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,
* Department of Biochemistry and Immunology and
School of Pharmacy, Federal University of Minas Gerais, Belo Horizonte, Brazil;
Centro de Pesquisas René Rachou, Fundacao Oswaldo Cruz, Belo Horizonte, Brazil;
Department of Parasitology, University of São Paulo, São Paulo, Brazil;
¶ Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037;
|| Discipline of Cell Biology, Federal University of São Paulo, São Paulo, Brazil; and
# Department of Molecular Biology, Princeton University, Princeton, NJ 08544
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-galactosylceramide-CD1 complex. However, these GPI anchors and
related structures were unable to activate NKT cells in vitro or in
vivo. We found that high titers of Ab anti-GPI mucins, but not
anti-GIPLs, were detected in sera from wild-type as well as in
TAP1-/-, CD1d-/-, and MHC class
II-/- mice after immunization. However, T-dependent
anti-GPI mucin Ab isotypes, such as IgG1, IgG2a, IgG2b, and IgG3,
were absent on MHC class II-/-, but were conserved in
CD1d-/- and TAP1-/- mice. Furthermore, we
found that CD1d-/- mice presented a robust cytokine as
well as anti-GPI mucins and anti-GIPL Ab responses, upon
infection with T. cruzi parasites. These results
indicate that, despite binding to CD1d, GPI mucins and related
structures expressed by T. cruzi appear not to evoke
dominant CD1d-restricted immune responses in vivo. In contrast, MHC
class II is critical for the production of the major Ig G isotypes
against GPI mucins from T. cruzi
parasites. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin association (3, 4), and MHC class II, such as endosomal trafficking (5, 6). As expected, given its specialization for lipid Ag binding,
CD1 is functionally independent of the TAP peptide transporters
(7, 8).
CD1 proteins are nonpolymorphic and comprise five isotypes. CD1a, -b,
-c, and -e constitute group 1, found in humans but not in rodents, and
CD1d constitutes group 2, which is conserved in rodents and humans.
Group 1 isotypes can present mycobacterial lipids to
CD4-CD8- (double negative
(DN)3),
CD8+, or CD4+ T cell lines
or clones expressing apparently diverse TCRs (9, 10, 11, 12). CD1d
presents -galactosylceramide (-GalCer) to DN or
CD4+ NK1.1+ NKT cells,
which express an invariant, germline-encoded TCR -chain
(V14-J18 or J281 by older nomenclature in mice, and
V24-J18 or JQ by older nomenclature in humans) paired with
restricted V-chains (mainly V8 in mice and V11 in humans)
(13, 14, 15, 16). NKT cells promptly release IL-4 and IFN-
upon
TCR engagement and without prior sensitization (17). They
exert regulatory functions in tumor rejection (18, 19) and
autoimmune diseases such as type I diabetes (20, 21). They
also regulate various infectious conditions, such as LPS-induced shock
(22) and Plasmodium yoelii and
Listeria monocytogenes infections (23, 24).
Although the importance of CD1d and NKT cells is well established, the
origin and identity of their natural ligands remain unknown.
-GalCer, a glycolipid extracted from marine sponges
(25), binds to CD1d and strongly stimulates mouse and
human NKT cells (26, 27, 28, 29). However, -GalCer may not be
the natural ligand for CD1d because ceramides with -linked dextro
sugars have not been found in mammalian cells. A candidate
natural ligand might be self GPI anchors (30). In fact, it
has been suggested that GPI anchors of P. falciparum and
Trypanosoma brucei induce Ab production in a CD1d-dependent
manner (31). However, evidence for the role of GPI is
controversial, because Ab responses to P. falciparum were
found to be MHC class II rather than CD1d restricted by two other
groups (32, 33).
The surface of T. cruzi, like that of other protozoa,
contains abundant GPI-related structures. T. cruzi expresses
107 copies/cell comprising GPI-anchored
mucin-like glycoproteins (GPI mucins) and glycoinositolphospholipids
(GIPLs) that together coat a significant extension (60–80%) of
parasite plasma membrane (34, 35). The -galactosyl
terminal residues of O-linked oligosaccharides on GPI mucins
represent the major targets for the lytic human Ab response against
T. cruzi (36, 37, 38). The GIPLs are exotic GPI
structures with no attached proteins and a conserved glycan core whose
galactofuranose residues appear to be highly immunogenic
(39). In this study, we investigated the ability of GPI
mucins and GIPLs from T. cruzi to bind CD1d and activate NKT
cells. We also tested the possibility that CD1d or NKT cells could
promote the production of Abs against glycolipid Ags in vivo. We
observed that GPI mucins, GIPLs, and their phosphatidylinositol (PI)
moieties bound to CD1d, but were unable to activate NKT cells.
Furthermore, the in vivo Ab responses against GPI mucins or GIPLs were
found to be independent of CD1d and NKT cells. Rather, MHC class II
appeared to be crucial for class-switching to Ab anti-GPI mucins of
the IgG1, IgG2, and IgG3 isotypes. Together, our results indicate that
during infection with T. cruzi, GPI mucins and GIPLs elicit
innate and adaptive immune responses in a CD1/NKT cell-independent
manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type (WT) and I-Ab-/- C57BL/6
mice were obtained from The Jackson Laboratory (Bar Harbor, ME),
TAP1-/- mice were obtained from H. Ploegh
(Harvard University, Boston, MA) and backcrossed to C57BL/6 for nine
generations (40), and
C57BL/6.CD1d-/- mice were produced in our
laboratory and checked in a routine basis with a fluorescein-labeled
anti-CD1d mAb (41). All mice were raised in a specific
pathogen-free barrier environment and kept according to institutional
animal care and use guidelines. C57BL/6 mice used for infection were
obtained and maintained from Centro de Pesquisas René Rachou,
Oswaldo Cruz Foundation (Fiocruz, Belo Horizonte, MG, Brazil). In
experiments for T. cruzi infection, 8- to 10-wk-old male and
female CD1d-/- and C57BL/6 mice were
used.
Purification of glycoconjugates
GPI mucins and GIPLs were purified as described (37, 42). In brief, parasite pellets containing 1 x
1010 trypomastigotes or epimastigotes (Y strain)
were freeze dried and sequentially delipidated with chloroform:methanol
partition, followed by butanol:water partition. The extracts were
purified by hydrophobic-interaction chromatography in octyl-Sepharose
column (Amersham Pharmacia Biotech, Uppsala, Sweden) eluted with
propanol gradient (5–60%). The GPI mucins were detected by Western
blotting, chemiluminescent ELISA (CL-ELISA) using
anti--galactosyl Abs (37, 42), and silver staining
of SDS-PAGE gels (43). GIPLs were identified by
electrospray ionization-mass spectrometry (ES-MS) analysis.
Isolation of PI moiety
T. cruzi GPI mucins and GIPLs were deaminated, as previously described (42, 44). In brief, GPI mucins were extracted with water-saturated butanol (91% 1-butan-1-ol), dried, and redissolved in 0.1 M sodium acetate buffer, pH 4.0, and deaminated by three additions of 0.5 M sodium nitrite at 60°C. The samples were mixed with 9% butanol, and the released PIs were recovered by butanolic extractions. Epimastigote GIPLs were submitted to the same protocol without pre-extraction with butanol.
Mass spectrometry analysis
GPI anchors, released from GPI mucins by proteinase K treatment (42), and GIPLs were analyzed by ES-MS. Spectra were obtained in a Finnigan LCQDuo ion-trap mass spectrometer (Finnigan; ThermoQuest, San Jose, CA). Samples were dissolved in 50% propan-1-ol, containing 10 mM ammonium acetate and 0.1% formic acid, and introduced into the electrospray source by injection through a 30-µm (internal diameter) fused silica capillary at a flow rate of 5 µl/min. Electrospray capillary voltage was set to 36–46 V, and temperature to 200°C. Spectra were acquired in negative ion mode at 3 s/scan over a mass range of m/z 200-2000. Collision-induced dissociation ES-MS of parent ions was conducted at a relative collisional energy of 25–35% (2.5–3.5 V). Source parameters were optimized using previously well-characterized T. cruzi GIPLs (42).
Competition for lipid binding to rCD1d
Binding to CD1d was determined using a competition assay, as
described (45), with some modifications. Purified mouse
rCD1d1 molecules were coated at 5 µg/ml in PBS overnight at 4°C on
96-well tissue culture plates. After washing three times with PBS,
CD1d-coated wells were preincubated with various concentrations of
competitors in PBS at room temperature for 18 h before addition of
-GalCer in PBS (1 µM; a gift from Y. Koezuka, Pharmaceutical
Research Laboratory, Kirin Brewery, Takasaki, Japan) for 3 h at
room temperature. Both competitors and -GalCer were sonicated before
use. After washing the plates, 5 x 104
DN32D3 cells (V14-J18/V8 NKT hybridoma) (15) per
well were added in 1:1 EHAA/RPMI mixture (Biofluids, Rockville, MD)
supplemented with 50 µm 2-ME, penicillin-streptomycin-gentamicin,
glutamine (endoplasmic reticulum (ER) medium), and 5% heat-inactivated
FCS (ER-5), and incubated for 18 h at 37°C. Supernatants were
harvested, and IL-2 was released and measured by CTLL assay, as
previously described (46).
Macrophage stimulation
Murine peritoneal thioglycolate-elicited macrophages were
harvested and cultured, as described elsewhere (43, 47).
WT or CD1d-/- C57BL/6 peritoneal exudate cells
were harvested in cold FCS-free DMEM (Life Technologies, Grand Island,
NY), centrifuged, and resuspended in DMEM supplemented with 5%
heat-inactivated FCS and 40 mg/ml gentamicin at a final concentration
of 2 x 106 cells/ml. Cells were incubated
for adherence to 96-well plates for 3 h, and unattached cells were
washed away. Macrophages were primed overnight with IFN- (25 U/ml;
PharMingen) and were subsequently incubated with trypomastigote GPI
mucins. Supernatants were collected after 24 and 48 h for TNF-
and NO measurements, respectively (43, 47). TNF- was
quantified using ELISA kit (Duoset ELISA Development System mouse
TNF-, catalogue DY410; R&D Systems, Minneapolis, MN), and NO was
measured using Griess reagents (48).
Immunization with GPI mucins and GIPLs
Ags (0.8 nmol GPI mucins or 0.8 nmol GIPLs) were diluted in saline and mixed 1:1 with Alum 2% (Alhydrogel 2%; Accurate Chemical and Scientific, Westbury, NY) by vortexing for 3 min and incubated for 2 h at room temperature before injection. One hundred microliters of the mixture was injected into the footpad and tail-base sides of C57BL/6 WT and mutant mice. After 2 wk, mice were boosted with the same dose of Ag mixed with Alum. At the end of the third week, mice were killed, and sera, as well as inguinal and popliteal lymph nodes, were harvested for Ab detection and T cell purification assays.
T cell enrichment and proliferation
Murine lymph nodes were harvested in HBSS (Life Technologies, Grand Island, NY) containing 5% FCS, and the cell suspensions were passed through nylon wool columns for T cell enrichment, as described (49). A total of 3 x 105 T cells and 1 x 105 2000 rad-irradiated splenocytes was cultured at 200 µl final volume in ER medium supplemented with 10% FCS (ER-10) at 37°C for 3 days before pulsing with 0.50 µCi/well [3H]thymidine for 8–18 h.
Infection with T. cruzi
Mice were infected i.p. with 5000 blood-form trypomastigotes Y strain of T. cruzi. Parasitemia levels were evaluated by counting parasites in 5 µl blood from the tail vein. Mortality was evaluated by daily inspection of the cages.
Chemiluminescent ELISA
Serum Abs to GPI mucins or GIPLs were assayed by CL-ELISA. In brief, microtiter black or white (Maxisorb Nunc 96 FluoroNunc Plate; catalog 237018 and 436110, respectively; Nunc, Albertslund, Denmark) 96-well plates were coated with 6 pmol GPI mucins or GIPLs per well diluted in 50 µl PBS for 18 h at 4°C. The wells were washed three times with PBS-0.05% Tween 20 (PBS-T) and blocked with PBS-1% BSA for 2 h at 37°C. After washing with PBS-T, serially diluted serum samples in PBS-1% BSA were added to the wells and incubated for 1 h at 37°C. The plates were then washed three times with PBS-T, and biotinylated goat anti-mouse IgG + IgM (H + L) (1:50,000), IgM (1:20,000) (Jackson ImmunoResearch Laboratories, West Grove, PA), IgG1 (1:10,000), IgG2a (1:10,000), IgG2b (1:2,000), or IgG3 (1:2,500) (0.5 mg/ml; Southern Biotechnology Associates, Birmingham, AL) was added for 1 h at 37°C, followed by streptavidin-peroxidase (1:5,000; Southern Biotechnology Associates). The wells were washed four times with PBS-T and once with carbonate buffer, pH 9.6. ECL reagent (Amersham Pharmacia Biotech, Piscataway, NJ) 100 µl/well was added, and the plates were immediately read using a Luminometer (program BB Lux, Wallac 1450 Microbeta Plus Liquid Scintillation Counter; PerkinElmer, Wellesley, MA).
Cytokines and Ab produced in infected mice
Murine spleen cells from infected and noninfected C57BL/6 or CD1d-/- mice were obtained on day 8 after infection, as previously described (50), and cultured at 5 x 106 cells/ml/well, in 24-well plates, with RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 0.05 mM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. Supernatants were harvested after 48 or 72 h at 37°C, and the levels of cytokines were measured by ELISA kits (R&D Systems). Mice were bled on days 0, 8, and 14 after infection, and the level of serum cytokines and Abs was measured by conventional and CL-ELISA, respectively, as described above.
Statistical analysis
Data are presented as means ± SEM. Statistical differences were determined by one-way ANOVA, followed by t test to evaluate differences between the experimental vs the control groups. The p values were determined using Student’s t test and considered significant if <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mucin-derived GPIs and free GIPLs purified from epimastigote forms
of T. cruzi yielded highly pure samples. The results
presented in Fig. 1
show the negative ion
mass spectra of GPI anchors purified from epimastigote-derived GPI
mucins (upper panel) and GIPLs (lower
panel). The former presented a major doubly charged
([M-2H]2-) pseudomolecular ion species at
m/z 917.1 (M = 1836.2), which could be assigned to a
GPI species containing four hexoses, glucosamine (GlcN),
ethanolaminephosphate, 2-aminoethylphosphonate (AEP), and
phosphatidylinositol
(myo-inositol-phosphate-C16:0-alkyl-C16:0-O-acylglycerol).
In contrast, major doubly charged ([M-2H]2-)
pseudomolecular ion species were observed for the GIPL preparation at
m/z 1066.1 (M = 2134.2), which could be assigned to a
compound containing six hexoses, GlcN, AEP, and
myo-inositol-phosphoceramide (C24:0-fatty
acid-C18:0-sphinganine). These two preparations were used in the
different experiments presented in this study. Both mucin-derived GPIs
and GIPLs, upon nitrous deamination, released phosphatidylinositol
alkylacylglycerol (GPI-derived PI) and inositol-phosphoceramide
(GIPL-derived PI), generating major ion species at m/z 795.6
and 892.8, respectively. These parent ion species were further
fragmented to confirm the proposed assignments given above (data not
shown).
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Competition assay for lipid binding to CD1d
Binding of GPI mucins, GIPLs, and PIs was tested in microwell
plates coated with soluble rCD1d. Binding was detected as a decreased
IL-2 release by DN32D3 hybridoma cells upon addition of -GalCer,
their cognate CD1d-binding ligand. The results were expressed as
percentage of inhibition of the IL-2 induced by -GalCer alone in the
absence of competitor. The GPI mucins and GIPL structures tested caused
nearly 100% inhibition of DN32D3 activation by -GalCer even at 3:1
and 1:1 competitor/-GalCer molar ratio (Fig. 2). The PI moieties derived from GPI
mucins or GIPLs also competed with -GalCer, although not as well,
perhaps because their solubility was inferior to that of the
glycoconjugates. We further titrated the inhibitor down in some
experiments and found reduction in the range of 30–50% of inhibition
for both GPI mucin and GIPL at 1:0.5 ratio. The PI portions of both
molecules did not inhibit at this ratio. Addition of GPI/GIPL after the
-GalCer and CD1d complex being formed did not result in
inhibition of NKT cell activation. Collectively, these results suggest
that T. cruzi GPI/GIPL structures compete with -GalCer
for binding to CD1d.
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Trypomastigote-derived GPI mucins are able to activate macrophages
(42, 43, 47). We have recently shown the involvement of
Toll-like receptor 2 on macrophage activation by GPI mucins and GIPLs
from T. cruzi parasites (51, 52). However, the
complete set of receptors required for macrophage activation by GPI
mucins still remains to be defined. Because GPI mucins bind to CD1d, we
investigated whether the previously described activity of these
molecules on macrophages was CD1d dependent. WT and
CD1d-/- macrophages primed with IFN- were
stimulated with trypomastigote-derived GPI mucins and TNF- and NO
production evaluated in the culture supernatants after 24 and 48
h, respectively (Fig. 3). Although
IFN- priming is not necessary for TNF- and NO release, it was
used in this study because it causes a significant increase in the
production of these mediators, 2- and 4- to 6-fold increase,
respectively (43, 47). Identical results were obtained for
both CD1d-deficient and CD1d-sufficient macrophages, demonstrating that
CD1d expression is not required for GPI mucin activity on macrophages.
Similar results were observed with LPS (data not shown).
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Next, we asked whether T. cruzi-derived GPI structures
could stimulate T cell proliferative responses in vivo. WT mice were
immunized with GPI mucins or GIPLs s.c., and cell suspensions of lymph
node were separated by nylon column. T cell-enriched preparations were
cultured in vitro with irradiated spleen cells from unimmunized mice
and stimulated with GPI structures. T cells from GPI mucin-immunized
mice responded well to Ag restimulation in vitro, whereas those from
GIPLs did not (Fig. 4, upper
panel). To characterize the Ag presentation pathway involved in
the response to GPI mucins, we immunized WT,
CD1d-/-, or MHC class
II-/- mice. There was a marked decrease in T
cell proliferation in MHC II-deficient mice, whereas
CD1d-/- mice responded normally (Fig. 4, lower left panel).
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14-J18 V8 TCR (16), to
respond to GPI structures presented by CD1d. CD1d complexed with GPI
mucins or GIPLs did not induce IL-2 release by DN32D3 cells; neither
did the PI moieties of these glycolipids (data not shown). In contrast,
NKT cells exposed to -GalCer-loaded CD1d produced high levels
of IL-2. Ab response to GPI mucins
To characterize the MHC or CD1 Ag presentation pathway involved in
helper activity for Ab response in vivo, WT,
CD1d-/-, MHC class
II-/-, or TAP-/- mice
were immunized with GPI mucins or GIPLs, and their serum Ab (IgM + IgG)
titers were measured by CL-ELISA. WT and mutant mice produced similar
levels of Abs, although MHC class II-/- mice
exhibited a reduction in total Ab production against GPI mucins (Fig. 5A, upper panel).
In contrast, GIPL immunization did not elicit Ab production even in WT
mice (Fig. 5A, lower panel). Fig. 5B
shows that IgM was produced by all WT and mutant mice. IgG1, IgG2a, and
IgG3 were normal in WT and CD1d-/- mice, but
severely decreased in MHC II-/- mice.
Altogether, our results show that MHC class II expression, rather than
CD1d, is crucial for Ig class-switching in the Ab response to GPI
mucins.
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Finally, we infected WT as well as CD1d-/-
mice with the Y strain of T. cruzi and we determined
parasitemia and mortality rates as well as the levels of several
cytokines and Ab isotypes. Our results show that both parasitemia and
mortality are similar (Fig. 6), when
comparing the two different mouse lineages. The results presented in
Table I show no difference in the levels
of IFN-, IL-4, IL-10, IL-12, and TNF- in the sera or supernatants
from splenocytes derived from WT and CD1d-/-
mice infected with T. cruzi. Similarly, Ab responses to GPI
mucins and GIPL were mostly preserved in the
CD1d-/- mice (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-GalCer to P. yoelii- or
Plasmodium berghei-infected mice results in strong
antimalaria activity mediated by IFN--secreting NKT cells
(55). GPI anchors were shown to be natural ligands of CD1d (30), and are abundantly expressed in the surface of protozoan parasites (34, 35). Thus, it is tempting to speculate that GPI anchors and related structures may be the main targets for early NKT cell responses during acute infection with protozoan parasites. In fact, CD1d-restricted IL-4-secreting CD4+ NKT cells specific for GPI anchors have been implicated in mediating Ab production against GPI-anchored proteins of P. falciparum or T. brucei (31). In this context, T. cruzi parasites arise as an interesting model, as they express large amounts of GPI-anchored proteins (mainly GPI mucins) and GIPLs on their surface. It is noteworthy that GPI mucins are heavily glycosylated (37, 38) and highly polymorphic (56), whereas GIPLs on their own are of glycolipid nature and have no protein covalently attached to them. Therefore, GPI mucins and GIPLs may not be suitable for presentation via the conventional pathways involving MHC class I and class II. Nevertheless, in humans and experimental models, GPI mucins and GIPLs have been shown to be major targets for IgG Ab responses elicited during T. cruzi infection (35, 36, 37, 38, 39, 57).
In this study, we investigated whether T. cruzi-derived GPI
anchors and GIPLs can elicit an Ab production in an NKT cell-dependent
manner. Initially, we examined the interaction with CD1d and found that
GPI mucins, GIPLs, and derived PI moieties indeed bind to CD1d.
However, we were unable to detect any NKT cell activation by the
T. cruzi-derived glycolipids. Although those GPI anchors
have the basic structure required for interaction with CD1, such as
lipid tail and polar head group, they may not be appropriated for
recognition by NKT cells bearing the TCR V14-J281 chain.
According to the current view, acyl chains of the lipid tails are
involved in binding to CD1 molecules, whereas Ag specificity is
determined by TCR recognition of the polar head group
(58). Indeed, -GalCer analogs with shortened acyl
chains or sphingosine lipid chains (27), but not
-GalCer or -mannosyl-ceramide (26), have their NKT
cell-stimulatory activity preserved. Monosialoganglioside (GM1) and
lipoarabinomannan, likewise, bind to CD1d, but do not stimulate DN32D3
hybridomas (58). The TCR antigenic specificity seems to be
conferred by residues of the CDR3 loop encoded by the D segment, J,
or by N-region addition. In agreement with this, 24.8 hybridoma, which
expresses V14-J281 associated with V8.2/J2.5, does not
respond to -GalCer, but is stimulated by phospholipids with several
polar head groups (59). DN32D3 instead do not recognize
those phospholipids. Different from 24.8 hybridoma, DN32D3 expresses
V14-J281 associated with V8.2/J2.4. Thus, despite their
limited diversity, we cannot rule out that another CD1d-restricted NKT
cell population may be recognizing T. cruzi GPI structures
in vivo. However, our data indicate that the major NKT cell subset,
which bears V14-J18, does not recognize T.
cruzi-derived GPI anchors and related structures (i.e., GIPLs and
PI) tested in this study.
Next, we investigated the NKT cell involvement in T cell-mediated response to highly purified T. cruzi GPI mucins and GIPLs in vivo. Different from previous studies, in this study we used a molecularly defined protocol, immunizing animals with highly purified molecules, instead of whole parasites, which reduces the risk of dubious results due to the cross-reactivity with other parasite molecules. We found that both proliferative and Ab responses were similar in WT and CD1d-/- mice immunized with GPI mucins. In contrast, both responses were largely reduced in MHC class II-/- mice. These results indicate that NKT cells are not required for in vivo response to GPI mucins, contrasting with data described elsewhere, suggesting that CD1d-NKT cell interactions are required for providing cognate help for IgG production anti-circumsporozoite protein of Plasmodium via GPI anchors (31). Our results rather indicate that IgG production to T. cruzi GPI mucins is mediated through classical MHC class II-CD4+ T cell interaction. Together, these findings suggest that the T cell epitope involved in providing help to B cells to respond to GPI mucins is a peptide instead of glycolipid. Similar results were reported by Molano et al. (32), who showed that MHC class II, rather than CD1d, is crucial for anti-circumsporozoite IgG responses during immunization with irradiated Plasmodium parasites.
Different studies have demonstrated the ability of T.
cruzi-derived GIPLs to act as polyclonal activators as well as
adjuvants for B cell activation and Ab production (60, 61). Although anti-GIPL IgM and IgG Abs can be found in
serum of patients with Chagas’ disease (39, 55), the
ability of purified GIPLs to elicit a specific Ab response is less
documented. Unlike GPI mucins, immunization with GIPLs was not
successful in mice. We did not detect any GIPL-specific Abs after
immunization. It is possible that IgM anti-GIPL Abs found during
infection with T. cruzi parasites both in humans and mice
are a result of a T cell-independent production. Furthermore, the
cross-reaction with T. cruzi glycoproteins could be used to
explain the IgG production specific for GIPLs observed during T.
cruzi infection. In fact, the B cell epitope -galactofuranose,
the main Ab target in the GIPLs, is also shared by other parasite
glycoproteins (39).
We also tested whether the macrophage activation by GPI mucins requires
CD1d. In fact, recent data from our laboratory show that the activity
of GPI mucins on macrophage involves Toll-like receptor 2 (51, 52). However, the complete requirement of receptors for
macrophage activation has not been defined. Regardless, our results
demonstrate that CD1d is not required for the macrophage activation by
T. cruzi-derived GPI mucins, as indicated by the similar
levels of TNF- and nitrite in the supernatants of macrophages from
WT and CD1d -/- stimulated with GPI mucins.
Finally, a recent study has demonstrated a consistent enhancement of
parasitemia in CD1d-/- and
J18-/- mice infected with the CL strain of
T. cruzi. However, no differences in terms of mortality were
observed between the WT and knockout mice (62). When we
used the Y strain of T. cruzi to infect WT and
CD1d-/-, we did not observe any difference in
terms of mortality either. No differences between WT and
CD1d-/- were observed either in the levels of
IgM, IgG1, or IgG2a responses against GPI mucins/GIPLs or in those
of the cytokines produced by macrophages (i.e., TNF-, IL-10, and
IL-12) or NKT cells (i.e., IFN- and IL-4) in response to infection
with T. cruzi. Thus, even if NKT cells of diverse TCR may be
activated in vivo by T. cruzi, they seem to have a minor
importance for the progress of the infection, as, in our case, neither
parasitemia and mortality, nor cytokine and Ab production are altered
in CD1d deficient.
In conclusion, we demonstrated in this study that T.
cruzi-derived GPI-related structures compete for -GalCer
binding to CD1d, but this interaction is not important either for Ab
production or for cellular responses to GPI mucins or GIPLs elicited
during infection with T. cruzi. Rather, IgG production to
GPI mucins seems to be mediated through classical MHC class
II-CD4+ T cell interaction. Furthermore, our data
indicate the CD1d and NKT cell pathway has no major role in host
resistance to T. cruzi infection. However, the relevance of
CD1 for response to GIPLs or GPI anchors cannot be totally discarded.
It is possible that other CD1 isoforms found in humans, but absent in
mice, may present GPI anchors and related structures to T cells.
Additional experiments to answer these questions are an important
matter of future investigation.
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Acknowledgments |
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-GalCer. We thank members of Bendelac Lab and Jabri Lab
for helpful discussion, and Oscar Bruna-Romero for critically reading
the manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Ricardo T. Gazzinelli, Department of Biochemistry and Immunology, Federal University of Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG, 31270-910, Brazil. E-mail address: ritoga{at}dedalus.lcc.ufmg.br
3 Abbreviations used in this paper: DN, double negative; AEP, 2-aminoethylphosphonate; CL-ELISA, chemiluminescent ELISA; ER, endoplasmic reticulum; ES-MS, electrospray ionization-mass spectrometry; GalCer, galactosylceramide; GIPL, glycoinositolphospholipid; GlcN, glucosamine; PI, phosphatidylinositol; RLU, relative luminescence unit; WT, wild type.
Received for publication April 1, 2002. Accepted for publication August 7, 2002.
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) and GPI-derived PI (
).
Lower panel, GIPLs (
) and GIPL-derived PI (•).
Asterisk indicates no significant inhibitory activity as compared with
NKT hybridoma cells stimulated with 
)- or GIPL (
)-immunized or unimmunized (
), and MHC class
II-/-
(
) were
incubated with medium or GPI mucin. Cell proliferation was evaluated by
[3H]thymidine incorporation. Different letters
mean statistically different results (p <
0.05). Lower right, NKT hybridomas were added to a
CD1d-coated plate preincubated with PBS, 
), MHC II-/- (
) or
CD1d-/-
(
