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(Journal of Leukocyte Biology. 2003;73:127-136.)
© 2003 by Society for Leukocyte Biology

Interleukin-4 biases differentiation of B cells from Trypanosoma cruzi-infected mice and restrains their fratricide: role of Fas ligand down-regulation and MHC class II-transactivator up-regulation

E. V. Acosta Rodriguez, E. Zuñiga, C. L. Montes and A. Gruppi

Inmunología, Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Argentina

Correspondence: Dr. Adriana Gruppi, Inmunologia, Departamento de Bioquímica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Ciudad Universitaria, Haya de la Torre y Medina Allende, Cordoba (5000), Argentina. E-mail: agruppi{at}bioclin.fcq.unc.edu.ar


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present work, we demonstrate that interleukin (IL)-4 is able to rescue B cells from Trypanosoma cruzi-infected mice, counteracting the strong apoptotic signals that these cells received in vivo. We have observed that IL-4 restrains the apoptosis of immunoglobulin (Ig)M+ and IgG+ B cells from infected and normal mice without inducing them to proliferate. In addition, IL-4 does not modify the quantity or quality of the antibodies secreted by B cells from infected mice, as it blocks their terminal differentiation to plasma cells and favors memory pathway. It is interesting that the protective effect of IL-4 over B cells from infected mice is mediated, at least partly, by the down-regulation of Fas ligand (FasL) expression, which leads to interference in the apoptosis executed by these B cells through the Fas/FasL death pathway. Accordingly, a marked up-regulation of the "FasL gene repressor" class II transactivator was observed, suggesting that this would be one mechanism underlying the IL-4-mediated FasL down-regulation.

Key Words: apoptosis • memory B cell • plasma cell • control mechanism • survival


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chagas’ disease (American trypanosomiasis), caused by Trypanosoma cruzi, is a chronic and transmissible disease that affects nearly 20 million people in South and Central America [1 ]. During the acute phase of infection, a massive T and B cell polyclonal activation coexists with a markedly depressed cellular and humoral-immune response [2 ]. This functional deficiency, also reported in other infections, has been explained by triggering immunoregulatory mechanisms, which trying to control the extensive lymphocyte proliferation, induce apoptosis of T and B cells [3 4 5 6 ]. Particularly, the highly activated B lymphocytes isolated from mice undergoing the acute phase of T. cruzi infection have been proven to coexpress Fas and Fas ligand (FasL) and to commit fratricide mediated by the death pathway triggered by these molecules [7].

Cell death of proliferating lymphocytes is necessary to preserve a healthy and balanced immune system [8 ]. Nevertheless, if during an infectious process, lymphocyte apoptosis occurs previously to the pathogen elimination, it could restrict the magnitude of the effector response, facilitating the establishment of the microorganism and the chronicity of the infection [9 ]. In this case, cell death of activated lymphocyte would play a deleterious role for the host. Accordingly, we have reported [7 ] that during T. cruzi infection, the Fas/FasL-mediated apoptosis targets parasite-specific immunoglobulin G (IgG)+ B cells, and the blockage of this pathway, in vivo and in vitro, leads to the emergence of a parasite-specific IgG response. Therefore, the Fas/FasL pathway seems to be one of the mechanisms underlying B cell apoptosis, which in turn, would delay the rising of a protective, humoral-immune response. Considering that several studies [10 11 ] have demonstrated the importance of antibodies (Ab) for host survival and parasite clearance during T. cruzi infection, the identification of signals that rescue B lymphocytes from apoptosis would provide data to design strategies to enhance humoral, protective immunity and eradicate this infectious agent.

It is largely known that mature, resting B cells are inexorably programmed to die by apoptosis when cultured in vitro. The apoptosis of these lymphocytes is regulated by several B cell growth stimuli, including lipopolysaccharide (LPS) and anti-IgM Ab, which inhibit cell death [12 ]. Conversely, in contrast to its effects on normal B cells, LPS fails to revert the increased B cell apoptosis observed in acutely T. cruzi-infected mice [13 ]. This finding suggests that each stimulus acts in a different way depending on the cell type, cell activation state, and mainly on the kind of signals previously received by the cell. Hence, the cytokine interleukin (IL)-4 has been implicated not only in preventing apoptosis of small, dense, naïve B cells in vitro [14 ] but also in inducing Fas resistance in B cells activated through anti-CD40 engagement [15 ] and in protecting B cell receptor-stimulated B cells from protein kinase A-mediated apoptosis [16 ]. However, the intracellular signaling pathway induced by IL-4 that culminates in B cell apoptosis inhibition has been only partially elucidated [17 ]. Indeed, many reports [14 15 16 17 18 19 20 ] have described several antiapoptotic stimuli for naïve or in vitro-activated B cells, there are few data about the effect of B cell survival stimuli in the context of an infectious process where B cells are activated by a "storm" of microbial mitogens, cytokines, chemokines, and pathogen interactions.

Considering this, we have investigated the effect of IL-4 on the life/death decision of B cells from T. cruzi-infected mice. In this paper, we document that IL-4 is able to rescue B cells from T. cruzi-infected mice from in vitro, spontaneous apoptosis. Strikingly, IL-4-induced survival does not rebound in increments of Ig secretion, as this cytokine blocks the final differentiation of B cells to plasma cells and favors the memory pathway. Moreover, we demonstrate that IL-4 induces a down-regulation in FasL expression, suggesting that this could be one of the mechanisms underlying its survival effect on B cells from infected mice. Finally, we report the up-regulation of the "FasL gene repressor" major histocompatibility complex class II (MHC-II) transactivator (CIITA) upon IL-4 treatment, indicating that it could be involved in the decreased FasL expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Infection with T. cruzi
BALB/c mice, 6–8 weeks old (Comisión Nacional de Energía Atómica, Buenos Aires, Argentina), were intraperitoneally infected with 500 trypomastigotes from T. cruzi (Tulahuén strain) diluted in phosphate-buffered solution (PBS) as described previously [13 ]. After 15 days postinfection (acute phase), mice were killed by cervical dislocation, and spleens were obtained by surgical act. Noninfected, normal littermates were processed in parallel.

IL-4
Concentrated supernatants from transfected LT-1-4 cells that secrete IL-4 were titered with a specific enzyme-linked immunosorbent assay (ELISA) assay, and concentration was adjusted at 5 µg/ml. Then, the supernatant was filtered for sterilization and used in culture at a final concentration of 25 ng/ml.

Reagents
RPMI 1640, protease inhibitors, 2-mercaptoethanol (2-ME), molecular weight markers, LPS from Escherichia coli serotype 0127:B8, nitrocellulose membranes, and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO). Electrophoretic reagents were from BioRad (Richmond, CA). L-Glutamine was purchased from Life Technologies (Paisley, UK). Fetal bovine serum (FBS) was from Natocor (Cordoba, Argentina). All other chemical reagents are commercially available in analytical grade.

Ab
PE-labeled anti-mouse FasL, Syndecan (Synd)-1, MHC-II, and CD19 Ab; fluorescein isothiocyanate (FITC)-labeled anti-mouse Fas, CD3, Mac-1, and CD38 Ab; and biotin-labeled anti-mouse B7.2 (CD86) Ab as well as cychrome-labeled streptavidin (St-Cy) were purchased from BD PharMingen (Palo Alto, CA). Anti-mouse CIITA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-mouse IgM, anti-mouse IgG, or anti-goat IgG and isotype-specific Ab for murine IgG1, IgG2a, IgG2b, IgG3, and IgM were purchased from Sigma-Aldrich.

Cell preparations
Splenocytes from infected or normal mice were obtained by homogenization in a tissue grinder. Erythrocytes were lysed in red blood cell lysis buffer (Sigma-Aldrich). For B cell purification, monocytes were removed by adherence to plastic (1 h incubation at 37°C in 10 cm Petri dishes), and T cells were depleted by magnetic cell-sorting using anti-Thy 1.2-coated magnetic beads (Dynal Biotech, Compiégne, France) following the manufacturer’s instructions. This procedure yielded an enriched B cell population with <2% CD3+ cells and <3% Mac-1+ cells, as determined by flow cytometry (FCM) analysis (data not shown) and >95% of viable cells, as determined by trypan blue exclusion. The B cell-enriched population was resuspended in complete RPMI medium containing 10% FBS, 50 µM 2-ME, and 40 µg/ml gentamicin to a final concentration of 2 x 106cell/ml.

FCM analysis and apoptosis assay
B cells isolated from normal or T. cruzi-infected mice were incubated with IL-4 (25 ng/ml) or medium alone at a density of 1 x 106cell/ml in a volume of 1 ml for the indicated times (see Figs. 1 2 3 4 5 ). After culture, B cells were harvested, and their suspensions were washed twice in ice-cold fetal calf serum buffer [Hanks’ balanced saline solution (HBSS), 1% FBS, 0.1% NaN3] and were preincubated with anti-mouse CD32/CD16 monoclonal Ab (Fc block, clone 2.4G2) at 4°C for 30 min. Cells were then incubated with PtdEtn, FITC, or biotin-conjugated Ab at 4°C for 30 min and were washed with FCM buffer. When biotin-labeled Ab were used, a third step involving an extra 30-min incubation was performed with St-Cy. For apoptotic cell detection, PI staining was performed. Briefly, harvested cells were washed twice with HBSS and fixed overnight in 1 ml 70% ethanol at 4°C. Cell pellets were gently resuspended in 1 ml hypotonic fluorochrome solution (50 µg/ml PtdIns diluted in sodium citrate, 4 mM, plus 0.3% Nonidet P-40) and were kept at 4°C for 18 h in the dark. Data were acquired on a Cytoron Absolute® cytometer (Ortho Diagnostic System, Raritan, NJ) and were analyzed using WinMDI 2.8 software (Joseph Trotter, Scripps Institute, La Jolla, CA). In all cases, cell debris was eliminated through gating live cells from forward-scatter versus side-scatter dot plots [21 ].



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  		Figure 1. IL-4 rescues from spontaneous apoptosis B cells from T. cruzi-infected mice but does not induce their proliferation. B cells obtained from normal (BN) or T. cruzi-infected mice (BI) were cultured for 18–20 h with medium alone (+ medium) or with 25 ng/ml IL-4 (+ IL-4). (A) After culture, B cell nuclei were stained with PI, and the cells were subjected to apoptosis analysis by FCM. This figure shows the histograms of the DNA content obtained in one representative experiment. M1 indicates the percentage of B cells from infected (upper panels) or normal (lower panels) mice with hypodiploid DNA content. (B) The percentage of apoptotic B cells represented as bars with statistical analysis. Data are representative of five identical experiments performed with separated groups of animals. (C) B cells were assessed for proliferation by [3H]-TdR uptake. Proliferation of LPS-stimulated B cells from normal or infected mice was included. B cell proliferation is expressed as the mean cpm + SD. Unless indicated, the differences found were not statistically significant (P>0.1).

 


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  		Figure 2. Blockade in Ig secretion and reduced percentage of Synd-1+ plasma cells after IL-4 treatment. Normal or T. cruzi-infected mice B cells (BN and BI, respectively) were cultured during 96 h with medium alone (+ medium) or IL-4 (+ IL-4). (A) After culture, cell-free supernatants were harvested, and Ig isotype concentrations were determined in triplicate by ELISA. Data are presented as ng/ml + SD. In all cases, the differences found were not statistically significant, and P values > 0.3. (B) B cells from normal (left panels) and infected (right panels) mice cultured with medium (upper panels) or IL-4 (lower panels) were stained with PE-labeled anti-mouse Synd-1 and were analyzed by FCM. Staining with isotype control is shown as a black line. Data representative of three independent experiments are presented as percentage of Synd-1+ cells ± SD.

 


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  		Figure 3. Increased percentage of B cells with memory phenotype after IL-4 treatment. Normal or T. cruzi-infected mice B cells (BN, upper panels, and BI, lower panels, respectively) were cultured during 48 h with medium alone (+medium) or IL-4 (+ IL-4). After culture, B cells were triple-stained with FITC-labeled anti-mouse CD38, biotin-labeled anti-mouse B7.2 plus St-Cy, and PE-labeled anti-mouse Synd-1 and were analyzed by FCM. A region of CD38hiB7.2+ was identified, and the percentage (±SD) of this population was registered in the table below the density plots. The percentage of Synd-1+ plasma cells after 48 h of culture is also depicted.

 


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  		Figure 4. IL-4 down-regulates FasL expression but does not modify Fas levels in B cells obtained from T. cruzi-infected mice. B cells from normal or T. cruzi-infected mice were processed for FasL detection when freshly explanted (upper histograms in A) or for FasL and Fas detection after 18-h culture with medium alone (+ medium, filled, light-gray histogram) or with 25 ng/ml IL-4 (+ IL-4, open, black histogram). B cells were double-stained with PE-labeled anti-mouse FasL and FITC-labeled anti-mouse CD19 or FITC-labeled anti-mouse Fas and PE-labeled anti-mouse CD19 and were analyzed by FCM. CD19+ population was gated for further analysis of FasL (A) or Fas (B) expression. Isotype-control staining is shown in each histogram as a dark-gray line. The results are expressed as percentage of positive cells ± SD. Data are representative of three independent experiments.

 


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  		Figure 5. IL-4 induces the expression of MHC-II molecules and CIITA on B cells. (A) B cells from normal (BN) or T. cruzi-infected mice (BI) were cultured for 18 h with medium alone (+ medium, open, black histogram) or with 25 ng/ml IL-4 (+ IL-4, filled, gray histogram). After culture, B cells were double-stained with PE-labeled anti-mouse MHC-II and FITC-labeled anti-mouse CD19 and were analyzed by FCM. Data are presented as the percentage of cells MHC-II+ and the mean of fluorescence ± SD in the CD19+-gated population. (B) CIITA expression was determined by Western blot after specific CIITA immunoprecipitation from lysates of 18-h cultured B cells. In this experiment, B cells from two infected mice (BI-1 and BI-2) were processed in parallel. As control of specificity, a mixture containing the primary antibody and the Agarose-Protein A/G (Ab + Ag-Prot AG) was blotted. The arrow indicates the specific band of CIITA (140–145 kDa).

 
Cell proliferation assay
Cells (2x105)/well of B cell-enriched population from normal and T. cruzi-infected mice were cultured in quadruplicate in a volume of 200 µl in flat-bottom 96-well tissue-culture plates (TPP, Germany) for 18–20 h with 25 ng/ml IL-4, 20 µg/ml LPS (positive proliferation control), or medium alone. Cells were harvested, and proliferation was measured by the incorporation of 1 µCi [3H]-thymidine (TdR)/well during the entire culture period (18 h) using a ß-scintillation counter. Results are expressed as incorporation of radioactivity (cpm±SD).

Ab isotypes determination
For the determination of Ig secretion, B cells isolated from normal and T. cruzi-infected mice were incubated with IL-4 (25 ng/ml) or medium alone at a density of 2 x 106cell/ml in a volume of 2 ml. After 4 days of culture, the supernatants were collected and assayed in an isotype-specific ELISA. Ninety-six-well ELISA plates were coated with 10 µg/ml isotype-specific goat anti-mouse Ab (IgM, IgG1, IgG2a, IgG2b, and IgG3) diluted in PBS overnight at 4°C, extensively washed, and blocked by the addition of bovine serum albumin (BSA), 1%, for 1 h at room temperature. Plates were emptied, and supernatants from IL-4- or nonstimulated cell cultures were added in triplicate at 37°C for 2 h in a humidified atmosphere. After washing three times with PBS containing 0.05% Tween 20, peroxidase-conjugated anti-mouse IgG or anti-mouse IgM was added and incubated at 37°C for 1 h. The reaction was developed with o-phenylendiamine, and the concentration was measured with reference to standard curves using known amounts of the respective murine Ig isotypes (Sigma-Aldrich).

Immunoprecipitation
Following Santa Cruz research application procedures, an immunoprecipitation technique was performed to detect CIITA expression. Briefly, 200 µg total cell lysates of B cells cultured with or without IL-4 during 18 were incubated at 4°C for 2 h with 1.5 µg anti-mouse CIITA (Santa Cruz Biotechnolgy). Then, 30 µl Agarose-Protein A/G Plus (Santa Cruz Biotechnology) was added to each tube and incubated overnight at 4°C with constant shacking. After that, each tube was subjected to centrifugation at 2500 rpm for 5 min and washed twice with RIPA buffer and twice with PBS buffer. Finally, 40 µl sample buffer, 1x, was added to the pellet, and each sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a Miniprotean II electrophoresis apparatus (BioRad). Briefly, each sample was resolved on a 7.5% separating polyacrylamide slab gel. After electrophoresis, the separated proteins were transferred onto nitrocellulose membranes and probed with anti-mouse CIITA Ab. Blots were then incubated with peroxidase-conjugated secondary antibody, developed by using the enhanced chemiluminescence system, and finally exposed to Amersham Hyperfilm (Amersham Biosciences, UK) for 3–5 min. Prestained protein molecular weight markers (Sigma Chemical Co.) were run in parallel. Protein concentration was estimated by the method of Bradford, using the BioRad protein assay. BSA was used as protein standard.

Statistical analysis
Unpaired Student’s t-test was used for the comparison of means, and values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-4 rescues B cells from T. cruzi-infected mice from spontaneous apoptosis but does not induce their proliferation
To determine the effects of IL-4 in the survival of B cells from T. cruzi-infected mice, purified, splenic B cells from infected animals were cultured with 25 ng/ml IL-4 or medium alone. B cells from normal animals were assayed in parallel. After 18 h, cultured B cells were harvested and processed for apoptotic cell detection by PI staining. As shown in Figure 1A (left panels) and as we have described previously [13 ], B cells from T. cruzi-infected animals undergo increased apoptosis (47.1% of apoptotic cells vs. 19.4% in B cells from normal animals). In addition, as clearly illustrated in Figure 1A (upper panels, left vs. right), IL-4 was able to decrease this percentage of spontaneous apoptosis in B cells from infected mice. As reported [14 15 ], we have also observed that IL-4 restrains the apoptosis of normal B cells (Fig. 1A , lower panels, left vs. right). Figure 1B shows the mean percentages of apoptotic B cells from normal or infected mice cultured with or without IL-4 obtained after five independent experiments. The apoptosis inhibition induced by IL-4 was statistically significant in B cells from normal (17.0% with medium vs. 7.6% with IL-4, P=0.01) and infected (45.9% vs. 24.0%, P=0.0002) mice.

To ensure that the decrease in the percentage of apoptotic cells is not an artifact as a result of proliferation of nonapoptotic B cells, we have tested whether IL-4 induces B cell proliferation. To do this, B cells from normal or infected mice were cultured for 18–20 h with 25 ng/ml IL-4 or medium alone, and the cell proliferation was measured by [3H]-TdR uptake. As described previously [13 ], we have observed that B cells from T. cruzi-infected mice cultured with medium alone proliferate spontaneously, revealing their in vivo stimulation (Fig. 1C) . Furthermore, provided that IL-4 does not modify the level of [3H]-TdR uptake in B cells from normal or infected mice, it can be concluded that IL-4 is unable to induce B cell proliferation. Conversely, B cells from normal mice proliferated in response to LPS-mitogenic stimulus (P<0.01), and B cells from T. cruzi-infected mice failed to do this as a consequence of their suppressed immune status. These results were further confirmed by cell-cycle analysis with PI staining (data not shown).

Altogether, these data indicate that IL-4 is able to revert the apoptotic process on B cells from normal and infected mice without inducing proliferation.

IL-4 does not induce an increment of Ig secretion
As IL-4 favors B cell survival, we thought it would rebound in an increase in the secretion of Igs, as there are higher numbers of competent B cells to differentiate to plasma cells. Surprisingly, the levels of IgM (Fig. 2A , left panel) or any of the IgG isotypes (Fig. 2A , right panel) were not significantly increased in the supernatants of normal or infected mice B cells cultured during 96 h in the presence of IL-4. Consistently with their in vivo activation, B cells from infected animals secrete more Igs than B cells from normal mice. However, this "spontaneous" Ab secretion of B cells from infected mice is not further raised by IL-4 in any of the isotypes tested. Therefore, we attempted to determine whether IL-4 is able to modify the reactivity of the Ab released, although it does not result in an increase in the whole Ig production. To do that, we have evaluated Ab reactivity in the supernatants of B cells from infected or normal mice cultured during 96 h with or without IL-4. No differences were found regarding Ab reactivity against a T. cruzi lysate (specific antigens) or against murine skeletal muscle or heart autoantigens (common targets of Chagas’ disease autoimmunity; data not shown).

Therefore, IL-4 does not modify the secretion of Ab (i.e., in quantity or quality), although it signals B cells for survival. The simplest explanation for this behavior is that even when IL-4 rescues B cells from apoptosis, it also blocks their differentiation to plasma cells. To address this issue, after the 96-h culture of B cells from normal or infected mice with or without IL-4, we evaluated the percentage Synd-1+ B cells, described previously as plasma cells [22 ], by FCM. As illustrated in Figure 2B (upper vs. lower panels), Synd+ cells were markedly decreased upon IL-4 treatment, in normal and infected mice B cells. Thus, by analyzing the results concerning Ab production and the percentage of Synd+ cells together, it could be concluded that IL-4 blocks the terminal differentiation of B cells to plasma cells.

IL-4 directs B cell differentiation to the memory pathway
Upon activation, mature B cells have to make the choice between the plasma cell and the memory cell pathways [23 ]. Taking into account that IL-4 blocks the differentiation to plasma cells, we next attempted to study whether IL-4 favors the differentiation to memory B cells by evaluating the phenotype of IL-4-treated B cells. Memory B cells can be distinguished from other subpopulations of B cells on the basis of their phenotype. In this sense, memory B cells have been described to express surface Ig, B7.1/CD80, B7.2/CD86, and Fas [24 ], but in contrast to their counterparts in humans, murine memory B cells express high levels of CD38 [25 26 ]. Thus, to evaluate the phenotype of IL-4-treated B cells, normal or T. cruzi-infected mice B cells were cultured with medium alone or with 25 ng/ml IL-4 during 48 h. After that, B cells were triple-stained with FITC-labeled anti-mouse CD38, biotin-labeled anti-mouse B7.2, and PtdEtn-labeled anti-mouse Synd-1 and were analyzed by FCM. In accordance with the results obtained after 96-h culture, the 48-h IL-4 treatment was sufficient to avoid B cell differentiation to plasma cells as illustrated by the reduction in the percentage of Synd+ cells (table in Fig. 3 , left column). Furthermore, a region enclosing a CD38hiB7.2+ (memory cells) subpopulation was defined in CD38 versus B7.2 density plots (Fig. 3) . As shown in the table of Figure 3B cells from infected mice exhibited a higher percentage of cells with memory phenotype in comparison with B cells from normal mice, in agreement with their high degree of activation. It is interesting that after IL-4 treatment, the subpopulation of B cells with "memory phenotype" was significantly increased, in normal (0.2% in unstimulated vs. 5.1% in IL-4-treated B cells) or infected (2.2% vs. 12.9%) mice B cells.

FasL is down-regulated by IL-4 in B cells from infected mice
We have recently demonstrated that B cells from T. cruzi-infected mice express Fas and FasL and commit fratricide mediated by this death pathway [7 ]. Considering this and the fact that IL-4 has been shown to induce Fas resistance in B cells [15 ], we have attempted to evaluate whether the down-regulation of Fas or FasL is one of the mechanisms by which IL-4 rescues infected mice B cells from apoptosis. To test that, Fas and FasL expressions were assessed by FCM in freshly explanted B cells and after an 18-h culture with or without IL-4. As expected, unstimulated, cultured B cells from normal mice expressed background levels of Fas (Fig. 4B , left histogram) and FasL (Fig. 4A , lower left histogram), whereas unstimulated B cells from infected mice expressed Fas (Fig. 4B , right histogram) and FasL (Fig. 4A , lower right histogram) proteins. IL-4 treatment was unable to modify Fas expression on B cells from infected or normal mice (Fig. 4B) . In contrast, as shown in Figure 4A (lower panels), IL-4 induced a marked down-regulation (more than 50%) in FasL expression on B cells. This effect is well-appreciated in B cells from infected mice (12.4% in IL-4-treated vs. 25.8% in unstimulated) but at a lesser extent in B cells from normal mice as a result of their lower degree of FasL expression (3.7% vs. 7.8%). Thus, by comparing the percentages of FasL+ B cells when freshly explanted (Fig. 4A , upper panels) and after culture (Fig. 4A , lower panels), it could be concluded that the percentage of FasL-expressing B cells remained essentially unchanged after an 18-h culture with medium alone, but it is significantly decreased upon IL-4 treatment.

IL-4 induces up-regulation of the "FasL gene repressor" CIITA
CIITA expression appears to be a nearly absolute requisite for expression of MHC-II whether constitutive or inducible [27 28 ]. Recent reports have shown that CIITA interacts with DNA-binding proteins to selectively bind the MHC-II promoters and induce the expression of MHC-II [29 ] but simultaneously sequesters these DNA-binding proteins from their other gene targets [30 31 ]. Accordingly, it has been demonstrated that CIITA can down-regulate nuclear factor of activated T cells (NFAT)-mediated activation of the FasL gene in T cells [32 ].

To analyze whether IL-4 induced the expression of CIITA in our system, we first determined the expression of MHC-II in B cells after an 18-h culture with IL-4. By FCM analysis, we observed that consistently with their in vivo activation, B cells from infected animals presented a higher mean of fluorescence compared with B cells from normal mice (2576 vs. 2440), indicating a higher level of MHC-II expression per cell (Fig. 5A ). Furthermore, after IL-4 treatment, we observed an increment in the mean of fluorescence of B cells from normal (from 2440 to 2766) and infected mice (from 2576 to 2810), indicating that IL-4 is able to enhance MHC-II expression in B cells.

To directly confirm the increment of CIITA expression mediated by IL-4, an immunoprecipitation technique was performed with cellular lysates of infected mice B cells cultured with or without IL-4 for 18 h. As shown in Figure 5B , CIITA expression is markedly up-regulated in the IL-4-stimulated B cells from infected mice in comparison with the nonstimulated ones.

In conclusion, IL-4 is able to up-regulate CIITA expression in B cells from T. cruzi-infected mice, and this up-regulation would rebound not only in MHC-II gene transcription induction but also in FasL-promoter negative regulation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously reported [7 ] that B cells from T. cruzi-infected mice undergo apoptosis in vivo during the infection. Moreover, we have described that this B cell-sufficient apoptosis (or fratricide) is mediated by the Fas/FasL pathway and selectively eliminates IgG+-activated B cells reactive against parasite antigens. This Fas-mediated elimination of parasite-specific B cells during the acute infection added to the death of effector T lymphocytes [33 ] seems to be responsible for the immunosuppression observed. Furthermore, this extensive death of lymphocytes would limit the ability of the host to achieve complete elimination of the parasite and favor the chronicity of the infection [7 34 ]. In this regard, studying signals that could avoid or delay B cells apoptosis during the acute phase of T. cruzi infection would provide new insights to improve the protective, humoral response.

In the present study, we have demonstrated that IL-4, a stimulus that signals B cells differently from LPS, is able to protect the activated B cells from infected mice and the resting B cells from normal mice from spontaneous apoptosis. In addition, after an 18-h IL-4 treatment, we detected at the same time apoptosis inhibition and a down-regulation in the expression of the proapoptotic protein FasL. However, in our system, IL-4 treatment during 18 h does not modify the expression of the antiapoptotic proteins Bcl-xL or Bcl-2 (unpublished), even when by this time, IL-4 was clearly able to rescue B cells from apoptosis. Recently, it has been reported [35 ] that IL-4 is able to up-regulate Bcl-xL determined at the mRNA level by reverse transcriptase-polymerase chain reaction after 7 h of culture and also at protein level by Western blot but only after 48 h of culture. Therefore, although this protein does not seem to be involved in the early antiapoptotic role of IL-4 described, we do not exclude that IL-4 can up-regulate Bcl-xL expression in our system as well.

FasL down-regulation has been recently reported to be a common mechanism used by some T cell activation-induced cell death (AICD) inhibitors (i.e., transforming growth factor-ß1, cyclosporine A, retinoic acid, and glucocorticoids) to protect T cells from death [36 37 38 ]. Moreover, vasoactive intestinal peptide and pituitary adenilate cyclase-activating polypeptide have also been shown to constrain the AICD of the CD4+ T cell stimulated with anti-CD3 plus IL-2 by inhibiting the expression and/or DNA-binding activity of several transcriptional factors involved in FasL regulation, such as c-myc, nuclear factor (NF)-{kappa}B, NF-ATp, and early growth factors 2/3 [39 ]. In this study, we have demonstrated that this mechanism of FasL-negative regulation is also working in a B cell system in which the fratricide mediated by the Fas/FasL pathway is an ongoing process. In addition, this negative modulation occurs when B cells are stimulated with IL-4, a cytokine widely described to enhance humoral-immune response and to protect B cells from cell death [14 15 16 17 18 19 20 ]. Regarding the intracellular mechanisms underlying the FasL-negative regulation, it has been recently reported [40 ] that CIITA-deficient mice have aberrant expression of FasL in T and B cells. Moreover, B cells from these mice show higher levels of FasL upon activation and kill the Fas-bearing target cells more efficiently. The authors suggest that the modulation of FasL expression is at least partially controlled by CIITA. Sustaining this, we have demonstrated that in our system, IL-4 up-regulates CIITA and concomitantly enhances MHC-II expression and decreases FasL expression in B cells from T. cruzi-infected mice. Our data strongly suggest that CIITA increment would be responsible, at least partly, for the IL-4-induced FasL down-regulation on T. cruzi-infected mice B cells, which finally rebound in a reduced B cell fratricide. Furthermore, this mechanism may reduce the levels of parasite-specific B cell fratricide in the course of the T. cruzi infection.

A recent study has shown that IL-4 knockout (KO) mice are more resistant to the infection with T. cruzi [41 ], and this may argue against our hypothesis that IL-4 would rescue parasite-specific B cells. However, this is not so dramatic if it is considered that IL-4 is a pleiotropic cytokine [42 ] that modulates several cell populations such as T helper cell type 1 (Th1) and Th2 subsets as well as macrophage [43 ]. In this regard, it has been reported that IL-4-treated macrophages present accelerated parasite replications because of a biased metabolism to the arginase pathway [44 ]. Accordingly, it is likely that the macrophages from IL-4 KO mice exhibit a marked inhibition of the arginase pathway, which would be a critical factor for inducing resistance to infection, obscuring the effect of IL-4 on the B cell compartment.

The final goal of inhibiting B cell death should be to enhance humoral-immune response. In this work, we have demonstrated that IL-4 enhances B cell survival but also restrains the terminal differentiation to Ab-secreting plasma cells, as determined by unmodified Ab secretion (Fig. 2A) and the reduced percentage of Synd-1+ plasma cells (Fig. 2B) . Indeed, IL-4 favors the differentiation to memory B cells, as determined by the increase in the percentage of CD38hiB7.2+ B cells (Fig. 3) . This result, interesting in the context of an infection, is in line with two reports that have demonstrated that IL-4 promotes the differentiation of LPS-activated normal B cells to the memory pool [45 46 ]. Consequently, IL-4 would not appear to be important for enhancing the "short-term" effector humoral-immune response, but instead, it would favor the differentiation to memory B cells, allowing the renewing and maintenance of this B cell pool. In this regard, the newest vaccination strategies to fight against chronic diseases are focused at inducing and maintaining memory B and T cells to provide larger numbers of effector lymphocytes upon subsequent boosting or infection, which have been shown to gradually decrease the number of memory cells [47 ].

In conclusion, this work provides evidence about the down-regulation of FasL expression induced by IL-4 in B cells activated during the course of a natural infection. Concomitantly to FasL down-modulation, we have detected a marked up-regulation of CIITA that could mediate the negative regulation observed. Additionally, our report further characterizes the importance of IL-4 in modulating B lymphocyte differentiation. Therefore, these contributions could be relevant to many infectious diseases.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the "Consejo Nacional de Investigaciones Científicas y Técnicas" (CONICET), Fundación Antorchas, "Agencia Córdoba Ciencia", and "Agencia Nacional de Promoción Científica y Técnica (FONCYT)" given to A. G. E. Z. and E. V. A. R. thank CONICET for the fellowships granted. A. G. and C. L. M. are members of the Scientific Career of CONICET. We thank Dr. Claudia C. Motran for her continuous support.

Received July 9, 2002; accepted October 22, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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