Skip Navigation


International Immunology Advance Access originally published online on October 27, 2007
International Immunology 2007 19(12):1395-1402; doi:10.1093/intimm/dxm107
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
19/12/1395    most recent
dxm107v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Pellegrini, A.
Right arrow Articles by Gea, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pellegrini, A.
Right arrow Articles by Gea, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Spleen B cells from BALB/c are more prone to activation than spleen B cells from C57BL/6 mice during a secondary immune response to cruzipain

Andrea Pellegrini1, Natalia Guiñazú1, Maria Pilar Aoki1, Isabel Chico Calero2, Eugenio Antonio Carrera-Silva1, Nuria Girones2, Manuel Fresno2 and Susana Gea1

1 Inmunología, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, CIBICI-CONICET, Universidad Nacional de Córdoba, Córdoba, Argentina
2 Centro de Biología Molecular Severo Ochoa, CSIC-Universidad Autónoma de Madrid, España

Correspondence to: S. Gea; E-mail: sgea{at}mail.fcq.unc.edu.ar


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
There is an increasing interest in the study of roles that B cells may play in regulating immune responses both in protection and in pathogenesis. However, little is known about additional immune functions of B cells independently of antibody production. In this study, we have assessed how the immunization with T-dependent antigens in different host genetic backgrounds affects several parameters of B cells during secondary immune responses. We have previously reported that BALB/c immunized with cruzipain, induced heart autoimmunity, whereas C57BL/6 mice were resistant. In a comparative study employing the same experimental model, we demonstrated that BALB/c-enriched spleen B cells presented higher ability to proliferate releasing elevated levels of IL-4. Moreover, spleen of immune BALB/c mice presented an increased number of germinal center and plasma cells as well as higher expression of B-cell activation markers (MHC class II, CD40, CD86). These findings demonstrate the influence of genetic background on B-cell activation and emphasize the importance of examining B-cell behavior in the context of the specific immunogens.

Keywords: animal models, B cells, cytokines


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
B cells are typically characterized by their ability to produce antibodies. In addition, B cells possess regulatory functions that may play protective or pathogenic roles. The regulatory functions may be carried out either through antibodies themselves or by cellular interactions and cytokines. B cells also may act as antigen-presenting cell (APC) (1).

Many of the critical events leading to B-cell activation and differentiation have been proposed to occur in germinal center (GC), among them somatic mutation, production of plasma cell precursors and generation of memory B cells (2). Perturbation of B-cell maturation may lead to generation, activation and clonal expansion of B cells that secrete pathogenic antibodies. An overt activity of GC has been reported in patients with active lupus (3). Moreover, reciprocal interactions between B and T cells are initiated by antigen capture through the B cell receptor (BCR), followed by antigen processing and presentation of peptide–MHC class II (MHC-II) complexes, along with costimulatory signals to antigen-specific T cells (4, 5). CD40 interaction on B cells with soluble or cell-bound CD40L or expressed by activated T cells is essential for B survival and proliferation, GC and memory B-cell formation, and upregulation of surface molecules among others (6).

The best-characterized costimulatory molecules belong to the family of B7 proteins (B7-1/CD80 and B7-2/CD86) which are ligands for the T-cell membrane proteins CD28 and CTLA4 (7). The relative expression of CD80 and CD86 on APC may have different functional consequences, such as driving T-cell differentiation into either the Th1 or the Th2 pathway, respectively (5, 8, 9).

Hyperexpression of MHC-II molecules and induction of CD86 on B cells are a consequence of ligation of the BCR (4). To date, few studies have been addressed to investigate the costimulatory and MCH-II molecules in the secondary immune response to T-dependent antigen.

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease which is highly prevalent in Latin America (10). Autoimmunity is one of the mechanisms proposed to explain the pathogenesis of disease. Although it is supported by several experimental evidences it is still controversial (11–13). Cruzipain (Cz) is a major cysteine protease expressed in all developmental forms of the parasite (14, 15). This glycoprotein is highly immunogenic in humans (16) and murine infections as well as in experimental models (17). The immunization with Cz generated heart autoimmune response and damage only in BALB/c but not in C57BL/6 (B6) mice (18, 19). However, the study of the B-cell compartment during the secondary immune responses to Cz in these mouse strains has not been explored.

In a comparative study employing this experimental model, we demonstrated that only BALB/c-enriched spleen B cells presented higher ability to proliferate in vitro to several stimuli and released elevated levels of IL-4. An increased number of GC B cells and plasma cells as well as a higher expression of activation markers (MHC-II, CD40, CD86) on B cells was detected in BALB/c.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Cz purification
Epimastigote forms of T. cruzi Tulahuen strain were grown at 28°C in brain heart infusion (Britania, Bs As, Argentina) supplemented with 0.5% tryptose, 10% FSC, 200 mg ml–1 hemin, 100 U ml–1 penicillin and 100 mg ml–1 streptomycin. Parasites were harvested during the exponential growth phase centrifuged at 5000 x g for 10 min at 4°C and washed with PBS. The parasites were resuspended in three volumes of 0.25 M sucrose, 5 mM KCl and 1 mM each of the protease inhibitors, TLCK and phenylmethylsulphonylfluoride (Sigma Chemical Co., St Louis, MO). Epimastigotes were disrupted by three cycles of freezing (–20°C) and thawing (4°C). The homogenates were centrifuged at 7000 x g for 15 min at 4°C. Saturated ammonium sulfate solution, adjusted to pH 7 with NH4OH, was added to the supernatant while stirring in an ice bath to 50% saturation. The precipitate obtained after centrifugation of this suspension was carefully dissolved and dialyzed in 50 mM Tris-ClH, 150 mM NaCl, pH 7.4. CaCl2, MgCl2 and MnCl2 were added to a final concentration of 5 mM each. Subsequently, the samples were submitted to affinity chromatography and the absence of enzyme activity was checked by 10% SDS-PAGE containing 0.1% gelatin as a substrate. Purity was tested as previously described Giordanengo et al. (20).

Mice immunization
Inbred female BALB/c and B6 mice, aged 6–8 weeks, were purchased from Faculty of Veterinary Sciences (National University of La Plata, Bs As, Argentina) and 129/Sv/Ev mice WT, 129/Sv/Ev IL-4 KO (21) or 129/Sv/Ev IFN-{gamma}R KO (22) mice were kindly provided by Dr Michel Aguet. Mice were immunized by intradermal injection with inactivated Cz or OVA (Sigma Chemical Co.) emulsified in CFA (Sigma Chemical Co.) at the base of the tail and along the back. Immune mice were injected with 10 µg of Cz or OVA in 0.1 ml of PBS mixed with 0.1 ml of CFA, on days 0, 14 and 28. The studies were performed 14 days after the third immunization. Mice were maintained according to National Research Council's guide for the care and use of laboratory animals.

Spleen cell preparation
Spleens from BALB/c, B6, 129/Sv/Ev WT, IL-4 KO and IFN-{gamma}R KO mice were removed and cell suspensions were prepared by homogenization in a tissue grinder. Erythrocytes were lysed by incubation in lysing buffer (Sigma Chemical Co.). Spleen cells were resuspended in complete RPMI-1640 medium (Sigma Chemical Co.) containing 10% FSC, 50 µM ß-mercaptoethanol and 40 µg ml–1 gentamycin.

B-cell-enriched suspensions were obtained using the following procedure. Briefly, macrophages from spleen cells were depleted by selective adherence to glass Petri dishes for 2 h at 37°C. Non-adherent cell suspensions were depleted of T cells by magnetic cell sorting using anti-Thy-1.2-coated magnetic beads (Dynal Biotech, Compiègne, France), as indicated by the manufacturer's instructions. This procedure yielded an enriched B-cell population >86% CD19+ cells with <1% CD3+ cells, <6% CD11c+ cells and <5% F4/80+ cells, as determined by flow cytometry analysis and >95% of viable cells, as determined by trypan blue exclusion (data not shown).

B-cell phenotypic analysis by flow cytometry
Briefly, 1 x 106 red-cell-depleted splenocytes were incubated with anti-mouse CD32/CD16 antibody (control antibody) in order to block non-specific Ig trapping through Fc receptors. The following PE-conjugated anti-mouse mAbs were used for analysis: anti-CD19, anti-IgM and anti-CD138 (BD PharMingen, San Diego, CA). FITC-conjugated anti-mouse mAbs: anti-CD3, anti-IgD, anti-CD40 and anti-I-Ad/I-Ed (BD PharMingen). Biotin-conjugated anti-mouse mAbs: anti-CD23, anti-CD5, anti-peanut agglutinin (PNA), anti-CD80 and anti-CD86. All conjugated antibodies were purchased by eBioscience (San Diego, CA) unless otherwise indicated. Steptavidin–FITC (eBioscience) was used to detect biotinylated antibodies. Data from stained samples were acquired using Orthodiagnostics, Raritan, NJ, flow cytometry. A total of 20 000 events were analyzed using WinMDI 2.8 software (J. Trotter, Scripps Research Institute, La Jolla, CA).

Lymphoproliferation assay
Proliferation assays were set up in triplicate, in 96-well flat-bottomed plates (Nunc, Naperville, IL). Each culture consisted of 2 x 105 purified B cells in a final volume of 200 µl. LPS (20 µg ml–1), IL-4 (100 U ml–1) plus anti-CD40 (5 µg ml–1), Cz (5 µg ml–1) and synthetic peptide (10 µg ml–1) derived from Cz: P2: aa 291–302 (YNDSAAVPYWII) (GenBank: M84342 [GenBank] ) were used. A non-related peptide-NRP-(SDDDMGFGLFDD) was used as control. After incubation at 37°C in 5% CO2 for 48 h, the proliferative response was monitored by adding 1 µCi [3H] thymidine. Cultures were stopped 16 h later by automated harvesting and processed for measurement of incorporated radiolabelling using a liquid scintillation counter.

Cytokine assays
Enriched B cells (2 x 106/ml) were cultured separately in presence of 5 µg ml–1 Cz or medium alone in 24-well plates (Nunc). Culture supernatants were collected after 48 h and IL-4, IL-6, IL-10, IFN-{gamma} and TNF{alpha} were assayed by capture ELISA, using mAb pairs purchased from BD PharMingen and eBioscience. Briefly, ELISA plates (Corning) were coated with anti-cytokine antibody overnight at 4°C. Plates were washed and blocked with 10% FSC for 2 h at room temperature. Supernatants (100 µl) from different groups were added to the plates and incubated overnight at 4°C. Plates were washed and incubated with biotinylated anti-cytokine antibody for 1 h at room temperature. After washing, streptavidin–peroxidase was added to the wells and incubated for additional 30 min. Plates were washed, developed using O-phenylendiamine and H2O2 substrate; and read at 490 nm in an ELISA plate reader (BioRad). Standard curves were generated using recombinant cytokines (BD PharMingen).

Statistical analysis
The Student's t-test for unpaired values were used to determine the levels of significance in flow cytometric, proliferation and ELISA assays; P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Characterization of spleen B-cell subpopulations in immune BALB/c and B6 mice
First, we analyzed the presence of CD19+ cells in spleens from Cz immune and non-immune mice. The B-cell population represented ~55% of total cells in both non-immune mouse strains. Fourteen days after the Cz immunization, the percentages of B lymphocytes diminished compared with non-immune groups and the values were similar between BALB/c and B6 mice. However, B-cell absolute numbers were significantly higher in immune BALB/c respect to B6 group (Fig. 1A).


Figure 1
View larger version (47K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Percentages and absolute numbers of spleen B-cell populations from BALB/c and B6 mice. Spleen suspensions from non-immune and immune mice were stained with combinations of mAb fluorescent conjugated and analyzed by flow cytometry. Data analyzed were obtained from live lymphocyte gate. (A) Total percentages and absolute numbers of CD19+ cells from non-immune and Cz immune mice from both BALB/c (white bars) and B6 (black bars) mouse strains. (B) Splenocytes were stained for IgM and IgD surface markers. Mature (IgMint/low IgDhigh), T1 (IgMbright IgDneg) and T2 (IgMbright IgDbright) cell gates were drawn according to previous report (23). (C) Percentages of mature, immature T1 and T2 B cells from OVA or Cz immune BALB/c (white bars) and B6 (black bars) mice. Data are representative of three separate experiments consisting of at least five mice per group. BALB/c versus B6; *P < 0.05.

 
With the aim to explore the phenotypic changes in spleen B cells induced by Cz immunization, we analyzed the following surface markers: IgMint/low IgDhigh CD23pos CD21pos—mature naive—, IgMbright IgDneg CD23neg CD21neg—immature transitional 1 (T1)—, IgMbright IgDbright CD23pos CD21bright—immature transitional 2 (T2)— and IgMbright IgDneg CD23neg CD21pos—marginal zone (MZ)—B cells (23). We also studied the spleen B-cell phenotypes of mice immunized with a non-related protein such as OVA. Flow cytometric analysis revealed that percentages of mature B cells were higher in BALB/c immunized with Cz or OVA compared with B6. Conversely, the percentages of IgMbright IgDneg (T1 and MZ) and IgMbright IgDbright (T2) B cells were significantly higher in B6 mice independently of antigen used for the immunization (Fig. 1B and C). We further characterized the percentages of mature and immature B-cell subsets in non-immune mice from both strains. Mature and IgMbright IgDbright (T2) cells showed a similar distribution, whereas IgMbright IgDneg (T1 and MZ) B cells were 2-fold higher in B6 than BALB/c mice. These results were similar when we assayed the combination IgM and CD23 markers (data not shown).

Even though, B1 lymphocytes are a distinct subset of B cells, predominant in peritoneal and pleural cavities, we decided to study it in spleen of immune mice analyzing double-positive CD5+CD19+ expressions. We found that independently of the antigen used for immunization, both mouse strains exhibited no significant differences in percentages of B1 cells (data not shown).

Antigen immunization increased GC and plasma B-cell numbers in BALB/c mice
Next, we analyzed the possibility that others B-cell subsets were differentially induced by antigen immunization in both mouse strains. GC B cells can be phenotypically identified by expression of PNA-binding sites (2). Thus, we investigated GC B-cell population by double stain flow cytometry B220+PNA+. As shown in (Fig. 2A), Cz or OVA induced in BALB/c a higher percentage of GC B cells than B6 mice. GC B-cell subset was confirmed analyzing the CD38 expression on mature non-naive B cells defined as CD19pos IgMneg (24) (data not shown).


Figure 2
View larger version (9K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Splenic GC and plasma B cells from BALB/c and B6 mice. Spleen suspensions from immune BALB/c (white bars) and B6 (black bars) mice were analyzed by flow cytometry. (A) Percentages of PNA+ B cells. B-cell populations were gated on using B220 antibody conjugated to PE and GC B cells were detected using biotinylated PNA plus streptavidin–FITC. (B) Percentages of splenic plasma B cells (CD138pos). Data are representative of three separate experiments consisting of at least five mice per group. BALB/c versus B6; **P < 0.001, *P < 0.05.

 
The analysis of plasma cells studying syndecan-1 marker (CD138) (25), showed that the percentages of these cells were significantly higher in BALB/c compared with B6 mice independently of the antigen used in the immunization (Fig. 2B).

B cells from Cz immune BALB/c showed higher proliferative response than B6 mice
To determine whether spleen B cells from Cz immune BALB/c and B6 mice differed in their proliferative ability, we measured B-cell response to different stimuli (LPS, rIL-4 plus anti-CD40) by [3H] thymidine incorporation. Of note, these cultures were essentially free of contaminating T cells (<1%). We found that B cells from both mouse strains displayed strong proliferative response to these stimuli. However, cells from BALB/c mice showed ~2-fold higher stimulation index compared with B6 mice (Fig. 3A). We next determined whether Cz or a derived peptide differentially affected the proliferative response of enriched B-cell suspensions. A strong proliferative response in cultures from both strains was observed, although the proliferation indexes were ~2-fold higher in BALB/c compared with B6 mice (Fig. 3B). In addition, when BALB/c and B6 cells were assayed with NRP as control, no proliferative responses were found.


Figure 3
View larger version (5K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Proliferative response of spleen B cells from Cz immune BALB/c and B6 mice. Cells from BALB/c (white bars) and B6 (black bars) mice were culture in presence of (A) LPS (20 µg ml–1), anti-CD40 (5 µg ml–1) plus rIL-4 (100 U ml–1) and (B) Cz (5 µg ml–1), P2 (10 µg ml–1), NRP (10 µg ml–1). The results obtained from triplicate assays were express as stimulation indexes (ratio between mean cpm from stimulated cultures and mean cpm from unstimulated cultures). Values are representative of at least three independent experiments. BALB/c versus B6; *P < 0.05.

 
Differential production of cytokines from enriched B cells in response to Cz
In order to investigate whether Cz immunization was capable to induce different B-cell cytokine profile in BALB/c and B6 mice, we assayed IL-4, IL-6, IL-10, TNF{alpha} and IFN-{gamma} cytokines by ELISA. In supernatants of enriched B cells stimulated with Cz or unstimulated high levels of IL-4 were detected in BALB/c, while this cytokine was undetectable in similarly stimulated B6 mice (Fig. 4).


Figure 4
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Production of IL-4, IL-6 and IL-10 by enriched spleen B cells from Cz immune BALB/c and B6 mice. (A) Cytokines were determined by ELISA in culture supernatants from enriched spleen B cells from immunized BALB/c (white bars) and B6 (black bars) mice. Supernatants were obtained after 48 h stimulation with 5 µg ml–1 Cz or medium alone. Results are representative of three independent experiments. *P < 0.05, ** P < 0.002 compared with the group indicated, ND (not detectable). (B) Side scatter versus forward scatter plot of enriched B-cell suspensions. Histograms show percentages of positively staining cells for CD19 and CD3 markers (filled gray histograms) and isotype controls (black lines). Data are representative of two independent experiments.

 
Cz stimulation increased IL-6 and IL-10 levels in both mouse strains, although only the IL-6 was significantly higher in BALB/c compared with B6 mice (Fig. 4). Enriched B cells stimulated with Cz or maintained with medium alone produced no measurable TNF{alpha} or IFN-{gamma} (data not shown).

B-cell activation molecules are differentially modulated in immune BALB/c and B6 mice
In attempt to analyze whether activation molecules are differentially modulated on B cells from immune mice, we studied CD40, CD80 and CD86 cell-surface expression. For this, total spleen cells from BALB/c and B6 mice were specifically restimulated with OVA or Cz during 24 h, and the percentages of double-positive cells were evaluated by flow cytometry. In BALB/c mice, the CD40 MFI was significantly higher on B lymphocytes from mice immunized with Cz compared with OVA. In B6 mice, CD40 MFI was lower than that in BALB/c either after specific antigen stimulation (Cz or OVA) or without stimulation (Fig. 5A). We also observed that the percentage of CD40+ B cells were significantly higher in Cz respect to OVA immune BALB/c mice, whereas B6 mice showed no modification (data not shown).


Figure 5
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Activation marker analysis on B cells from BALB/c and B6 mice. Spleen cells were maintained in medium alone or stimulated with Cz for 24 h. B-cell population were gated on using CD19 antibody conjugated to PE and CD40, MHC-II, CD86, CD80 expression were determined using antibodies conjugated to FITC or biotinylated reagents plus streptavidin–FITC. The data shown represent MFI of (A) CD40, (B) MHC-II, (C) CD86 and (D) CD80 in gated CD19+ cells. Spleen B cells from OVA immune (white bars) and Cz immune (black bars) mice are shown. Data are the mean ± SD of three parallel experiments, *P < 0.001 compared with the group indicated.

 
Besides, MHC-II MFI was higher on B cells from BALB/c compared with B6 mice independently of the antigen used in the immunization and there was no difference between specific antigen stimulation or not (Fig. 5B). In addition, the percentages of B cells that express MHC-II were significantly higher in Cz respect to OVA immune BALB/c, whereas B6 mice showed no modifications (data not shown).

CD86 costimulatory molecule was significantly upregulated on B cells upon stimulation with Cz while this effect was not observed with OVA stimulation in immune BALB/c mice. In the same way, B cells from B6 strain showed a significant increase of CD86 MFI only after Cz stimulation (Fig. 5C). We next analyzed the expression of CD80 costimulatory molecule and no significant differences in CD80 MFI were observed between Cz or OVA immune BALB/c or B6 mice (Fig. 5D).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
To date, few studies have addressed the mechanisms that could contribute to maintain or regulate secondary immune responses independently of antibody production. B cells can act as potent APC and also influence immune response by producing cytokines (26). However, most of the studies have been performed with naive B lymphocytes, B cells in primary immune response and in vitro through the exogenous addition of cytokines and/or anti-cytokines antibody (27–29). In contrast, we have characterized here the spleen B-cell populations in BALB/c and B6 mice immunized with Cz, a T. cruzi antigen, and OVA as control antigen in T-dependent secondary responses.

We found that the spleen B-cell absolute numbers were significantly higher in Cz immune BALB/c compared with B6 group. These data were compatible with the transient esplenomegaly in Cz immune BALB/c mice (18). In contrast, when using OVA as immunogen we did not detect changes in CD19+ cell number between both mouse strains (18, 19). We also found that independently of the antigen nature employed for the immunization, the mature B-cell percentages were higher in BALB/c than B6 strain. In contrast, the percentages of IgMbright IgDneg (T1 and MZ) and IgMbright IgDbright (T2) B cells were higher in B6 than BALB/c mice. Consequently, the B-cell subsets distribution is likely dependent on the mouse strain. Thus, non-immune mice were studied and we did not find differences in the mature and T2 B-cell percentages between the strains. However, T1 and MZ B-cell percentages were 2-fold higher for B6. In agreement with our results, Won et al. (30) reported that normal B6 have double percentage of MZ B cells than BALB/c mice. Together, these results suggest that naive mature and T2 B cells were the subpopulations mainly affected after of several immunizations.

Regarding GC B cells, our findings indicate that independently of antigen used for immunization, the percentages of these cells were higher in BALB/c. It is important to note, that many of the critical events leading to B-cell activation and differentiation have been proposed to occur in GC (31, 32) and exacerbated GC responses could lead to the deregulation in the development of plasma cells (33, 34). In fact, these latter cells were significantly increased in BALB/c respect to B6 mice independently of antigen used. This clearly indicates that the murine genetic background is an important factor involved in determining B-cell profile after immunization. A relevant issue is the potential impact of differences in the B-cell repertoires between the two mouse strains. In fact, the pattern of VH gene family expression in the mouse primary B-cell repertoire is strain dependent. The mouse strains B6 and BALB/c carry different IgH haplotypes. In addition, VH gene family usage is also influenced by genetic factors unlinked to the IgH locus (35).

The higher stimulation indexes to polyclonal activators and specific stimuli of enriched spleen B cells from Cz immune BALB/c compared with B6 mice suggests that B cells from Cz immune BALB/c mice are more prone to proliferate, indicating that differences in genetic background could influence B-cell response. The B-cell proliferative response to P2 peptide could due to a slightly APCs contamination in the enriched B-cell suspensions which could be adsorbing and presenting peptides in polyvalent arrays (36). In contrast with our results, Hussain et al. (37) reported that purified B cells from non-immune BALB/c and B6 elicited similar proliferative response after anti-IgM stimulation. It is note that anti-IgM is different to specific antigen stimulation in B-cell proliferation. Regarding the mechanism, we cannot rule out the possibility that the different proliferative responses observed would be a consequence of differences in B-cell division and/or cell death. This important issue is in progress in our laboratory.

The ability of different T-cell cytokines to influence B-cell differentiation is well recognized (38). However, activated B cells themselves can produce significant quantities of cytokines that may contribute to environment (28). An aspect poorly explored is the B-cell cytokine production in a secondary immune response. Interestingly, we demonstrated that IL-4 was synthesized only by BALB/c enriched B-cell suspensions ex vivo. It has been reported that IL-4 production is an intrinsic feature of GC B cells (32). Though this topic was not studied by us, GC B-cell subpopulation was greater expanded in immune BALB/c. Thus, we postulated that the type-2 polarizing cytokine environment during Cz immune response (18) supports the B lymphocyte differentiation into IL-4 secreting cells only in BALB/c. In agreement with our results, IL-4 producing B cells have been found following infection with pathogens that preferentially induce a type-2 immune response (28, 29).

Even though the study of the activation markers on B lymphocytes revealed that the in vitro-specific antigen stimulation did not modify the CD40 expression in both mouse strains, the CD40 MFI was higher on B cells from BALB/c immunized with Cz than OVA; respect to B6 mice. In accord with our results, it has been reported that IL-4 is able to induce an increase of CD40 expression on human B cells (39). It is important to note that CD40 levels on B cells from non-immune mice were also higher in BALB/c than in B6 (data not shown). Therefore, this suggests that antigen nature and murine background are related with CD40 expression on B cells. Taking into account that CD40 is a potential growth receptor in B-cell malignancies (40), we postulated that augmented CD40 expression could be implicated in the increased number of GC and plasma cells observed in Cz immune BALB/c mice.

In B cells, MHC-II synthesis is dramatically increased after encounter with antigen, by T-cell-derived signals and by microbial products (4, 41). Even though, specific antigen stimulation did not modify the MHC-II expression, we demonstrated that MHC-II expression on B cells ex vivo was related with murine genetic background.

CD80 and CD86 molecules are differentially expressed on populations of APC and regulated by cytokines and others factors (8, 42). The requirement of these molecules in the context of the specific immunogens has been scarcely explored (7). Cz but not OVA induced an upregulation of CD86 but not CD80 in both mouse strains. In this regard, it has been suggested that CD86 costimulation is capable to activate Th2 cytokine production, while CD80 costimulation seems to be less efficient (5, 9). The dominant role of CD86 costimulation to Th2 cells was supported by studies demonstrating that the specific IL-4 production was inhibited by mouse anti-CD86 treatment (8). The different activation pattern of B cells from BALB/c and B6 mice is likely an effect of the difference in the Th subsets induced by the antigen. Thus, Cz induced a Th2 response in BALB/c mice (18), whereas OVA induced a Th1 immune response in both mouse strains (43, 44).

Even though, there are some papers studying the IL-4 or IFN-{gamma} direct effect on the B-cell activation state (45–47), studies of the modulation of the B-cell activation markers in secondary response to T-dependent antigen are to our knowledge scare. In addition, studies with IL-4 KO or IFN-{gamma}R KO Sv129 mice demonstrated the relevance of the polarizing cytokine signals in the regulation of activation markers on B cells during Cz secondary immune response (data not shown).

In summary, in the present study we reported marked differences in B-cell activation during the secondary immune response related to murine genetic background and antigen nature. We demonstrated that an immunodominant antigen of T. cruzi generates a higher spleen B-cell activation state in BALB/c, a strain susceptible of developing autoimmune response favoring the autoantibody production, whereas it does not occur in B6, a resistant strain.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT), Consejo Superior de Investigaciones Científicas (CSIC-Spain), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and Secretaría de Ciencia y Tecnología (SECYT-UNC).


   Acknowledgements
 
S.G. and P.A. are Research Career Investigators from CONICET. A.P., N.G. and E.A.C.-S. thank CONICET for the fellowships granted.


   Abbreviations
 
APC, antigen-presenting cell
BCR, B cell receptor
Cz, cruzipain
GC, germinal center
MHC-II, MHC class II
MZ, marginal zone
PNA, peanut agglutinin
T1, immature transitional 1 B cells
T2, immature transitional 2 B cells

   Notes
 
Transmitting editor: S. Izui

Received 11 April 2007, accepted 1 October 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

  1. Mizoguchi A, Bhan AK. A case for regulatory B cells. J. Immunol. (2006) 176:705.[Abstract/Free Full Text]
  2. Shinall SM, Gonzalez-Fernandez M, Noelle RJ, Waldschmidt TJ. Identification of murine germinal center B cell subsets defined by the expression of surface isotypes and differentiation antigens. J. Immunol. (2000) 164:5729.[Abstract/Free Full Text]
  3. Grammer AC, Slota R, Fischer R, et al. Abnormal germinal center reactions in systemic lupus erythematosus demonstrated by blockade of CD154-CD40 interactions. J. Clin. Invest. (2003) 112:1506.[CrossRef][Web of Science][Medline]
  4. Marshall-Clarke S, Tasker L, Heaton M, Parkhouse R. A differential requirement for phosphoinositide 3-kinase reveals two pathways for inducible upregulation of major histocompatibility complex class II molecules and CD86 expression by murine B lymphocytes. Immunology (2003) 109:102.[CrossRef][Web of Science][Medline]
  5. Hussain S, Delovitch TL. Dysregulated B7-1 and B7-2 expression on nonobese diabetic mouse B cells is associated with increased T cell costimulation and the development of insulitis. J. Immunol. (2005) 174:680.[Abstract/Free Full Text]
  6. Fecteau JF, Neron S. CD40 stimulation of human peripheral B lymphocytes: distinct response from naive and memory cells. J. Immunol. (2003) 171:4621.[Abstract/Free Full Text]
  7. Zhang P, Martin M, Yang Q-B, Michalek SM, Katz J. Role of B7 costimulatory molecules in immune responses and T-helper cell differentiation in response to recombinant HagB from Porphyromonas gingivalis. Infect. Immun. (2004) 72:637.[Abstract/Free Full Text]
  8. Nakajima A, Watanabe N, Yoshino S, Yagita H, Okumura K, Azuma M. Requirement of CD28-CD86 co-stimulation in the interaction between antigen-primed T helper type 2 and B cells. Int. Immunol. (1997) 9:637.[Abstract/Free Full Text]
  9. Freeman G, Boussiotis V, Anumanthan A, et al. B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4. Immunity (1995) 2:523.[CrossRef][Web of Science][Medline]
  10. WHO. Control of Chagas Disease. Report of a WHO Expert Committee. WHO Tech. Rep. Ser. (2002) 905:1.
  11. Girones N, Fresno M. Etiology of Chagas disease myocarditis: autoimmunity, parasite persistence, or both? Trends Parasitol. (2003) 19:19.[CrossRef][Web of Science][Medline]
  12. Leon J, Engman D. The significance of autoimmunity in the pathogenesis of Chagas heart disease. Front. Biosci. (2003) 8:e315.[Web of Science][Medline]
  13. Kierszenbaum F. Views on the autoimmunity hypothesis for Chagas disease pathogenesis. FEMS Immunol. Med. Microbiol. (2003) 37:1.[Web of Science][Medline]
  14. Cazzulo J, Cazzulo Franke M, Martinez J, Franke de Cazzulo B. Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi. Biochim. Biophys. Acta. (1990) 1037:186.[CrossRef][Medline]
  15. Souto-Padron T, Campetella O, Cazzulo J, de Souza W. Cysteine proteinase in Trypanosoma cruzi: immunocytochemical localization and involvement in parasite-host cell interaction. J. Cell Sci. (1990) 96:485.[Abstract/Free Full Text]
  16. Arnholdt A, Piuvezam M, Russo D, et al. Analysis and partial epitope mapping of human T cell responses to Trypanosoma cruzi cysteinyl proteinase. J. Immunol. (1993) 151:3171.[Abstract]
  17. Laderach D, Cerban F, Motran C, Vottero de Cima E, Gea S. Trypanosoma cruzi: the major cysteinyl proteinase (cruzipain) is a relevant immunogen of parasite acidic antigens (FIII). Int. J. Parasitol. (1996) 26:1249.[CrossRef][Web of Science][Medline]
  18. Giordanengo L, Guinazu N, Stempin C, Fretes R, Cerban F, Gea S. Cruzipain, a major Trypanosoma cruzi antigen, conditions the host immune response in favor of parasite. Eur. J. Immunol. (2002) 32:1003.[CrossRef][Web of Science][Medline]
  19. Guinazu N, Pellegrini A, Giordanengo L, et al. Immune response to a major Trypanosoma cruzi antigen, cruzipain, is differentially modulated in C57BL/6 and BALB/c mice. Microbes Infect. (2004) 6:1250.[CrossRef][Web of Science][Medline]
  20. Giordanengo L, Maldonado C, Rivarola H, et al. Induction of antibodies reactive to cardiac myosin and development of heart alterations in cruzipain-immunized mice and their offspring. Eur. J. Immunol. (2000) 30:3181.[CrossRef][Web of Science][Medline]
  21. Kopf M, Le Gros G, Bachmann M, Lamers M, Bluethmann H, Kohler G. Disruption of the murine IL-4 gene blocks Th2 cytokine responses. Nature (1993) 362:245.[CrossRef][Medline]
  22. Huang S, Hendriks W, Althage A, et al. Immune response in mice that lack the interferon-gamma receptor. Science (1993) 259:1742.[Abstract/Free Full Text]
  23. Carsetti R, Rosado M, Wardmann H. Peripheral development of B cells in mouse and man. Immunol. Rev. (2004) 197:179.[CrossRef][Web of Science][Medline]
  24. Ridderstad A, Tarlinton DM. Kinetics of establishing the memory B cell population as revealed by CD38 expression. J. Immunol. (1998) 160:4688.[Abstract/Free Full Text]
  25. Calame K. Plasma cells: finding new light at the end of B cell development. Nat. Immunol. (2001) 2:1103.[CrossRef][Web of Science][Medline]
  26. Lund F, Garvy B, Randall T, Harris D. Regulatory roles for cytokine-producing B cells in infection and autoimmune disease. Curr. Dir. Autoimmun. (2005) 8:25.[Medline]
  27. Duddy ME, Alter A, Bar-Or A. Distinct profiles of human B cell effector cytokines: a role in immune regulation? J. Immunol. (2004) 172:3422.[Abstract/Free Full Text]
  28. Harris D, Haynes L, Sayles P, et al. Reciprocal regulation of polarized cytokine production by effector B and T cells. Nat. Immunol. (2000) 1:475.[CrossRef][Web of Science][Medline]
  29. Harris DP, Goodrich S, Mohrs K, Mohrs M, Lund FE. Cutting edge: the development of IL-4-producing B cells (B effector 2 cells) is controlled by IL-4, IL-4 receptor {alpha}, and Th2 cells. J. Immunol. (2005) 175:7103.[Abstract/Free Full Text]
  30. Won W-J, Kearney JF. CD9 is a unique marker for marginal zone B cells, B1 cells, and plasma cells in mice. J. Immunol. (2002) 168:5605.[Abstract/Free Full Text]
  31. Wolniak K, Shinall S, Waldschmidt T. The germinal center response. Crit. Rev. Immunol. (2004) 24:39.[CrossRef][Web of Science][Medline]
  32. Johansson-Lindbom B, Borrebaeck CAK. Germinal center B cells constitute a predominant physiological source of IL-4: implication for Th2 development in vivo. J. Immunol. (2002) 168:3165.[Abstract/Free Full Text]
  33. Noelle R, Erickson L. Determinations of B cell fate in immunity and autoimmunity. Curr. Dir. Autoimmun. (2005) 8:1.[Medline]
  34. William J, Euler C, Primarolo N, Shlomchik MJ. B cell tolerance checkpoints that restrict pathways of antigen-driven differentiation. J. Immunol. (2006) 176:2142.[Abstract/Free Full Text]
  35. Viale A, Coutinho A, Freitas A. Differential expression of VH gene families in peripheral B cell repertoires of newborn or adult immunoglobulin H chain congenic mice. J. Exp. Med. (1992) 175:1449.[Abstract/Free Full Text]
  36. Clark M, Massenburg D, Siemasko K, Hou P, Zhang M. B-cell antigen receptor signaling requirements for targeting antigen to the MHC class II presentation pathway. Curr. Opin. Immunol. (2004) 16:382.[CrossRef][Web of Science][Medline]
  37. Hussain S, Salojin KV, Delovitch TL. Hyperresponsiveness, resistance to B-cell receptor–dependent activation-induced cell death, and accumulation of hyperactivated B-cells in islets is associated with the onset of insulitis but not type 1 diabetes. Diabetes (2004) 53:2003.[Abstract/Free Full Text]
  38. Baumgarth N. Two-phase model of B-cell activation. Immunol. Rev. (2000) 176:171.[CrossRef][Web of Science][Medline]
  39. Valle A, Zuber C, Defrance T, Djossou O, De Rie M, Banchereau J. Activation of human B lymphocytes through CD40 and interleukin 4. Eur. J. Immunol. (1989) 19:1463.[Web of Science][Medline]
  40. Sasaki T, Hoshida Y, Xu J, et al. Prognostic significance of CD40 expression in malignant lymphoma developing in rheumatoid arthritis. J. Cancer Res. Clin. Oncol. (2005) 131:797.[CrossRef][Web of Science][Medline]
  41. Bartlett W, Noelle R. A cell surface ELISA to detect interleukin-4-induced class II MHC expression on murine B cells. J. Immunol. Methods. (1987) 105:79.[CrossRef][Web of Science][Medline]
  42. Crow M. Modification of accessory molecule signaling. Springer Semin. Immunopathol. (2006) 27:409.[CrossRef][Web of Science][Medline]
  43. Yip HC, Karulin AY, Tary-Lehmann M, et al. Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response. J. Immunol. (1999) 162:3942.[Abstract/Free Full Text]
  44. Maletto B, Ropolo A, Moron V, Pistoresi-Palencia MC. CpG-DNA stimulates cellular and humoral immunity and promotes Th1 differentiation in aged BALB/c mice. J. Leukoc. Biol. (2002) 72:447.[Abstract/Free Full Text]
  45. Lombard-Platet S, Meyer V, Ceredig R. Both IFN-gamma and IL-4 induce MHC class II expression at the surface of mouse pro-B cells. Dev. Immunol. (1997) 5:115.[Web of Science][Medline]
  46. Whitley M, Cheng H, Tomasi T, Boothby M. Distinct IL-4 response mechanisms of the MHC gene A alpha in different mouse B cell lines. Mol. Immunol. (1993) 30:821.[CrossRef][Web of Science][Medline]
  47. Roos A, Schilder-Tol E, Chand M, et al. Differential regulation of expression of the MHC class II molecules RT1.B and RT1.D on rat B lymphocytes: effects of interleukin-4, interleukin-13 and interferon-gamma. Immunology (1998) 93:33.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
19/12/1395    most recent
dxm107v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Pellegrini, A.
Right arrow Articles by Gea, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pellegrini, A.
Right arrow Articles by Gea, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?