology, VIB-VUB, Paardenstraat 65, B-1640 St-Genesius-Rode, Belgium. E-mail: abeschin{at}vub.ac.be
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, type I) versus alternatively activated
macrophages (aaM, type II). Recently, we showed that although mice
infected with phospholipase C-deficient
(PLC-/-) Trypanosoma brucei
brucei exhibit a clear shift from type I to the type II cytokine
production, wild type (WT)-infected mice remain locked in a type I
cytokine response. In the present study, phenotype and accessory cell
function of macrophages elicited during WT and
PLC-/- T. b. brucei infection
were compared. Results indicate that caM develop in a type I
cytokine environment in the early phase of WT and
PLC-/- trypanosome infection, correlating
with inhibition of T cell activation triggered by a mitogen, a
superantigen, or an antigen. In the late stage of infection, only
PLC-/--infected mice resisting the infection
develop type II cytokine-associated aaM correlating with impaired
antigen- but not mitogen- or superantigen-induced T cell
activation.
Key Words: antigen presentation • immunosuppression • phospholipase C • trypanosome infection
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Macrophages are among the first host immune cells to encounter the infecting trypanosomes and/or their products. Consequently, these cells may play a protective or pathogenic role, depending on their state of activation during the course of infection. A protective role of macrophages may rely on the clearance of African trypanosomes by phagocytosis and/or by secreting products such as tumor necrosis factor (TNF) and nitric oxide (NO), which were documented in vitro to be trypanolytic and trypanostatic, respectively [3 4 5 6 7 8 9 10 11 ]. Besides that, macrophages contribute to the pathology associated with African trypanosomosis by erythrophagocytosis, resulting in the host’s anemia [12 13 14 15 16 ]. Conversely, macrophages are the central effector cells involved in the inhibition of T cell blastogenic response to mitogens and antigens occurring during African trypanosome infection [17 18 19 20 21 22 23 24 ]. However, whether this suppressive activity of macrophages limits the development of harmful immune responses or allows the parasite to establish a chronic infection remains unclear.
According to recent studies, the type I/type II cytokine balance may
influence the development of different subsets of suppressive
macrophages that are antagonistically regulated
[25
26
27
28
]. Classically activated macrophages (caM)
occur in a type I cytokine environment [interferon-
(IFN-),
TNF] and are inhibited by type II cytokines [interleukin (IL)-4,
IL-13, IL-10]. In contrast, alternatively activated macrophages
(aaM) develop in a type II cytokine environment and are inhibited by
type I cytokines. caM, possessing cytotoxic, antimicrobial, and
antiproliferative function based on their ability to secrete NO, play a
defensive role in several diseases. However, caM secreting
inflammatory mediators (TNF, IL-1, IL-6, NO) are also involved in the
setting of immunopathologies. It was proposed that aaM secreting
anti-inflammatory molecules [IL-10, transforming growth factor ß
(TGF-ß)] down-regulate inflammatory processes and counteract NO
synthesis by expressing arginase that compete with inducible NO
synthase for L-arginine as substrate. During African trypanosome
infection, phenotypic analyses of macrophages suggest the occurrence of
caM. Indeed, macrophages from Trypanosoma brucei
brucei-infected animals express increased levels of major
histocompatibility complex (MHC) class II molecules and release
prostaglandin (PG) E2 and reactive oxygen intermediates
[18
19
20
21
, 24
, 29
], which are
typical of a classical activation state. Moreover, IFN- was shown to
sensitize macrophages to produce TNF, IL-1, and NO in response to VSG
[18
, 30
31
32
]. Furthermore, NO and IFN-
participate in the inhibition of T cell proliferative response
occurring in African trypanosome-infected animals [17
,
18
, 22
, 23
,
33
34
35
36
].
The occurrence of aaM during African trypanosomosis remains
speculative. This may be because in most murine models of African
trypanosomosis, a predominant type I cytokine environment is created,
fueling the generation of caM [18
, 32
,
36
37
38
]. However, a recent study showed that a T.
b. brucei mutant [phospholipase C null mutant
(PLC-/-)] induces the production of type I
cytokines during the early stage of infection, followed by the
secretion of type II cytokines in the late/chronic phase of the disease
[39
]. Although wild type (WT) T. b. brucei
kill mice within 5 weeks, PLC-/- trypanosomes
induce a chronic infection. It is interesting that
PLC-/--infected mice recover from inhibition
of mitogen-induced T cell proliferation. This recovery correlates with
the absence of macrophages inhibiting mitogen-induced proliferation,
i.e., caM.
Because a sequential induction of type I and type II cytokine
environment occurs in the PLC-/- trypanosome
model, this model was adopted to evaluate the capacity of T. b.
brucei to elicit aaM versus caM. Moreover, a comparative
analysis of the accessory cell function of macrophages elicited during
WT and PLC-/- T. b. brucei
infection was performed. Results indicate that during the early phase
of infection, WT and PLC-/- parasites elicit
caM that develop in a type I cytokine environment, correlating with
the inhibition of T cell activation triggered by a mitogen
[concanavalin A (Con A)], a superantigen [staphylococcal enterotoxin
B (SEB)], or an antigen [hen egg lysozyme (HEL)]. During the late
stage of infection, only PLC-/--infected mice
resisting the infection develop type II cytokine-associated aaM,
which correlates with the impairment of antigen- but not mitogen- or
superantigen-induced T cell activation.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Experimental design
At each time interval post-infection, levels of cytokines, NO,
or arginase activity were quantified in peritoneal exudate cells (PEC),
plasma, or blood mononuclear cells of three infected mice. For each
parameter, results were expressed as the mean response of the three
infected animals tested individually (±SE) and compared
with the same parameters assessed in three noninfected mice. Results
are representative of at least five similar, independent experiments
performed. Statistical analyses were assessed by Student’s
t-test to validate the data.
Cells and T cell hybridoma preparations
Nonadherent lymph node cells from naïve control mice
(LNCn) were prepared as described [17
].
Adherent cell population from the peritoneal cavity of noninfected or infected mice, prepared as described [17 ], was resuspended in RPMI 1640 (Gibco BRL, Grand Island, NY) and supplemented with fetal calf serum (10%), penicillin-streptomycin (100 U–100 µg/ml), L-glutamine (2 mM), and 2-mercaptoethanol (5x10-5 M; complete medium). Plastic adherent cells, containing 95% MAC-1-positive cells as determined by fluorescence-activated cell sorter (FACS) analysis, were considered as macrophages and used as antigen-presenting cells.
The mouse 1E5.11 T cell hybridoma (I-Ed-restricted) reactive to HEL, kindly offered by Dr. A. Darji (GBF, Braunschweig, Germany), or the mouse 13.26.8-H6 Vß8-expressing T cell hybridoma reactive to SEB, kindly provided by Dr. Muriel Moser (Free University Brussels, Gosselies, Belgium), was propagated in complete medium.
To obtain HEL-specific T cells, mice were injected intra-footpad with 25 µg HEL emulsified in complete Freund’s adjuvant. After 8 days, nonadherent cells were prepared from draining popliteal lymph nodes and used as HEL-responding T cells.
Mononuclear cells from blood collected on heparin (20 U/mL; Sigma Chemical Co., St. Louis, MO) were purified on Ficoll-Paque (Amersham Pharmacia Biotech AB, Uppsala, Sweden) as described [41 ], and plastic-adherent cells were prepared [17 ].
Con A-induced T cell activation
Macrophages from uninfected or infected mice
(1.25x105/ml) were co-cultured with nonadherent LNCn
(2x106/ml) in 96-well plates (Falcon®, Becton Dickinson,
Rutherford, NJ) in the presence of Con A (10–0.3125 µg/ml; Sigma
Chemical Co.) at 37°C in a humidified atmosphere containing 5%
CO2. T cell activation was determined by measuring IL-2
concentration in 24-h co-culture supernatants using a specific
enzyme-linked immunosorbent assay (ELISA; PharMingen Europe,
Erembodegem-Aalst, Belgium) as described [24
].
SEB-induced T cell activation
Macrophages from noninfected or infected mice
(5x105/ml) were co-cultured with the 13.26.8-H6 T cell
hybridoma (105/ml) in complete medium in the presence of
serial dilutions of purified SEB (4–0.125 µg/ml; Toxin Technology,
Sarasota, FL). T cell activation was determined as described for Con
A-induced T cell activation.
Antigen-specific T cell activation
The 1E5.11 T cell hybridoma (2x105/ml) or
HEL-specific T cells (2x106/ml) were co-cultured in
complete medium with macrophages from noninfected or infected mice
(5x105/ml) in the presence of HEL (50 µg/ml; Merck &
Co., Rahway, NJ). T cell activation was monitored as described for Con
A-induced T cell activation.
Uptake and catabolism of HEL protein
HEL was radiolabeled with 125I as described
[24
] to a specific activity of 4.0 x
106 cpm/µg. Macrophages were allowed to adhere on 35-mm
tissue-culture plates (2 h, 37°C) and then incubated for 3 h at
37°C with radiolabeled HEL (6 µg/5x105 cells/ml).
Cells were washed [1% bovine serum albumin (BSA) in
phosphate-buffered saline (PBS)] until no radioactivity could be
detected in the washing medium, lysed in Triton X-100 (0.5% in PBS),
and the total amount of incorporated radioactivity was measured. The
radioactivity released from macrophages incubated with
125I-labeled HEL in the presence of 0.2 mM sodium azide to
block endocytosis was subtracted to calculate the net HEL uptake.
To determine the antigen catabolism, macrophages were loaded with 125I-labeled HEL and washed as described above. Cells were then chased for 90 min at 37°C in HEL-free medium. The 10% trichloroacetic acid (TCA)-soluble and -insoluble radioactivity present in the cell lysate or culture medium supernatants was measured. In control experiments, macrophages incubated with 125I-labeled HEL were chased in medium containing sodium azide. The radioactivity released from the appropriate controls was subtracted to obtain the net HEL catabolism.
Expression of surface molecules on macrophages
Biotinylated anti-I-Ed, anti-intercellular adhesion
molecule-1 (ICAM-1), anti-CD40, anti-MAC-1, and isotype-control
antibodies used for cytofluorimetry analysis were purchased from
PharMingen Europe. CTLA-4/immunoglobulin (Ig) used to detect the
surface expression of B7 was a kind gift of Dr. K. Thielemans
(Allergy/Immunology, Internal Medicine, Free University Brussels).
Fluorescein isothiocyanate (FITC)-conjugated streptavidin and
FITC-labeled anti-(human) IgG were obtained, respectively, from
Amersham Life Sciences (Bucks, UK) and Serotec (Oxford, UK). Cells were
stained as described [24
] and analyzed on a Becton
Dickinson FACStar using the CELLQuest program. Isotype-control
antibodies did not show significant background staining.
Plasma and PEC-culture supernatant collections
At different time intervals following infection, blood collected
by heart-puncture on heparin (20 U/mL) was centrifuged (10,000
g, 10 min) and frozen at -80°C until analysis. Peritoneal
exudate cells from noninfected or infected animals (5x105)
were stimulated with 2.5 µg Con A or 1 µg SEB in 1 mL complete
medium at 37°C in a humidified atmosphere containing 5%
CO2. Culture supernatants were collected after 24 h
(IL-4, IL-13 quantification) or 72 h (IFN-, IL-10, NO
quantification) and frozen at -80°C until analysis.
Quantification of cytokines
Cytokines were quantified using specific sandwich ELISAs for
IFN-, IL-4, IL-10 (PharMingen Europe), or TNF and IL-13 (R&D
Systems, Abingdon, UK). They were performed in accordance with the
manufacturers’ protocols.
Quantification of NO
Levels of plasma NO were determined by quantifying nitrites as
described previously [8
]. All reagents were obtained
from Sigma Chemical Co. Briefly, nitrate was stoichiometrically reduced
to nitrite by incubating 100 µL plasma sample (1 h, 37°C) in the
presence of Aspergillus nitrite reductase (NAD[P]H, EC
1.6.6.2, 0.1 U/mL), reduced nicotinamide adenine dinucleotide phosphate
(NADPH; 120 µM), and flavine adenine dinucleotide (FAD; 5 µM).
Subsequently, excess NADPH was oxidized with L-lactic dehydrogenase (EC
1.1.1.27, type XI from rabbit muscle, 10 U/ml) and sodium pyruvate (10
mM) for 30 min at 37°C. Nitrite concentration in plasma was then
assayed by a standard Griess reaction as described [17
].
Nitrite concentration in PEC supernatants was assayed without reducing
nitrates.
Determination of arginase activity
Arginase activity was measured in PEC or adherent blood
mononuclear cell lysates as described by Munder et al.
[26
, 27
]. Briefly, cells were lysed with
100 µl Triton X-100 (0.1%). After 30 min on a shaker, arginase was
activated in the presence of 100 µl Tris-HCl (25 mM) and 35 µl
MnCl2 (10 mM, 10 min, 56°C). L-arginine hydrolysis was
conducted by incubating the cell lysate with 100 µL L-arginine (0.5
M, pH 9.7) at 37°C up to 1 h. The reaction was stopped with 800
µL H2SO4 (96%)/H3PO4
(85%)/H2O (1 v/3 v/7 v). The produced urea was quantified
at 540 nm after addition of 40 µL 
-isonitrosopropiophenone
(dissolved in 100% ethanol) followed by heating at 100°C for 20 min.
One unit of enzyme is defined as the amount that catalyzes the
formation of 1 µmol urea/min.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
 View larger version (21K): [in a new window] |
Figure 1. Parasitemia in Balb/c mice infected with WT or
PLC-/- T. b. brucei. Data
(mean±SE; n=5) are representative of six
independent experiments.
|
T cell activation by macrophages from WT or
PLC-/- T. b. brucei-infected mice
in the presence of Con A
The Con A-induced activation of LNCn co-cultured with peritoneal
macrophages collected during the early (first peak of parasitemia),
late (5 weeks), or chronic (5 months) stages of infection with WT or
PLC-/- T. b. brucei was
investigated in vitro by measuring IL-2 secretion in
co-culture supernatants. Nonstimulated LNCn or macrophages from
uninfected or WT- or PLC-/--infected mice
(pM-N, pM-WT, pM-PLC-/-) did not
produce detectable levels of IL-2. Similarly, macrophages from
noninfected or WT- or PLC-/--infected mice
activated with Con A did not produce detectable amounts of IL-2. Upon
activation with optimal concentration of Con A (2.5 µg/ml), LNCn
secreted about 550 pg/ml IL-2, which markedly increased to 3900 pg/ml
when co-cultured with pM-N (Fig. 2
). In contrast, Con A-induced IL-2 production was greatly reduced
in co-cultures of pM-WT and LNCn (p<0.01), to a similar
extent throughout the entire course of infection. IL-2 secretion was
also reduced following Con A stimulation in co-cultures of
pM-PLC-/- from early stage-infected mice
and LNCn but to a lesser extent than in co-cultures containing pM-WT
(p<0.01). Moreover, the accessory cell function of
pM-PLC-/- recovered in the course of
infection and tended to be up-regulated during the chronic stage of
infection compared with pM-N (Fig. 2)
. These results indicate that
whereas pM-WT exert an active, suppressive activity impairing Con
A-induced T cell activation during the entire course of T. b.
brucei infection, this defect is transient in
pM-PLC-/-, occurring mainly during the
early stage of infection.
 View larger version (22K): [in a new window] |
Figure 2. T cell activation by macrophages from WT or
PLC-/- T. b. brucei-infected mice
in the presence of Con A. During early (first peak of parasitemia),
late (5 weeks), or chronic (5 months) phase of infection, pM-N,
pM-WT, or pM-PLC-/- were co-cultured
with LNCn in the presence of various concentrations of Con A for
24 h. IL-2 concentrations were determined in co-culture
supernatants.
|
-WT and
pM-PLC-/- to stimulate a SEB-responding T
cell hybridoma was evaluated during the entire course of infection.
SEB-stimulated pM-N, pM-WT, or
pM-PLC-/- did not produce detectable
levels of IL-2 in the absence of the hybridoma nor did the latter
alone. PM-WT were impaired in their capacity to present SEB to the
hybridoma to a similar extent during the whole period of infection
compared with pM-N (p<0.01; Fig. 3
). PM-PLC-/- were also inhibited
significantly in their ability to present SEB during the early stage of
infection (p<0.01). However, from the late stage of
infection onward, SEB-presenting capacity of
pM-PLC-/- was restored. Hence, as observed
with Con A, although SEB activation of T cells by macrophages from
WT-infected mice is inhibited throughout the entire course of
infection, this defect is restored in macrophages from late-stage
PLC-/--infected animals.
 View larger version (25K): [in a new window] |
Figure 3. T cell activation by macrophages from WT- or
PLC-/--infected mice in the presence of SEB.
At different times post-infection, pM-N, pM-WT, or
pM-PLC-/- were co-cultured with LNCn in
the presence of various concentrations of SEB for 24 h. IL-2
concentrations were determined in co-culture supernatants.
|
-WT and pM-PLC-/-
to present HEL to a specific T cell hybridoma was analyzed in
vitro by measuring IL-2 production in culture supernatants. The
hybridoma, pM-N, pM-WT, or pM-PLC-/-
alone did not secrete detectable amounts of IL-2 in the presence of
HEL. Figure 4
shows that, compared with pM-N, pM-WT and
pM-PLC-/- had a significantly reduced
capacity to present HEL throughout the course of infection
(p<0.01). However, the ability of
pM-PLC-/- to present HEL was reduced to a
lesser extent than that of pM-WT during the whole period of
infection (p<0.05).
 View larger version (9K): [in a new window] |
Figure 4. HEL-specific T cell activation by macrophages from WT or
PLC-/- T. b. brucei-infected mice.
At different times post-infection, pM-N, pM-WT, or
pM-PLC-/- were co-cultured with
HEL-specific T cell hybridoma or HEL-specific T cells in the presence
of HEL (50 µg/ml) for 24 h. IL-2 concentrations were determined
in co-culture supernatants. Dashed lines indicate levels of IL-2 in
co-cultures containing pM-N.
|
-WT and pM-PLC-/- to
present HEL was further analyzed using lymph node cells from
HEL-immunized mice. The ability of HEL-specific T cells to produce IL-2
was impaired greatly by pM-WT collected during the entire course of
the disease compared with pM-N (p<0.01; Fig. 4
). The HEL
presentation ability of pM-PLC-/- to
HEL-specific T cells was reduced to a similar extent as pM-WT
throughout the infection period.
Collectively, these observations indicate that protein antigen-specific
T cell activation by pM-WT and
pM-PLC-/- remains impaired throughout the
whole course of the disease.
Phenotypic characterization of PEC from WT- or
PLC-/--infected mice
The phenotype of PEC-WT and PEC-PLC-/-
was analyzed in the course of infection. Microscopic examination
revealed that PEC-WT contained up to 80% enlarged cells with abundant
cytoplamic vacuoles or inclusions. This population was absent from
PEC-N and only represents 2% of PEC-PLC (not shown). Table 1
illustrates experiments performed in the late stage of infection.
Compared with pM-N, pM-WT and
pM-PLC-/- exhibited an increased uptake
and catabolism of HEL and expressed similar or increased levels of MHC
class II and ICAM-1 molecules. Compared with pM-N, the expression of
B7 and CD40 molecules was down-regulated on pM-WT and to a lesser
extent on pM-PLC-/-.
|
View this table: [in a new window] |
Table 1. Phenotype Analysis of Peritoneal Macrophages from Mice in Late Stage of
Infection with WT or PLC-/- T. b. brucei
|
-N but to a lower level than by pM-WT. During the
late/chronic phase of infection, PEC-PLC-/-
clearly exhibited a reduced ability to secrete TNF upon Con A
stimulation compared with PEC-N (p<0.01). Similar
modulations of TNF secretion were observed upon SEB stimulation of
PEC-WT and PEC-PLC-/- (not shown).
 View larger version (18K): [in a new window] |
Figure 5. Levels of TNF, nitrite, and arginase activity in PEC of WT or
PLC-/- T. b. brucei-infected mice.
At different time points post-infection, TNF and nitrite levels were
determined in PEC-N, PEC-WT, or PEC-PLC-/-,
cultured without or with Con A (2.5 µg/ml). Arginase activity was
determined in PEC-N, PEC-WT, or PEC-PLC-/-
lysates from WT- or PLC-/--infected mice
(mean±SE, n=3). Dashed lines indicate TNF and
nitrite levels or arginase activity in the cell cultures or cell
lysates from noninfected mice.
|
Arginase activity was not induced following T. b. brucei infection in PEC-WT at any moment post-infection, as well as in PEC-PLC-/- during the early phase of infection (Fig. 5) . In contrast and corresponding to reduced TNF and NO productions, PEC-PLC-/- exhibited increased arginase activity during the late and chronic stages of infection.
Collectively, these observations suggest that WT or PLC-/- T. b. brucei infection elicits phenotypically different macrophages in the peritoneal cavity of infected mice.
Production of IFN-, IL-10, IL-4, and IL-13 by PEC from WT or
PLC-/- T. b. brucei-infected mice
Because macrophage activity is regulated by cytokines
[25
], the cytokine environment in the peritoneal cavity
of mice was analyzed in vitro in culture supernatants of
PEC-WT or PEC-PLC-/- collected at different
time points post-infection. A spontaneous secretion of cytokines
occurred only in cell supernatants from late-stage, WT-infected mice
(0.4 ng/ml IFN-) and late/chronic-stage,
PLC-/--infected animals (0.1 ng/ml IL-10).
Figure 6
shows that, compared with PEC-N, Con A-induced IFN- production
by PEC-WT was significantly reduced only during the early stage of
infection (p<0.01). In contrast, Con A-induced IFN-
production by PEC-PLC-/- decreased
progressively from the late stage of infection onward compared with
PEC-N (p<0.01). The secretion of IL-10 by PEC-WT tended to
increase during the early phase of infection compared with Con
A-activated PEC-N but was decreased during the late stage of infection
(p<0.05). IL-4 and IL-13 were not detected in Con
A-activated PEC-WT throughout the course of infection. Infection with
PLC-/- parasites induced a progressive
increase in Con A-induced secretion of IL-10, IL-4, and IL-13, reaching
maximal values during the chronic stage of infection. Similar cytokine
modulations were observed in PEC-WT and
PEC-PLC-/- stimulated with SEB (not shown).
 View larger version (25K): [in a new window] |
Figure 6. IFN-, IL-10, IL-4, or IL-13 productions by PEC of WT or
PLC-/- T. b. brucei-infected mice.
At different times post infection, IFN-, IL-10, IL-4, or IL-13
productions were quantified in PEC-N, PEC-WT, or
PEC-PLC-/- activated with Con A (2.5 µg/ml;
mean±SE, n=3). Dashed lines indicate cytokine
levels in PEC-N. IL-4 or IL-13 levels were not detected in noninfected
or WT-infected mice except in IL-4 in late-stage WT-infected animals.
|
-WT and
pM-PLC-/- differs, because type II
cytokines are present mainly in
PLC-/--infected mice from the late stage of
infection.
NO levels and arginase activity in the blood of WT or
PLC-/- T. b. brucei-infected mice
We demonstrated previously that WT and
PLC-/- trypanosomes induce a systemic type I
cytokine response (IFN-) in the plasma of infected mice in the early
stage of infection [39
]. In addition, the levels of TNF
were increased in the plasma of WT-infected mice, reaching maximal
value in the late stage of infection. In
PLC-/--infected mice, TNF was only detected
in the early stage of the disease to lower levels than in WT-infected
mice. Finally, type II cytokines (IL-4, IL-10) were only observed in
the plasma of late-stage PLC-/--infected
animals. IL-13 could not be detected in the plasma of WT- or
PLC-/--infected mice (not shown).
To further investigate the phenotype of activated macrophages, NO levels were quantified as accumulation of nitrites in the plasma of WT- or PLC-/--infected mice. In parallel, arginase activity was quantified in blood monocytes. Increased levels of NO were observed in the plasma of WT-infected mice compared with uninfected animals (Fig. 7 ). These levels reached maximal values during the early stage of infection but remained elevated during the late stage of infection. NO plasma levels were increased in PLC-/--infected mice only in the early stage of infection to a lower extent than in WT-infected mice (p<0.05). No significant arginase activity could be detected throughout the infection with WT parasites (Fig. 7) . Arginase activity was, however, elicited from the late stage of infection in PLC-/--infected animals.
 View larger version (14K): [in a new window] |
Figure 7. Plasma nitrite levels or arginase activity in blood monocytes of WT or
PLC-/- T. b. brucei-infected mice.
At different time points post-infection, plasma levels of nitrite or
arginase activity in blood monocyte lysates were determined in WT- or
PLC-/--infected mice (mean±SE,
n=3). Dashed lines indicate nitrite levels in the plasma or
arginase activity in blood monocyte lysates from noninfected mice.
|
and possibly
reflect the presence of classically activated macrophages in
susceptible animals. In addition, an increased arginase activity
suggesting the development of alternatively activated macrophages
occurs exclusively in PLC-/--infected mice
showing a systemic type II cytokine environment. |
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-WT and pM-PLC-/-
[42
]. However, the activation status of pM-WT and
pM-PLC-/- differs in their L-arginine
metabolic pathways. pM-WT secreted NO progressively in the course of
infection but did not display arginase activity. The NO secretion by
pM-WT was paralleled with the synthesis of TNF. A similar modulation
of L-arginine metabolism and TNF also occurred in the blood of
WT-infected mice. These data confirm previous studies suggesting the
development of caM in WT T. b. brucei-infected mice
[18
, 32
, 36
37
38
]. The
occurrence of caM was also evidenced in
PLC-/--infected mice but only during the
early stage of infection. From the late stage of
PLC-/- trypanosome infection, macrophages
secreted neither TNF nor NO but showed arginase activity paralleled by
the production of type II cytokines (IL-4, IL-10, and IL-13) in the
peritoneal cavity and in the blood. These observations suggest the
induction of aaM in late/chronic-stage
PLC-/--infected animals. Because the latter
survived longer (>150 days compared with 30 days for WT-infected mice)
[39
], the sequential development of caM and aaM
may be beneficial to the host during T. b. brucei infection.
During the early stage of infection, caM-secreting inflammatory
mediators, as observed in WT and
PLC-/--infected mice, may be required to
control the aggressive parasitemic waves [36
37
38
,
43
, 44
]. The initiation of type II cytokine
production in the late stage of PLC-/-
trypanosome infection may down-regulate the development of caM and
induce the emergence of aaM [25
26
27
28
], which may, in
turn, decrease the host’s susceptibility to T. b. brucei.
Consistently, we observed that IL-10-/- mice
are extremely susceptible to WT and PLC-/-
infections, showing respective survival times of 8 and 13 days, and
display a classical macrophage hyperactivation status (unpublished
results).
How do the PLC-/- T. b. brucei
induce caM? The lower parasite burden (i.e., reduced antigenic load)
in PLC-/--infected animals compared with
WT-infected mice may be responsible for the induction of type II immune
response, as suggested in other parasite models [45
].
The absence of PLC activity may facilitate the shift from caM to
aaM induced by the PLC-/- T. b.
brucei. Indeed, the PLC enzyme may be involved in the release of
glycosylphosphatidylinositol (GPI)-anchored proteins [46
,
47
]. Moreover, GPI anchors prime macrophages to secrete
TNF, IL-1, IL-6, IL-12, and NO [31
, 48
] and
natural killer cell antigen 1.1 helper T (NKT) cells to produce IFN-
rather than IL-4 [49
]. Hence, the lower amount of GPI
that anchors in PLC-/--infected mice may
impair a sustained classical macrophage activation. In addition,
because the functional activation of macrophages is cytokine
dose-dependent and temporary [50
], the lower production
of TNF and the increased production of type II cytokines in the early
stage of PLC-/- parasite infections may favor
the development of aaMP in the late stage of infection.
pM-WT inhibited trypanosome-unrelated antigen-specific, and
aspecific T cell activation throughout the period of infection.
pM-PLC-/- also suppressed T cell
activation in response to antigen during the whole course of infection,
and the impairment of aspecific T cell activation only occurred during
the early phase of infection. Hence, the mechanisms by which
macrophages inhibit IL-2 secretion by T cells in response to antigens
and aspecific activators differ in WT- and
PLC-/--infected mice and through the course
of infection. During the early stage of WT T. b. brucei
infection, macrophages were shown to inhibit Con A-induced T cell
proliferation in the spleen and the lymph nodes, down-regulating IL-2
secretion and IL-2 receptor (IL-2R) expression via PG/NO-dependent
mechanisms [17
, 18
, 20
,
21
, 23
, 33
]. However, during
the late stage of infection, PG/NO-independent mechanisms emerge in
both lymphoid compartments [17
, 18
,
22
, 33
, 35
36
37
38
]. At this
moment, IFN- and TNF act synergistically to block Con A-induced T
cell responsiveness in the lymph nodes [17
,
22
, 36
]. Accordingly, during the early stage
of infection, pM-WT and pM-PLC, being caM, may inhibit T cell
activation induced by Con A and SEB by producing PG and NO
[25
]. In the late stage of infection, chronic TNF
production by WT-infected animals may take part in the inhibition of
aspecific T cell activation through attenuation of TCR signaling
[51
]. This mechanism should not be operative in
late-stage, PLC-/--infected animals because
pM-PLC-/- do not secrete TNF at this
moment.
As far as antigens are concerned, we demonstrated [24
]
that the impaired T cell activation during WT T. b. brucei
infection results from a defect in the step leading to the formation of
[antigenic peptide-MHC class II molecule] complex on the macrophage
surface, although the surface expression of MHC class II molecules
tended to be up-regulated. Moreover, the decreased expression of B7 and
CD40 co-stimulatory molecules on pM-WT could contribute to the
inhibition of antigen-specific T cell activation. The inhibition of
antigen-specific T cell activation does not result from the production
of PG, NO, or reactive oxygen intermediate by macrophages. However,
IL-10 contributes marginally to the impairment of antigen-induced T
cell activation observed in WT T. b. brucei-infected mice.
Such mechanisms, including a similar involvement for IL-10 in the
inhibition of antigen-specific T cell activation, also hold true for
macrophages of PLC-/--infected mice
(unpublished results). Moreover, as for aspecific T cell activation,
TNF produced by pM-WT all through the infection and by
pM-PLC-/- in the early stage of infection
may contribute to the inhibition of antigen-induced T cell activation,
down-regulating TCR signaling [51
]. An eventual role of
cytokines produced by aaM in the peritoneal cavity of
PLC-/--infected mice such as IL-10 and
TGF-ß on the inhibition of antigen-, mitogen-, or
superantigen-induced IL-2 secretion by T cells requires further
elucidation. To this regard, it was demonstrated that IL-10
participates in the inhibition of Con A-induced T cell proliferation in
T. congolense-infected mice [52
].
In conclusion, this study shows that WT and
PLC-/- T. b. brucei elicit
pro-inflammatory caM during the early phase of infection paralleled
with the impairment of antigen-specific and -aspecific T cell
activation. Importantly, only PLC-/-
parasites induce the development of anti-inflammatory aaM during the
late/chronic stages of infection, which correlated only with the
inhibition of antigen-induced T cell activation. The sequential
activation of caM in a type I cytokine environment at the beginning
of infection, followed by activation of aaM in a type II cytokine
environment during the late/chronic stage of infection, may be
responsible for the increased resistance of mice to
PLC-/- T. b. brucei infections.
Alternatively, aaM, by inhibiting antigen-specific immune response,
may favor the persistence of a chronic infection in
PLC-/--infected animals and prevent cure. It
is interesting that cattle, naturally tolerant to T.
congolense, exhibit increased transcription of IL-4 and IL-10
mRNAs with a concomitant reduction in NO secretion and TNF mRNA
expression [41
, 53
, 54
].
Hence, it might be fruitful to investigate whether infection
preferentially induces aaM in trypanotolerant cattle and whether
these cells contribute to host resistance.
Received July 1, 2000; revised October 16, 2000; accepted October 18, 2000.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
is involved in growth control of Trypanosoma brucei J. Cell Biol. 137,715-727 with a Th2-associated expression pattern J. Immunol. 160,1411-1418 are synergistic inducers of nitric oxide synthesis Eur. J. Immunol. 26,539-543[Medline] antibodies alters trypanosome-susceptible mice to a resistant-like phenotype J. Immunol. 161,5507-5515 receptor gene: nitric oxide-dependent and -independent suppression of T-cell proliferative responses and the development of anaemia Immunology 94,476-480[Medline]-dependent J. Immunol. 161,6775-6783-dependent nitric oxide production is not linked to resistance in experimental African trypanosomiasis Cell. Immunol. 192,24-32[Medline]-activated monocytes and increased transcription of interleukin-10 Parasite Immunol. (Oxf.) 20,421-429This article has been cited by other articles:
 |
|
 |
|
  M. Guilliams, K. Movahedi, T. Bosschaerts, T. VandenDriessche, M. K. Chuah, M. Herin, A. Acosta-Sanchez, L. Ma, M. Moser, J. A. Van Ginderachter, et al. IL-10 Dampens TNF/Inducible Nitric Oxide Synthase-Producing Dendritic Cell-Mediated Pathogenicity during Parasitic Infection J. Immunol., January 15, 2009; 182(2): 1107 - 1118. [Abstract] [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. Gangadharan, M. A. Hoeve, J. E. Allen, B. Ebrahimi, S. M. Rhind, B. M. Dutia, and A. A. Nash Murine gammaherpesvirus-induced fibrosis is associated with the development of alternatively activated macrophages J. Leukoc. Biol., July 1, 2008; 84(1): 50 - 58. [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]
|
|
|
|
|
|
M. Guilliams, G. Oldenhove, W. Noel, M. Herin, L. Brys, P. Loi, V. Flamand, M. Moser, P. De Baetselier, and A. Beschin African Trypanosomiasis: Naturally Occurring Regulatory T Cells Favor Trypanotolerance by Limiting Pathology Associated with Sustained Type 1 Inflammation J. Immunol., September 1, 2007; 179(5): 2748 - 2757. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Namangala, C. Sugimoto, and N. Inoue Effects of Exogenous Transforming Growth Factor {beta} on Trypanosoma congolense Infection in Mice Infect. Immun., April 1, 2007; 75(4): 1878 - 1885. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Hassanzadeh Ghassabeh, P. De Baetselier, L. Brys, W. Noel, J. A. Van Ginderachter, S. Meerschaut, A. Beschin, F. Brombacher, and G. Raes Identification of a common gene signature for type II cytokine-associated myeloid cells elicited in vivo in different pathologic conditions Blood, July 15, 2006; 108(2): 575 - 583. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Raes, L. Brys, B. K. Dahal, J. Brandt, J. Grooten, F. Brombacher, G. Vanham, W. Noel, P. Bogaert, T. Boonefaes, et al. Macrophage galactose-type C-type lectins as novel markers for alternatively activated macrophages elicited by parasitic infections and allergic airway inflammation J. Leukoc. Biol., March 1, 2005; 77(3): 321 - 327. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Manca, M. B. Reed, S. Freeman, B. Mathema, B. Kreiswirth, C. E. Barry III, and G. Kaplan Differential Monocyte Activation Underlies Strain-Specific Mycobacterium tuberculosis Pathogenesis Infect. Immun., September 1, 2004; 72(9): 5511 - 5514. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Gavrilescu, B. A. Butcher, L. Del Rio, G. A. Taylor, and E. Y. Denkers STAT1 Is Essential for Antimicrobial Effector Function but Dispensable for Gamma Interferon Production during Toxoplasma gondii Infection Infect. Immun., March 1, 2004; 72(3): 1257 - 1264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. B. Geldhof, J. A. Van Ginderachter, Y. Liu, W. Noel, G. Raes, and P. De Baetselier Antagonistic effect of NK cells on alternatively activated monocytes: a contribution of NK cells to CTL generation Blood, December 1, 2002; 100(12): 4049 - 4058. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Noel, G. Hassanzadeh, G. Raes, B. Namangala, I. Daems, L. Brys, F. Brombacher, P. D. Baetselier, and A. Beschin Infection Stage-Dependent Modulation of Macrophage Activation in Trypanosoma congolense-Resistant and -Susceptible Mice Infect. Immun., November 1, 2002; 70(11): 6180 - 6187. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Stempin, L. Giordanengo, S. Gea, and F. Cerban Alternative activation and increase of Trypanosoma cruzi survival in murine macrophages stimulated by cruzipain, a parasite antigen J. Leukoc. Biol., October 1, 2002; 72(4): 727 - 734. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Motran, F. L. Diaz, A. Gruppi, D. Slavin, B. Chatton, and J. L. Bocco Human pregnancy-specific glycoprotein 1a (PSG1a) induces alternative activation in human and mouse monocytes and suppresses the accessory cell-dependent T cell proliferation J. Leukoc. Biol., September 1, 2002; 72(3): 512 - 521. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Raes, P. De Baetselier, W. Noel, A. Beschin, F. Brombacher, and G. Hassanzadeh Gh. Differential expression of FIZZ1 and Ym1 in alternatively versus classically activated macrophages J. Leukoc. Biol., April 1, 2002; 71(4): 597 - 602. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
