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INFECTION AND IMMUNITY, Nov. 2001, p. 6588–6596 0019-9567/01/$04.00⫹0 DOI: 10.1128/IAI.69.11.6588–6596.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Vol. 69, No. 11

Dual Role of the Leishmania major Ribosomal Protein S3a Homologue in Regulation of T- and B-Cell Activation ANABELA CORDEIRO-DA-SILVA,1* MARGARIDA COUTINHO BORGES,1,2 ELIANE GUILVARD,2 ALI OUAISSI2

AND

Department of Biochemistry, Faculty of Pharmacy and Institute of Molecular and Cellular Biology, University of Porto, Porto, Portugal,1 and IRD UR 008 “Pathogeńie des Trypanosomatide´s”, 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 inhi-

bition of activating lymphokine gamma interferon (IFN-␥) production by macrophages (28). 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 initi-

* 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 @mail.telepac.pt. 6588

LmS3arp T- AND B-CELL REGULATOR IN L. MAJOR INFECTION

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ation 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. MATERIALS AND METHODS 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-␤-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). Treatment of mice. Twelve BALB/c mice were inoculated three times intraperitoneally (i.p.) at 10-day intervals with 50 ␮g 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 ␮g/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-␮l volume at 2 ⫻ 105 cells/well. The cells were stimulated with 5 ␮g ConA per ml in the presence of 50 ␮g of rLm3Sarp per ml or 100 ␮g of total Leishmania extract per ml. After 48 h of incubation at 37°C in 5% CO2, 1 ␮Ci 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

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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 fluorescenceactivated 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 ␮g of rLmS3arp per ml), total Leishmania antigens (10 ␮g/ml), fibronectin (10 ␮g/ml [Sigma]), bovine serum albumin (10 ␮g/ml [Calbiochem]), keyhole limpet hemocyanin (KLH) (10 ␮g/ml), ovalbumin (10 ␮g/ml [Sigma]), horseradish peroxidase (10 ␮g/ml [Sigma]), native double-stranded (DNA (10 ␮g/ml) [Sigma]), porcine type II thyroglobulin (10 ␮g/ml t[Sigma]), whale skeletal muscle type II myoglobulin (10 ␮g/ml [Sigma]). The plates were washed with PBS containing 0.1% Tween 20 (PBS-T [Calbiochem]) and blocked with PBS plus 1% gelatin (200 ␮l/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 ␮g/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 antiIgG2b) 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 ␮g 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-␥ (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 ␮l/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 (JES65H4 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). Immunoglobulin ELISPOT assays. Ninety-six-well flat-bottom plates were coated with unlabeled goat anti-mouse immunoglobulins (5 ␮g/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 (ELIS-

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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.

POTs) 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.

RESULTS 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

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[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). To rule out the possibility that the inhibition of T-cell proliferation was due to bacterial components present in rLS3arp, spleen cells from BALB/c mice were stimulated with ConA in the presence of E. coli total extracts. As shown in Fig. 2A, the level of T-cell proliferation in the presence of E. coli soluble extracts (SI ⫽ 108.5) was comparable to that observed in the control cells (SI ⫽ 124), indicating that the inhibition of T-cell proliferation is due to rLmS3arp. The reduction of T-cell proliferation might be due to a toxic effect of rLmS3arp on T cells. To test this possibility, we examined the in vitro activity of rLmS3arp against spleen cells. To do this, spleen cells from three mice were incubated separately at 37°C for 2, 12, and 24 h with RPMI 1640 medium supplemented with FCS and antibiotics as described in Materials and Methods, with or without 50 ␮g of rLmS3arp per ml. Aliquots of the cells were harvested at different time points and examined for viability by microscopic inspection and a trypan blue dye exclusion test. The results showed more than 98% living cells over the incubation period, which was not different from the viability in controls without rLmS3arp. Therefore, it is reasonable to assume that the down-regulation of T-cell proliferation by rLmS3arp is not due to a toxic effect on spleen cells. It is noteworthy that different preparations of rLmS3arp were examined in the assay. Since they were found to have similar pattern of T- and B-cell suppression and activation (see below), for clarity the data corresponding to a standard preparation of rLmS3arp are shown in this study.

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 ␮g/ml) with or without rLmS3arp (50 ␮g/ml) or with E. coli extract (0.17 ␮g/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.

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FIG. 3. B cells express CD69 in response to rLmS3arp. Expression of the CD69 activation marker in spleen cells from BALB/c (A and C) and BALB/c nude (B) mice after culture with rLmS3arp. In some experiments, rLmS3arp and LPS were incubated for 10 min with 10 ␮g of polymixin B per ml before incubation with spleen cells (B and C). A total of 2.5 ⫻ 105 cells per well were cultured in the presence of rLmS3arp (17 ␮g/ml) or LPS (10 ␮g/ml). After 20 h of culture CD69 was measured by FACS analysis. To determine the percentage of CD69 in B cells or CD4 or CD8 T cells, the different cell populations were positively gated. The data represent the means from triplicate cultures of spleen cells from three mice analyzed individually and are representative of three experiments done independently.

Furthermore, experiments using another L. major His6 tag fusion protein named LmSIR2, described in our previous studies (42, 43), showed no inhibitory effect on ConA-induced T-cell proliferation (SI ⫽ 124 in the presence of LmSIR2 protein and SI ⫽ 131 in the absence of LmSIR2, with less than 10% variation between three independent experiments). To determine whether rLmS3arp has the same activity toward T cells in vivo as in vitro, spleen cells from rLmS3arptreated or untreated-BALB/c mice, as described in Materials and methods, were cultured with or without ConA for 48 h. As shown in Fig. 2B, ex vivo spleens cells from rLmS3arp-treated mice showed significantly reduced levels of T-cell proliferation compared to the controls (P ⬍ 0.01). Moreover, when spleen cells from rLmS3arp-treated mice were stimulated in vitro with either Leishmania total antigens or LmS3arp, the SIs were not significantly different from those of the control nonstimulated cells (SI ⫽ 1.36, 1.24, and 0.81, respectively). 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. The Limulus amebocyte assay detected less than 0.02 ng of endotoxin per ml in our standard preparation of rLmS3arp. However, to rule out the possibility that the rLmS3arp biological activity was due to residual contaminating LPS, complementary experiments were done using polymyxin B and our standard preparation of rLmS3arp. As shown in Fig. 3C, treatment of BALB/c mice spleen cells with rLmS3arp in the presence of 10 ␮g of polymyxin B per ml had no significant effect on the up-regulation of CD69 expression by B cells, whereas the enhancing effect of LPS was significantly abrogated by drug treatment. Similar results were also obtained in the case of spleen cells from nude mice (Fig. 3B). Theses observations

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TABLE 1. Reduction of IFN-␥, IL-2, and IL-12 production by spleen cells from rLmS3arp-treated BALB/c mice Cytokine

IFN-␥ IL-2 IL-10 IL-4 IL-12

Cytokine level (ng/ml) in supernatantsa of spleen cells from untreated and rLmS3arp-treated BALB/c mice

rLmS3arp treatment

Control

ConA (5 ␮g/ml)

rLmS3arp (50 ␮g/ml)

Leishmania extract (100 ␮g/ml)

⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹

0.018 ⫾ 0.010 0.017 ⫾ 0.005 ⬍0.012 0.174 ⫾ 0.016 0.497 ⫾ 0.106 0.435 ⫾ 0.088 0.105 ⫾ 0.019 0.082 ⫾ 0.005 ⬍0.489 ⬍0.489

14.500 ⫾ 0.200 2.920 ⫾ 0.570b 3.995 ⫾ 0.145 1.367 ⫾ 0.087b 2.445 ⫾ 0.374 1.570 ⫾ 0.414 0.222 ⫾ 0.010 0.158 ⫾ 0.032 5.756 ⫾ 0.775 2.660 ⫾ 0.550b

0.396 ⫾ 0.006 0.024 ⫾ 0.005 ⬍0.012 ⬍0.012 0.096 ⫾ 0.003 ⬍0.098 0.020 ⫾ 0.005 0.018 ⫾ 0.001 0.728 ⫾ 0.031 0.527 ⫾ 0.344

0.016 ⫾ 0.006 0.006 ⫾ 0.008 ⬍0.012 ⬍0.012 0.190 ⫾ 0.054 ⬍0.098 0.031 ⫾ 0.006 ⬍0.012 1.007 ⫾ 0.468 ⬍0.489

a The levels of IFN-␥, IL-2, IL-10, IL-4, and IL-12 in the supernatants of spleen cells (2 ⫻ 105 cells/well) from rLmS3arp-treated or untreated BALB/c mice cultured with ConA, rLmS3arp, or Leishmania extract for 48 h. We have determined by ELISA the levels of these ILs in comparison with a standard curve using the recombinant ILs. The data represent means and standard deviations for triplicate cultures of spleen cells from three mice. The results are from a representative experiment of four carried out independently. b A significant difference in the IFN-␥, IL-2, and IL-12 levels (P ⬍ 0.0001, P ⬍ 0.02, and P ⬍ 0.05, respectively) was observed between samples from rLmS3arp-treated and untreated BALB/c mice after ConA stimulation.

strengthen the notion that the observed effects are due to rLmS3arp. 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. 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). To evaluate whether the increased IgM surface immunoglobulins could be linked to the amounts of IgM present in the sera, we used ELISA to measure the isotype immunoglobulin concentrations in mice sera after i.p. rLmS3arp injection. As shown in Fig. 4B, a significant increase (P ⬍ 0.005) in the total IgM levels in sera of BALB/c mice injected with rLmS3arp was

observed compared to the levels in untreated controls. No difference was observed between the two groups when the other immunoglobulin isotypes were examined (data not shown). These data are in agreement with the above results showing increased IgM-secreting B-cell numbers. 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. Having found a lack of specificity of IgM antibodies against rLmS3arp, we decided to assess more accurately the nature of the antibody reactivity by using a large panel of self and nonself molecules. As shown in Table 2, various degrees of reactivity of sera from rLmS3arp-treated mice were observed against different molecules. The optical density values recorded for rLmS3arp-treated mouse sera as well as those found for sera from nontreated mice showed that the most reactive molecules were DNA and KLH. Although the charge interactions could account for IgM binding to DNA or KLH, increasing the number of washing steps and the salt concentrations did not significantly modify the reactivity of either rLmS3arp-treated or nontreated mouse sera with DNA or KLH (data not shown). Moreover, an additional control using a commercially available mouse myeloma IgM showed low reactivity with the antigens. These observations indicate that when injected into mice without adjuvant, rLmS3arp could induce a strong B-cell activation leading to the production of IgM antibodies with a broad range of reactivity, although the level of immune reactivity with rLmS3arp was lower than those observed for other antigens. Hypergammaglobulinemia is often associated with the production of a variety of autoantibodies (8). Therefore, we attempted to examine whether rLmS3arp could be a target of antibodies present in sera from patients with autoimmune diseases (nuclear antigen positive). As shown in Fig. 6, sera from


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FIG. 4. In vivo stimulation of IgM-secreting spleen cells and IgM levels in the sera of rLmS3arp-treated mice. (A) Isotype immunoglobulinproducing spleen cells from untreated and rLmS3arp (50 ␮g/mouse)-treated BALB/c mice. Numbers of IgM-, IgG1-, IgG2a-, IgG2b-,and IgG3-secreting cells were determined by ELISPOT on day 15 after the last rLmS3arp injection. The data represent the mean and standard deviation for three animals analyzed individually and are representative of four experiments. (B) Levels of total IgM in the sera of rLmS3arp (50 ␮g/mouse)-treated or nontreated BALB/c mice. Total IgM levels were quantified by ELISA on day 15, after the last immunization with rLmS3arp, in comparison to standard curves using purified mouse IgM. Data represent the mean of triplicate samples and standard deviations and are representative of four experiments done independently.

patients exhibiting autoantibodies had a higher reactivity with rLmS3arp than did sera from healthy patients (P ⬍ 0.005). DISCUSSION 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 LmS3arpencoding gene was subcloned in an expression vector, and

FIG. 5. Titers of IgM antibodies in the sera of BALB/c mice 15 days after the last rLmS3arp injection. Total IgM, anti-rLmS3arp IgM and anti-Leishmania lysate antigen IgM were determined by ELISA. The results represent the mean for three mice and are representative of four independent experiments.

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TABLE 2. Lack of specificity of rLmS3arp-induced polyclonal B-cell activation Reactivitya (optical density at 492 nm) against: Treatment

FN

BSA

DNA

KLH

OVA

MYO

THY

None rLmS3arp Myeloma IgM controlb

0.144 ⫾ 0.022 0.305 ⫾ 0.031 0.034 ⫾ 0.012

0.247 ⫾ 0.038 0.49 ⫾ 0.084 0.067 ⫾ 0.024

0.612 ⫾ 0.052 0.976 ⫾ 0.156 0.069 ⫾ 0.002

0.384 ⫾ 0.075 0.718 ⫾ 0.137 0.064 ⫾ 0.003

0.169 ⫾ 0.016 0.354 ⫾ 0.028 0.050 ⫾ 0.003

0.207 ⫾ 0.014 0.514 ⫾ 0.008 0.064 ⫾ 0.004

0.143 ⫾ 0.032 0.388 ⫾ 0.032 0.022 ⫾ 0.006

a IgM antibody levels reacting against fibronectin (FN), bovine serum albumin (BSA), KLH, ovalbumin (OVA), native double-stranded DNA, porcine type II thyroglobulin (THY) and whale skeletal muscle type II myoglobulin (MYO) were determined by ELISA. Sera from rLmS3arp-treated and untreated mice were diluted 1/1,000 in PBS-T. The optical density was recorded at 492nm. Data represent the mean of triplicate samples from three mice ⫾ standard deviations and are representative of a set of four independently run experiments. b A mouse myeloma IgM immunoglobulin kappa chain was used to control for the background for each antigen; the dose employed (1.2 ␮g/ml) was identical to the arithmetic mean of IgM concentration found in the diluted sera from rLmS3arp-treated mice.

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. Fur-

FIG. 6. Reactivity of sera from patients with autoimmune diseases (antinuclear antigen positive) and healthy subjects against rLmS3arp. Each point represents individual serum diluted 1:100. Horizontal bars represent mean values. OD, optical density.

thermore, 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 differen-


VOL. 69, 2001

tial 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 cellmediated 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. ACKNOWLEDGMENTS This study received financial support from Fundação Calouste Gulbenkian-Programa Gulbenkian de estı´mulo à investigação, INSERM, and IRD UR 008. We thank Luı´s Delgado and Abı´lia Cunha from Saõ Joaõ 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. REFERENCES 1. Abranches, P., G. Santos-Gomes, and N. Rachamin. 1991. An experimental model for canine visceral leishmaniasis. Parasite Immunol. 13:537–550. 2. Andrade, C. R., L. V. Kirchhoff, J. E. Donelson, and K. Otsu. 1992. Recombinant Leishmania Hsp90 and Hsp70 are recognised by sera from visceral leishmaniasis patients but not Chagas’ disease patients. J. Clin. Microbiol. 30:330–335. 3. Angel, S. O., J. M. Requena, M. Soto, D. Criado, and C. Alonso. 1996. During canine leishmaniasis a protein belonging to the 83 kDa heat shock protein

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family elicits a strong humoral response. Acta Trop. 62:45–56. 4. Argov, S., C. L. Jaffe, M. Krupp, H. Slor, and Y. Shoenfeld. 1989. Autoantibody production by patients infected with Leishmania. Clin. Exp. Immunol. 76:190–197. 5. Avrameas, S. 1991. Natural autoantibodies: from “horror autotoxins” to “gnothi scauton.” Immunol. Today 12:154–159. 6. Badaro, R., E. Falcoff, and F. S. Badaro. 1990. Treatment of visceral leishmaniasis with pentavalent antimony and interferon gamma. N. Engl. J. Med. 322:16–21. 7. Badaro, R., T. C. Jones, E. M. Carvalho, D. Sampaio, S. G. Reed, A. Barral, R. Teixeira, and W. D. Johnson. 1986. New perspectives on a subclinical form of visceral leishmaniasis. J. Infect. Dis. 154:1003–1011. 8. Bendixen, G., T. Hadidi, R. Manthorpe, H. Permin, J. Struckmann, A. Whk, and M. Zaghloul. 1984. Antibodies against nuclear components in schistosomiasis. Allergy 39:107–113. 9. Berman, J. D. 1997. Human leishmaniasis: clinical, diagnostic and chemotherapeutic developments in the last 10 years. Clin. Infect. Dis. 24:684–703. 10. Bohme M. W., D. A. Evans, M. A. Miles, and E. J. Holborow. 1986. Occurrence of autoantibodies to intermediate filament proteins in human visceral leishmaniasis and their induction by experimental polyclonal B-cell activation. Immunology 59:583–588. 11. Bray, R. S. 1976. Immunodiagnosis of leishmaniasis, p. 66–76. In S. Cohen and E. Sadun (ed.), Immunology of parasitic infection. Blackwell Scientific Publications, Oxford, United Kingdom. 12. Bunn-Moreno, M. M., E. D. Madeira, K. Miller, J. A. Menezes, and A. Campos-Neto. 1985. Hypergammaglobulinaemia in Leishmania donovani infected hamsters: possible association with a polyclonal activator of B cells and with suppression of T cell function. Clin. Exp. Immunol. 59:427–434. 13. Burns, J. M., W. G. Shreffler, D. R. Benson, H. W. Ghalib, R. Badaro, Jr., and S. G. Reed. 1993. Molecular characterisation of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc. Natl. Acad. Sci. USA 90:775–779. 14. Cabral, M., J. O’Grady, and J. Alexander. 1992. Demonstration of Leishmania specific cell mediated and humoral immunity in asymptomatic dogs. Parasite Immunol. 14:531–539. 15. Carvalho, E. M., O. Bacellar, A. Barral, R. Badaro, and W. D. Johnson, Jr. 1989. Antigen specific immunosuppression in visceral leishmaniasis is associated with cell-mediated. J. Clin. Investig. 83:860–864. 16. Carvalho, E. M., R. Badaro, S. G. Reed, T. Jones, and W. D. Johnson, Jr. 1985. Absence of gamma interferon and IL-2 production during active visceral leishmaniasis. J. Clin. Investig. 76:2066–2069. 17. Carvalho, E. M., A. Barral, D. Pedral-Sampaio, M. Barral-Neto, R. Badaro, H. Rocha, and W. D. Johnson, Jr. 1992. Immunological markers of clinical evolution in children recently infectedd with L. chagasi. J. Infect. Dis. 165: 536–544. 18. Czerkinsky, C. C., L. A. Nilson, H. Nigren, O. Outcherlony, and A. J. Tarkowsky. 1983. A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody secreting cells. J. Immunol. Methods 65:109–121. 19. De Luna, R., M. L. Vuotto, M. T. L. Ielpo, R. Ambrosio, D. Piantedosi, V. Moscatiello, P. Ciaramella, A. Scalone, L. Gradoni, and D. Mancino. 1999. Early suppression of lymphoproliferative response in dogs with natural infection by Leishmania infantum. Vet. Immunol. Immunopathol. 70:95–103. 20. Dighiero, G., P. Lymberi, J. C. Mazie, S. Rouyre, G. S. Butler-Browne, R. G. Whalen, and S. Avrameas. 1983. Murine hybridomas secreting natural monoclonal antibodies reacting with self antigens. J. Immunol. 131:2267– 2272. 21. Gagnaire, M. H., C. Galambrun, and J. L. Stephan. 2000. Hemophagocytic syndrome: a misleading complication of visceral leishmaniasis in children—a series of 12 cases. Pediatrics 106:58–69. 22. Galvao-Castro, B., J. A. Sa Ferreira, K. F. Marzochi, M. C. A. Marzochi, S. G. Coutinho, and P. H. Lambert. 1984. Polyclonal B cell activation, circulating immune complexes and autoimmunity in human American visceral leishmaniasis. Clin. Exp. Immunol. 56:58–66. 23. Grimaldi, G., and R. B. Tesh. 1993. Leishmaniases of the New World: current concepts and importance for future research. Clin. Microbiol. Rev. 6:230–250. 24. Isakov, N., A. Tamir, and J. el-On. 1994. Suppression of antigen-specific T cell responses by Leishmania major excreted factor: inhibition of activation signals linked to the T cell antigen receptor and interleukin 2 receptor. Isr. J. Med. Sci. 30:673–679. 25. Kaye, P., A. J. Kurry, and J. M. Blackwell. 1991. Differential production of Th1-and Th2-derived cytokines does not determine the genetically controlled or vaccine-induced rate of cure in murine visceral leishmaniasis. J. Immunol. 146:2763–2770. 26. Lohman, K. L., P. J. Langer, and D. McMahon-Pratt. 1990. Molecular cloning and characterization of the immunological protective surface glycoprotein GP46/M-2 of Leishmania amazonensis. Proc. Natl. Acad. Sci. USA 87:8393–8397. 27. Lowry, O. H., N. J. Rosebrough, L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:267–269. 28. Murray, H. W., S. M. Carriero, and D. M. Donelly. 1986. Presence of a

6596

29.

30. 31. 32.

33.

34.

35.

36.


macrophage-mediated suppressor cell mechanism during cell-mediated immune response in experimental visceral leishmaniasis. Infect. Immun. 54: 487–493. Naora, H., I. Takai, M. Adachi, and H. Naora. 1998. Altered cellular responses by varying expression of a ribosomal protein gene: sequential coordination of enhancement and suppression of ribosomal protein S3a gene expression induces apoptosis. Cell. Biol. 141:741–753. Reina-San-Martıń, B., A. Cosson, and P. Minoprio. 2000. Lymphocyte polyclonal activation: a pitfall for vaccine design against infectious agents. Parasitol. Today 16:62–67. Requena, J. M., C. Alonso, and M. Soto. 2000. Evolutionary conserved proteins as proeminent immunogens during Leishmania infections. Parasitol. Today 16:246–250. Santos-Lima, E. C., R. Vasconcellos, B. Reina-San-Martin, C. Fesel, A. Cordeiro-da-Silva, A. Berneman, A. Cosson, A. Coutinho, and P. Minoprio. 2001. Significant association between the skewed natural antibody repertoire of Xid mice and resistance to Trypanosoma cruzi infection. Eur. J. Immunol. 31:634–645. Shreffler, W. G., J. M. Burns, R. Badaro, H. W. Ghalib, L. L. Button, W. R. McMaster, and S. G. Reed. 1993. Antibody response to visceral leishmaniasis patients to gp63, a major surface glycoprotein of Leishmania species. J. Infect. Dis. 167:426–430. Skeiky, Y. A. W., D. R. Benson, J. A. Guederian, J. A. Whittle, O. Bacelar, E. M. Carvalho, and S. G. Reed. 1995. Immune responses to leishmaniasis patients to heat shock proteins of Leishmania species and humans. Infect. Immun. 63:4105–4114. Squires, K. E., R. D. Schreiber, M. J. MacElrath, B. Y. Robin, S. L. Anderson, and H. W. Murray. 1989. Experimental visceral leishmaniasis: role of endogenous IFN-gamma in host defense and tissue granulomatous response. J. Immunol. 143:4244–4249. Sven, K., and T. Hofstad. 1990. Blastogenesis and polyclonal immunoglobulin synthesis in murine spleen cells stimulated with lipopolysaccharide, lipid A and acid degraded. FEMS Microbiol. Immunol. 2:29–32.

Editor: J. M. Mansfield

INFECT. IMMUN. 37. Ternynck, T., and S. Avrameas. 1986. Murine natural monoclonal autoantibodies: a study of their polyspecificities and their affinities. Immunol. Rev. 94:99. 38. Tolson, D. L., A. Jardim, L. F. Schnur, C. Stebeck, C. Tuckey, R. T. Beecroft, H. S. The, R. W. Olafson, and T. W. Pearson. 1994. The kinetoplastid membrane protein 11 of Leishmania donovani and African trypanosomes is a potent stimulator of T-lymphocyte proliferation. Infect. Immun. 62:4893– 4899. 39. Wool, I. G., Y. Endo, Y. L. Chan, and A. Gluck. 1990. Structure, function, and evolution of mammalian ribosomes, p. 203–214. In W. E. Hill, A. Dajhlberg, R. A. Garrett, P. B. Moore, D. Schlessinger, and J. R. Warner (ed.), The ribosome. American Society for Microbiology, Washington, D.C. 40. Wool, I. G., Y. L. Chan, and A. Gluck. 1995. Structure and evolution of mammalian ribosomal proteins. Biochem. Cell. Biol. 73:933–947. 41. Wool, I. G. 1996. Extraribosomal functions of ribosomal proteins. Trends Biochem. Sci. 21:164–165. 42. Yahiaoui, B., A. Taibi, and A. Ouaissi. 1996. Characterization of a Leishmania protein with extensive homology to silent information regulatory 2 protein of Saccharomyces cerevisiae. Gene 169:115–118. 43. Zemzoumi, K., D. Sereno, C. Francois, E. Guilvard, J. L. Lemesre, and A. Ouaissi. 1998. Leishmania major: Cell type dependent distribution of a 43 kDa antigen related to silent information regulatory-2 protein family. Biol. Cell. 90:239–245. 44. Zemzoumi, K., E. Guilvard, D. Sereno, A. Preto, M. Benlemlih, A. Cordeiro da Silva, J. Lemesre, and A. Ouaissi. 1999. Cloning of a Leishmania major gene encoding for an antigen with extensive homology to ribosomal protein S3a. Gene 240:57–65. 45. Zuckerman, A. 1975. Parasitological review. Current status of immunology of blood and tissue protozoa. Leishmania. Exp. Parasitol. 38:370–400. 46. Zwingenberger, K., G. Harms, C. Pedrosa, S. Omena, B. Sandkramp, and N. Neifer. 1990. Determinants of the immune response in visceral leishmaniasis: evidence for predominance of endogenous IL-4 over IFN-gamma production. Clin. Immunol. Immunopathol. 54:242–249.