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Infection and Immunity, November 2001, p. 6588-6596, Vol. 69, No. 11
Department of Biochemistry, Faculty of
Pharmacy and Institute of Molecular and Cellular Biology, University of
Porto, Porto, Portugal,1 and IRD UR 008 "Pathogénie des Trypanosomatidés", Centre IRD de
Montpellier, 34032 Montpellier, France2
Received 5 March 2001/Returned for modification 2 May 2001/Accepted 9 August 2001
We have recently characterized a novel Leishmania major
gene encoding a polypeptide of 30 kDa that was homologous to mammalian ribosomal protein S3a and was named LmS3a-related protein (LmS3arp). The protein was found to be expressed by all the Leishmania
species so far examined (L. infantum, L. amazonensis, and
L. mexicana). In the present study we have extended our
approach to the analysis of LmS3arp activity on T- and B-cell functions
in a murine model. The results presented in this report show that
LmS3arp plays a dual role in the regulation of T- and B-cell
reactivity. Indeed, we found that injection of the LmS3arp recombinant
protein (rLmS3arp) into BALB/c mice induces preferential activation of
B cells, as shown by the following criteria: (i) increased expression
of CD69 molecules on immunoglobulin M (IgM)-secreting spleen
cells, (ii) a considerable increase of IgM-secreting B cells, and (iii)
elevated levels of IgM antibodies in the sera of injected animals.
Moreover, the IgM antibodies are not specific to the
Leishmania antigens but preferentially recognize
heterologous antigens like myosin, thyroglobulin, DNA, and keyhole
limpet hemocyanin. Furthermore, the strong polyclonal expansion of
nonspecific, non-parasite-directed B-cell clones induced by rLmS3arp is
concomitant with a marked inhibition of T-cell proliferation. Analysis
of cytokine production revealed a significant downregulation of gamma
interferon, interleukin-2 (IL-2), and IL-12 secretion. Taken together,
our data suggest that rLmS3arp, through direct or indirect action
toward B and T cells and cytokine secretion, could participate in the
immunoregulatory processes that play a role in the balance of the Th1
and Th2 immune response.
Protozoan parasites of the genus
Leishmania result in a spectrum of human diseases that range
from self-healing cutaneous ulcers to potentially fatal visceral
infection, depending primarily on the species of parasites involved
(7, 9). The disease is prevalent in many tropical,
subtropical, and Mediterranean regions of the world and is transmitted
by the bite of the infected phlebotomine sandflies (Diptera:
Psychodidae). In Europe, visceral leishmaniasis is caused by
Leishmania infantum and is prevalent in various
Mediterranean countries. Domestic dogs constitute an important
reservoir of the infection (1, 11, 14, 19). Similar
disease symptoms develop in both humans and canines, including fever,
hypergammaglobulinemia, hepatosplenomegaly, and anemia (6,
45).
Leishmaniasis is characterized by a variety of immunopathological
disturbances (23). Both polyclonal B-cell activation and antigen-specific impairment of T-cell responses occur in certain circumstances. Indeed, it has been reported that spleen cells from
L. donovani-infected hamsters became unresponsive to
stimulation with concanavalir A (ConA). Furthermore, in susceptible
mice, systemic intracellular infection with L. donovani
resulted in the formation of adherent spleen cells which can suppress
both mitogen- and specific antigen-stimulated T-cell responses; this phenomenon is due in part to the inhibition of activating lymphokine gamma interferon (IFN- Paradoxically, there is a marked humoral response during active
disease, with elevated nonspecific immunoglobulin levels, mostly of the
immunoglobulin G (IgG) and IgM classes. Indeed, hypergammaglobulinemia,
rheumatoid factors, and circulating immune complexes suggesting
polyclonal activation of B cells occur during visceral leishmaniasis
(30). The parasite molecules which could be involved in
the development of these immunological alterations have not being fully characterized.
An increasing number of Leishmania antigens have been
identified. Some of them were considered Leishmania-specific
proteins playing a role in parasite development, (i.e., surface
protease gp63 [33], the surface glycoprotein gp46
[26], and the lipophosphoglycan-associated protein KMP11
[38]). Moreover, parasite genes with sequence homology
to eukaryotic genes of known function also appear to be involved in the
regulation of parasite growth and development (e.g., kinesin
[13] and heat shock proteins [2, 3, 34]). Other studies have reported that some Leishmania ribosomal
proteins function as immunoregulatory molecules (31, 34).
In fact, the eukaryotic ribosome is composed of four RNA molecules and more then 70 ribosomal proteins (39). There is increasing
evidence that ribosomal proteins are capable of extrachromosomal
functions (40, 41). Moreover, the acidic ribosomal
proteins (also called P-proteins) have been described as prominent
antigens during Leishmania infections (31).
Furthermore, a leishmanial protein homologous to the eukaryotic
ribosomal elongation initiation factor 4A induces a strong Th1 response
in peripheral blood mononuclear cells from leishmaniasis patients
(34).
In a previous study we have identified a novel L. major gene product with high sequence identity to the
eukaryotic ribosomal S3a protein (LmS3arp), a component of the small
ribosomal 40S subunit also involved in a number of cellular processes
including cell proliferation, differentiation, and apoptosis
(29). Moreover, using molecular and immunological
approaches, we demonstrated that LmS3arp is expressed by a number of
other Leishmania species including L. infantum, L. mexicana, and L. amazonensis.
However, other parasite components belonging to the large ribosomal
protein family such as S3a have not yet being examined for a possible
role in the host-parasite relationship. The purpose of our study was to
characterize the effects of a recombinant LmS3arp (rLmS3arp) on T- and
B-cell activation as well as on cytokine profiles. The data obtained
showed a dual role of rLmS3arp on T- and B-cell activation, being
suppressive and mitogenic toward T and B cells, respectively.
Mouse strains.
Six-week-old BALB/c male mice were obtained
from the Gulbenkian Institute of Science (Oeiras, Portugal). BALB/c
athymic (nude) mice were obtained from Harlan Iberica.
Subcloning and purification of LmS3arp.
The L. major promastigote cDNA insert encoding a 32,000-molecular-weight
protein (LmS3arp) (44) was subcloned into the
high-expression vector pQE31, resulting in the production of a
significant amount of LmS3arp containing six histidine residues at its
N terminal. Expression and purification of the
His6-rLm3Sarp protein were carried out essentially as
described by the manufacturer (QIA Expressionist System
Manual; Quiagen, Inc., Chatsworth, Calif.). Recombinant protein
production in Escherichia coli was induced by the addition
of 2 mM isopropyl- Treatment of mice.
Twelve BALB/c mice were inoculated three
times intraperitoneally (i.p.) at 10-day intervals with 50 µg of
rLmS3arp. Two weeks after the final inoculation, spleens and sera were collected.
Cell culture and proliferation assays.
After cervical
dislocation, the spleens were removed and homogenized in a petri dish.
After two or three washes in RPMI 1640 culture medium (Sigma Chemical
Co.), the cells were adjusted to 107/ml in RPMI 1640 culture medium supplemented with 2 mM glutamine (Sigma), penicillin and
streptomycin (100 U/ml and 100 µg/ml, respectively [Sigma]), 0.05 mM 2-mercaptoethanol, 20 mM HEPES (Gibco BRL), and 10% fetal calf
serum (FCS) (Gibco-BRL). Spleen cells were cultured in 96-well
flat-bottom plates in a 200-µl volume at 2 × 105
cells/well. The cells were stimulated with 5 µg ConA per ml in the
presence of 50 µg of rLm3Sarp per ml or 100 µg of total
Leishmania extract per ml. After 48 h of incubation at
37°C in 5% CO2, 1 µCi of [3H]thymidine
(Amersham, Arlington Heights, Ill.) was added to the wells. Pulsed
splenocytes were harvested on a glass filter using an automated
multiple-sample harvester and dried. Incorporation of radioactive
thymidine was then determined by liquid scintillation as specified in a
standard protocol. Assays were carried out in triplicate, and the
stimulatory index (SI) was calculated by dividing the arithmetic mean
of counts per minute (cpm) obtained from stimulated cultures by the
arithmetic mean of cpm obtained from control cultures without stimulation.
Immunofluorescence and flow cytometry analysis.
Spleen and
lymph nodes were gently dissected to obtain single-cell suspensions.
The cells were washed by centrifugation and ressuspended in PBS
supplemented with 10% FCS. A total of 106 splenocytes or
lymph node cells were washed three times with fluorescence-activated
cell sorter (FACS) buffer (PBS containing 0.1% FCS and 0.11% sodium
azide) and then distributed into a 96-well microtiter culture plate.
The cells were incubated with saturating concentrations of
R-phycoerythrin-conjugated hamster anti-mouse CD69 plus
fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD8 (Ly-2)
monoclonal antibody, FITC-conjugated rat anti-CD4 from Pharmingen, or
FITC-conjugated goat anti-mouse IgM from Southern Biotechnology. After
a 30-min incubation on ice, the cells were washed by three
centrifugations in FACS buffer and the flow cytofluorometric analysis
was done in a FACS apparatus (Becton Dickinson).
Enzyme-linked immunosorbent assays (ELISAs) for
immunoglobulins.
Ninety-six-well flat-bottom microtiter plates
(Immune Plate Maxisorp; Nunc) were coated overnight at 4°C with one
of the following reagents (in carbonate buffer [pH 8.5]): unlabeled
goat anti-mouse immunoglobulin (5 µg of rLmS3arp per ml),
total Leishmania antigens (10 µg/ml), fibronectin
(10 µg/ml [Sigma]), bovine serum albumin (10 µg/ml
[Calbiochem]), keyhole limpet hemocyanin (KLH) (10 µg/ml), ovalbumin (10 µg/ml [Sigma]), horseradish peroxidase (10 µg/ml [Sigma]), native double-stranded (DNA (10 µg/ml) [Sigma]),
porcine type II thyroglobulin (10 µg/ml t[Sigma]), whale skeletal
muscle type II myoglobulin (10 µg/ml [Sigma]). The plates were
washed with PBS containing 0.1% Tween 20 (PBS-T [Calbiochem]) and
blocked with PBS plus 1% gelatin (200 µl/well) for 1 h at room
temperature (25°C). The plates were incubated with serial dilutions
of each serum sample for 2 h at room temperature or a with mouse
myeloma IgM kappa chain (clone TEPC183 [Sigma]). The myeloma
IgM was used to control for the background for each antigen; the dose
used (1.2 µg/ml) was identical to the arithmetic mean of IgM
concentrations found in the diluted sera (1/1,000) from
rLmS3arp-treated mice. After being washed with PBS-T, the plates were
incubated for 1 h at room temperature with peroxidase-labeled goat
anti-mouse immunoglobulin isotypes (anti-IgM, anti-IgG, anti-IgG3,
anti-IgG2a, and anti-IgG2b) and developed with
o-phenylenediamine (OPD [Sigma]) in citrate buffer. The
concentration of nonspecific antibody was determined by comparison to a
standard curve generated with unlabeled purified isotypes. The specific
antibody was determined by the last dilution showing an optical density
at 492 nm higher than that of the negative control.
Cytokine ELISAs.
The cytokine production was determined by
two-site sandwich enzyme-linked immunosorbent assays in supernatants of
spleen and lymph node cells stimulated with 5 µg ConA per ml after a
48-h incubation at 37°C under 5% CO2. Ninety-six-well
flat-bottom microtiter plates were coated overnight at 4°C with
unlabeled rat antibodies to the cytokines IFN- Immunoglobulin ELISPOT assays.
Ninety-six-well flat-bottom
plates were coated with unlabeled goat anti-mouse immunoglobulins (5 µg/ml [Southern Biotechnologies]) in PBS (pH 8.5) buffer overnight
at 4°C as described previously (18). The plates were
blocked with PBS-1% gelatin for 1 h at room temperature and
washed with PBS-T. A single suspension of lymphocytes prepared in
sterile RPMI 1640 medium supplemented with 10% FCS was serially
diluted in immunoglobulin-coated plates at a starting concentration of
5 × 105 cells/well. The plates were incubated at
37°C for 6 h in a 5% CO2-in-air incubator. The
cells were lysed with 0.05% Tween 20, and the plates were washed three
times with PBS. Isotype-secreting cells were detected with
biotin-labeled goat anti-mouse isotypes (anti-IgM, anti-IgG1,
anti-IgG2a, anti-IgG2b and anti-IgG3 [Southern Biotechnologies]).
This complex was bound to avidin-alkaline phosphatase (Southern
Biotechnologies) for 2 h at 37°C. Enzyme-linked Immunospots (ELISPOTs) were developed after addition of BCIP
(5-bromo-4-chloro-3-indolylphosphate) substrate (Sigma) in
2-amino-2-methyl-1-propanol buffer (Sigma). The plates were incubated
for 2 h at 37°C, and the blue spots were counted
microscopically. The correlation between the number of spots developed
per well and the number of input cells per well was determined. Data
are presented as the number of spots per total number of spleen cells.
Statistical analysis.
The data were analyzed using
Student's t test.
Effects of rLmS3arp on T-lymphocyte activation.
In initial
experiments, we studied the effects of rLmS3arp on the activation of T
lymphocytes. Spleen cells from BALB/c mice were cultured with or
without affinity-purified rLmS3arp (Fig. 1) and
stimulated with ConA, a T-cell-specific mitogen. Unstimulated cells had
a low rate of replication and incorporated little
[3H]thymidine into DNA (161 ± 60 cpm). As shown in
Fig. 2A, addition of rLS3arp to spleen cells
dramatically inhibited ConA-induced T-cell proliferation (99%
inhibition of T-cell proliferation was observed in the presence of
rLmS3arp compared to the control).
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.11.6588-6596.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Dual Role of the Leishmania major
Ribosomal Protein S3a Homologue in Regulation of T- and B-Cell
Activation
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
) production by macrophages (28).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-D-thiogalactoside, and the cells were
cultured for an additional 3 to 5 h. The cells were harvested by
centrifugation for 3 min at 600 × g and resuspended in
8 M urea-0.1 M sodium phosphate-0.01 M Tris-HCl (pH 8.0)-2% sodium
dodecyl sulfate (SDS). The cellular debris were pelleted, and the
supernatant containing the recombinant protein was incubated with a
50% (vol/vol) Ni-nitrilotriacetic acid resin in buffer for 30 min at room temperature. The resin was washed three times with a buffer
containing 8 M urea, 0.1 M sodium phosphate, and 0.01 M Tris-HCl (pH
6.3), and the recombinant protein was eluted with the same buffer used
to wash the resin plus 100 mM EDTA. The recombinant protein was
analyzed before and after elution on 10% polyacrylamide gels
containing 0.2% SDS and visualized by staining with Coomassie blue.
For biological assays, the protein was dialyzed against PBS (10 mM
sodium phosphate [pH 7.2], 0.15M NaCl) in decreasing concentrations
of urea. The final dialysis was performed against PBS. The protein
concentration was determined using the Folin procedure
(27). As a parasite control protein carrying a
His6 tag made with the pQE31 plasmid and purified by the
same procedure, an L. major polypeptide with significant
homology to silent information regulator 2 protein (SIR2) of
Saccharomyces cerevisiae (rLmSIR2rp) was used in cellular
studies (42, 43).
(R4-6A2 cell line),
interleukin-2 (IL-2) (JES6-1A12 cell line), IL-4 (BVD4-1D11 cell line),
IL-10 (JES5-2A5 cell line), and IL-12 (9A5 cell line) in carbonate
buffer (pH 8.5). The plates were washed with PBS-T and blocked with
PBS-1% gelatin (200 µl/well) for 1 h at room temperature. They
were incubated with serial dilutions of each supernatant for 2 h
at room temperature. After being washed with PBS-T, the plates were
incubated for 1 h at room temperature with biotinylated rat
antibodies to the following cytokines: IFN- (XMG1.2 cell line), IL-2
(JES6-5H4 cell line), IL-4 (BVD6-24G2 cell line), IL-10 (SXC-1 cell
line), and IL-12 (C17.8 cell line). Antibody pairs specific for ILs
were provided by PharMingen, San Diego, Calif. After being washed with PBS-T, the plates were incubated for 1 h at room temperature with streptavidin-peroxidase (Sigma) and developed with OPD in citrate buffer. The optical densities were recorded at 492 nm. The
concentration of specific ILs were determined by comparison to a
standard curve generated with different recombinant interleukins:
rIFN-, rIL-2, rIL-4, rIL10, and rIL12 (R&D Systems).
RESULTS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (79K):
[in a new window]
FIG. 1.
SDS-polyacrylamide gel electrophoresis of different
preparations of rLmS3arp (A) and rLmSIR2rp (B) proteins fused
with six histidine residues purified on Ni-nitriloacetic acid resin.
Lanes 1 and 2 correspond to two different preparations.
View larger version (25K):
[in a new window]
FIG. 2.
Effects of rLmS3arp on T-cell activation. (A)
Proliferative responses of spleen cells from normal BALB/c mice after
ConA stimulation. The cells were cultured for 48 h (2.5 × 105/well) in the presence of ConA (5 µg/ml) with or
without rLmS3arp (50 µg/ml) or with E. coli extract (0.17 µg/ml). (B) Lymphocyte proliferation of spleen cells from
rLmS3arp-treated or nontreated mice stimulated in vitro with ConA. The
cells were pulsed with [methyl-3H]thymidine,
and the cpm were determined during the last 8 h of culture. The
data represent mean cpm and standard deviation from triplicate cultures
of spleen cells from three mice analyzed individually. One of four
independent experiments is depicted.
B cells express CD69 in response to rLmS3arp.
To examine more
accurately the in vivo effect of rLmS3arp on T- and B-cell suppression
and activation, we analyzed the expression of CD69, an early marker of
lymphoid cell activation. As shown in Fig. 3A, the
percentage of CD69 B splenocytes was markedly increased in BALB/c mice
after i.p. treatment with rLmS3arp and stimulation in vitro with
rLmS3arp compared with the percentage of unstimulated cells. As a
positive control, increased expression of CD69 was observed when using
Lipopolysaccharide (LPS) as a triggering agent. No significant increase
of the CD69 marker was observed in CD4+ and
CD8+ cells after in vitro stimulation with rLmS3arp. To
further examine whether stimulation of B cells by rLmS3arp does not
require the involvement of T-cell-dependent activities, we used
spleen cell suspensions from rLmS3arp-treated athymic BALB/c mice (nude
mice) cultured in vitro with rLmS3arp. As shown in Fig. 3B, the
percentage of B cells expressing CD69 increased to 60% compared to the
value obtained when using B cells from BALB/c mice (33%). This
observation may suggest that rLmS3arp preferentially triggers the
activation of B cells.
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rLmS3arp down-regulates the cytokine-producing capability of spleen
cells.
Complementary investigations were done to determine the in
vivo effect of rLmS3arp on the ex vivo cytokine production by spleen cells stimulation with ConA, rLmS3arp, or Leishmania
extract. Spleen cells from untreated or rLmS3arp-treated BALB/c mice
were cultured in the presence of ConA, rLmS3arp, or
Leishmania lysates for 48 h. The levels of IFN-,
IL-2, IL-10, IL-4, and IL-12 were measured by ELISA in supernatants
from cell cultures in comparison to a standard curve obtained using the
recombinant cytokines. As shown in Table 1 significant
reduction of IFN-, IL-2, and IL-12 production in spleen cells from
rLmS3arp-treated mice was observed compared to the cytokine production
in cells from nontreated mice (P < 0.001, P < 0.02, and P < 0.05, respectively), whereas the
variations observed for IL-10 and IL-4 production were not statistically significant (P > 0.4 and P > 0.3 respectively). No significant variations could be seen between
cell populations from rLmS3arp-treated or untreated mice cultured in
the presence of rLmS3arp or Leishmania extract.
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In vivo stimulation of IgM-secreting spleen cells by rLmS3arp.
To further examine the effect of rLmS3arp on the B-cell response, we
determined the number of B cells secreting IgM in the spleens of
rLmS3arp-treated and untreated BALB/c mice. Fifteen days following i.p.
rLmS3arp injection, the different isotype immunoglobulin-secreting
cells were determined by the ELISPOT assay. BALB/c mice treated with
rLmS3arp showed an increase (threefold) in the number of IgM-secreting
cells compared to that in untreated mice (Fig. 4A). No
significant increase was observed in the numbers of the different
isotypes of IgG-secreting spleen cells (IgG1, IgG2a, IgG2b, and IgG3).
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Lack of specificity of polyclonal B-cell activation induced by
rLmS3arp.
To assess the specificity of antibody response in sera
from rLmS3arp-treated mice, an ELISA using rLmS3arp and
Leishmania soluble extracts as antigens was applied.
Although large amounts of IgM could be detected in mice which received
rLmS3arp (Fig. 5), no specificity against rLmS3arp or
parasite lysates could be evidenced.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
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Discussion
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Among the evolutionarily conserved antigens of Leishmania, the ribosomal proteins LiP2a, LIP2b, LiP0/LcP0 and LeIF, have being shown to be recognized by the immune system of the host with high frequency (31). Other parasite components belonging to the large ribosomal protein family such as S3a have not yet been examined for their possible role in the host-parasite relationship. We have recently cloned a novel L. major gene encoding a parasite polypeptide with high sequence identity to the eukaryotic ribosomal S3a protein, which was termed LmS3a-related protein (LmS3arp) (44). The protein was expressed by all the Leishmania species so far examined (L. infantum, L. amazonensis, and L. mexicana). The LmS3arp-encoding gene was subcloned in an expression vector, and the corresponding recombinant protein was purified. This prompted us to investigate the effect of the recombinant LmS3arp (rLmS3arp) on T- and B-cell responses.
The results presented in this report show that rLmS3arp plays a dual role in the regulation of T- and B-cell reactivities. We have observed that rLmS3arp inhibited T-cell proliferation both in vitro and in vivo. Furthermore, up-regulation of CD69 cell surface marker occurred only in spleen B cells from rLmS3arp-treated mice, and this phenomenon was not abrogated by treatment with polymyxin B whereas LPS-induced CD69 surface expression was partially inhibited. These observations indicate that a possible contamination of rLmS3arp by LPS, which might account for the effect of rLmS3arp on B-cell activation, is unlikely. Furthermore, in vivo treatment of mice induced a significant increase in the number of B cells secreting immunoglobulins, predominantly of the IgM isotype. The IgM response is unrelated to the rLmS3arp or the total parasite extracts but seems to be directed against a large number of self and nonself antigens.
The occurrence of natural autoantibodies in normal BALB/c mice sera has been reported in a large number of studies (reviewed in reference (5)). Their reactivity is often directed against very highly conserved constituents such as DNA. Furthermore, recent comparative study of BALB/c and Xid mouse IgM repertoires showed greater dilution of sera in BALB/c than Xid mice. In addition, when serum samples were tested at the same IgM concentration, most IgM reactivity scored higher for BALB/c sera than for Xid sera in both self and nonself antigens (32). Moreover, hybridomas secreting antibodies against self antigens can be obtained by fusing spleen cells from normal adult animals; however, monoclonal antibodies of only the IgM isotype could be isolated (20). Interestingly, the affinities of these monoclonal antibodies for their antigens were of the same order of magnitude as those of some induced antibodies (37).
Thus, our data showing high levels of IgM antibodies in normal mouse sera reacting with DNA or KLH is in agreement with these observations. On rLmS3arp treatment of BALB/c mice, a significant increase of IgM levels with a broad range of reactivity was observed.
The strong polyclonal expansion of nonspecific, non-parasite-directed
B-cell clones induced by rLmS3arp suggests that the latter
resembles the classical mitogens such as lipid A (36). Moreover, the data obtained using euthymic and athymic mice support the
notion of a direct T-cell-independent mitogenic activity of rLmS3arp on
B cells. However, it is noteworthy that polyclonal nonspecific
responses are not an exclusive result of direct stimulation of B
lymphocytes by mitogenic factors but are also a result of T-cell
involvement through the release of cytokines which could modulate
B-cell differentiation and immunoglobulin secretion. This possibility
is in line with our results showing that rLmS3arp is able to modulate
cytokine secretion, and this may have implications for whether the Th1
or Th2 immune response will predominate. Indeed, a statistically
significant decrease of Th1 cytokines (IFN-, IL-2, and IL-12) was
observed in ConA-stimulated spleen cells from rLmS3arp-treated mice
compared to spleen cells from nontreated mice. In contrast, no
significant difference was observed in the amounts of Th2 cytokines
(IL-4 and IL-10) secreted by spleen cells from rLmS3arp-treated mice
and nontreated mice. Therefore, it is reasonable to assume that
rLmS3arp, through its action toward B and T cells and cytokine
secretion, could be involved in the immunoregulatory processes.
The mechanisms leading to preferential induction and/or expansion of
distinct Th-cell subsets are still not well understood. Although
IFN- seems to play a critical role in the early immune response that
both controls L. donovani infection and induces the tissue
granulomatous response (35), the differential production of Th1- and Th2-derived cytokines does not seem to determine the genetically controlled or vaccine-induced rate of cure in murine visceral leishmaniasis (VL) (25). In human VL,
antigen-specific immunosuppression during the acute phase of the
disease appears to be induced by a cell-mediated response
(15). Furthermore, the progression of VL is related to
markedly reduced lymphocyte proliferation and decreased IL-2 and
IFN- production by peripheral lymphocytes in response to leishmanial
antigens (16, 17). Evidence showing the predominance of
endogenous IL-4 over IFN- production during VL has also been
reported (46). In contrast to depression of the cellular
response, there is a strong humoral response during active disease,
with increased production of nonspecific immunoglobulins, mostly of the
IgM and IgG isotypes (22). Therefore, it is reasonable to
assume that LmS3arp, among other parasite molecules, through its direct
or indirect effect on IL-2 and IFN- production and B-cell
activation, could participate in the mechanisms that may play a role in
the balance of Th1 and Th2 immune response.
Evidence supporting the existence of parasite-derived molecules which could modulate the host cellular reactivity has been reported. Indeed, soluble parasite-derived antigens from L. major and L. donovani are mitogenic and trigger the production of immunoglobulins with autoantibody activity (10). Furthermore, crude extracts of L. donovani and L. mexicana amazonensis contain components which cause strong in vitro polyclonal activation of hamster spleen cells (12). Moreover, an excreted factor derived from the culture medium of L. major was found to suppress ConA-induced polyclonal activation of mouse T cells (23). Therefore, it is reasonable to assume that parasite soluble immunosuppressive factors and mitogenic molecules both lead to a state of transient immunosuppression that probably helps the parasite to establish chronic infection in animals and humans.
Parasites can elicit a complex series of cellular interactions which result in specific immune response or suppression depending on the immunoregulatory balance in the host. Our study provides, to our knowledge, the first evidence that a Leishmania ribosomal protein (S3a homologue) which belong to the ribosomal family could contribute to the host immune system dysfunction through its capacity to modulate T- and B-cell activities and cytokine release. The results indicate new levels of complexity in the pathogenesis of Leishmania infections.
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ACKNOWLEDGMENTS |
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This study received financial support from Fundação Calouste Gulbenkian-Programa Gulbenkian de estímulo à investigação, INSERM, and IRD UR 008.
We thank Luís Delgado and Abília Cunha from São João Hospital, Porto, Portugal, for autoimmune patient sera. A.O. is a head of research at INSERM, and E.G. is a research senior investigator at INSERM.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry, Faculty of Pharmacy, Rua Anibal Cunha, 164, Porto, Portugal. Phone: 351-22-2078906. Fax: 351-22-2003977. E-mail: mop62612{at}mail.telepac.pt.
Editor: J. M. Mansfield
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, November 2001, p. 6588-6596, Vol. 69, No. 11
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.11.6588-6596.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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