The role of anti-HSP70 autoantibody-forming VH1–JH1 B-1 cells in Toxoplasma gondii-infected mice

Mei Chen1, Fumie Aosai1, Kazumi Norose1, Hye-Seong Mun1 and Akihiko Yano1

1 Department of Infection and Host Defense, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan

Correspondence to: A. Yano; E-mail: yano{at}faculty.chiba-u.ac.jp.
Transmitting editor: S. Koyasu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Anti-heat shock protein 70 (HSP70) autoantibody formation was induced by B-1 cells (CD5+ B cells) in Toxoplasma gondii-infected mice. Here we report that VH1–JH1 B-1 cells from peritoneal exudate cells (PEC) of T. gondii-infected C57BL/6 mice (B6, a susceptible strain) increased predominantly. Moreover, the hybridoma lines producing anti-T. gondii HSP70 (TgHSP70) antibody cross-reactive with mouse HSP70 (mHSP70) expressed the VH1–JH1 gene, whereas the hybridoma lines producing anti-TgHSP70 antibody non-cross-reactive with mHSP70 expressed the VH11A–JH1 gene or VH12–JH1 gene. The avidity maturation of anti-TgHSP70 IgG antibody in the sera of BALB/c mice (a resistant strain) and that of anti-mHSP70 IgG autoantibody in the sera of B6 mice were observed 9 weeks after T. gondii infection. T. gondii numbers in the brains of T. gondii-infected B6 mice treated with anti-mHSP70 autoantibody were markedly higher than those in the brains of T. gondii-infected B6 mice treated with anti-TgHSP70 antibody. Furthermore, B-1 cells producing IL-10 down-regulated the IFN-{gamma} expression of PEC in T. gondii-infected mice. These results indicate that B-1 cells dominantly expressing VH1–JH1 mRNA, and producing anti-HSP70 autoantibody and IL-10 regulate susceptibility of mice to T. gondii infection.

Keywords: anti-HSP70 autoantibody, avidity maturation, B-1 cell, Toxoplasma gondii, VH1–JH1


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Toxoplasma gondii, an obligate intracellular protozoan parasite, is an important cause of morbidity and mortality, especially in congenital toxoplasmosis and immunocompromised hosts. Cellular immunity is essential for protection against T. gondii infection (1). We have demonstrated the existence of CD4CD8+ and CD4+CD8 cytotoxic T lymphocytes (CTL) specific for T. gondii-infected cells in patients with toxoplasmosis (26).

The proteins of heat shock protein 70 (HSP70) families have been shown to have important functions as molecular chaperones in peptide and protein transport between cell organelles (7,8). The role of HSP in antigen presentation and processing has been demonstrated (810). We have also reported that the induction of anti-T. gondii HSP70 (TgHSP70) antibody and anti-mouse HSP70 (mHSP70) autoantibody produced in T. gondii-infected BALB/c (a resistant strain) and C57BL/6 (B6, a susceptible strain) mice (11,12), and B-1 cells were responsible for anti-mHSP70 autoantibody formation in T. gondii-infected mice (11). In fact, the mechanisms of the autoimmunity induced by parasites are not yet well defined (13).

B-1 cells are a self-renewing population of B cells that differ from conventional B cells (B-2 cells) in that they are particularly predisposed to autoantibody production (1416). Although much is known about the signaling pathways that control B-1 cell growth and development (17), less is known about why these cells are prone to produce autoreactive antibodies. In the transgenic mice carrying the Ig heavy and light chain gene encoding an autoantibody against mouse red blood cells, about one-half of the mice developed autoimmune hemolytic anemia through the activation of peritoneal B-1 cells by enteric bacteria, whereas transgenic mice bred in germ-free or specific pathogen-free conditions neither produced autoantibodies nor suffered from anemia, indicating that tolerance was maintained in the latter environmental condition, and B-1 cell activation and autoantibody production did not occur (18).

Moreover, humoral immune response is achieved by the three processes of avidity maturation, class switching and memory formation. All three occur at about the same time after B lymphocyte activation. The basis of avidity maturation is the selection, in the germinal centers (GC), of antibodies that bind the antigen better. Early in an immune response, the selection is from the primary repertoire; later, it is from mutants generated by hypermutation at the Ig loci (19). Somatic hypermutation in GC may produce not only high-avidity antibodies, but also generate autoreactive B cell clones (20).

In this study, VH gene family expression in B-1 cells from peritoneal exudate cells (PEC) of T. gondii-infected mice and the hybridoma lines producing anti-TgHSP70 antibody cross-reactive with mHSP70 were detected. Moreover, the avidity maturation of anti-TgHSP70 IgG antibody and anti-mHSP70 IgG autoantibody in the sera of T. gondii-infected BALB/c and B6 mice was observed. Finally, the biological significance of anti-mHSP70 autoantibody produced by B-1 cells in the host defense to T. gondii infection was analyzed. Collectively, the present study elucidated the role of B-1 cells in the regulation of susceptibility of mice to T. gondii infection.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice and T. gondii strain
Eight-week-old female wild-type BALB/c and wild-type B6 mice were purchased from SLC (Hamamatsu, Japan). Cysts of an avirulent Fukaya strain of T. gondii were used for infection experiments as previously described (11,12,21).

Purification of B-1 cells in PEC of T. gondii-infected mice
BALB/c and B6 mice were sacrificed 1 week after T. gondii infection. To eliminate T cells, single-cell suspensions of PEC of BALB/c and B6 mice were incubated with microbeads conjugated with anti-mouse CD90 (Thy-1.2) (30-H12; Miltenyi Biotec, Auburn, CA) and passed through a magnetic field (VarioMACS separator system; Miltenyi Biotec). Subse quently, B-1 cells were positively enriched from T cell-depleted PEC using microbeads conjugated with anti-mouse CD5 (Ly-1) (53-7.3; Miltenyi Biotec). The purity of CD5+/B220+ for B-1 cells was determined by flow cytometry analysis (FACScan; Becton Dickinson, Mountain View, CA) with R-phycoerythrin (PE)-conjugated rat anti-mouse CD5 (Ly-1) (53-7.3; PharMingen, San Diego, CA)/FITC-conjugated rat anti-mouse CD45R (B220) (RA3-6B2; PharMingen).

Analysis of anti-mHSP70 autoantibody formation by B-1 cells
BALB/c and B6 mice were sacrificed 3 days after T. gondii infection. The B-1 cells from PEC of T. gondii-infected BALB/c and B6 mice purified as described above were resuspended in RPMI 1640 culture medium supplemented with 5% FCS, 2-mercaptoethanol and antibiotics, and were cultured for 3 days at 37°C in a 96-well microplate at 105 cells/well as previously described (11). Production of anti-TgHSP70 antibody and anti-mHSP70 autoantibody in the supernatants was tested by ELISA using recombinant TgHSP70 (rTgHSP70) and rmHSP70 as antigens.

Detection of the antigen specificity of anti-mHSP70 autoantibody produced by PEC B-1 cells from T. gondii-infected and uninfected B6 mice was performed by the adsorption of culture supernatant with rTgHSP70, rmHSP70 and BSA. B6 mice were sacrificed 0 and 3 days after T. gondii infection. The B-1 cells from PEC of uninfected and T. gondii-infected B6 mice were purified and incubated as described above. Three days after incubation, the culture supernatants were incubated for 1 h on ice in a plastic plate coated with 60 µg of either rTgHSP70 or rmHSP70 and then the preadsorbed supernatants were harvested. As control, the supernatants were similarly adsorbed in a plastic plate coated with 10 mg/ml of BSA. The preadsorbed supernatants were used for ELISA targeting rmHSP70 as previously described (11). The titrations of culture supernatants unadsorbed or preadsorbed with rTgHSP70, rmHSP70 or BSA against rmHSP70 were analyzed at dilutions of 1/2, 1/4, 1/8, 1/16 and 1/32 by ELISA.

Production of anti-TgHSP70 mAb cross-reactive with mHSP70
The spleen B-1 cells of T. gondii-infected B6 mice were purified as mentioned above. Then, the spleen B-1 cells of T. gondii-infected B6 mice were fused with hypoxanthine–aminopterin–thymidine-sensitive P3U1 cells at a 1:5 ratio using 45% polyethylene glycol (mol. wt 4000; Sigma, St Louis, MO). For cloning of hybridoma cell lines producing anti-HSP70 autoantibody (TgCRB 21 and TgCRB 28), the culture supernatants of the hybridomas were tested by ELISA using rTgHSP70 or rmHSP70 as target antigen. Hybridoma lines producing anti-TgHSP70 antibody (TgNCR A5 and TgNCR C2) and anti-HSP70 autoantibody (TgCR 16 and TgCR 20) were established as previously described (11).

Analysis of VH gene and IL-10 expression in B-1 cells
The expression of VH families in B-1 cells from PEC of uninfected and T. gondii-infected BALB/c and B6 mice and in various hybridoma lines producing anti-TgHSP70 antibody cross-reactive (CR) with mHSP70 (TgCR 16, TgCR 20, TgCRB 21 and TgCRB 28) and anti-TgHSP70 antibody non-cross-reactive (NCR) with mHSP70 (TgNCR A5 and TgNCR C2) (11) was analyzed. Total cellular RNA was extracted with TRIzol (Gibco/BRL, Grand Island, NY) from PEC B-1 cells of uninfected and T. gondii-infected mice and various hybridoma lines according to the manufacturer’s instructions. Then 1 µg of total RNA was reverse transcribed in a final volume of 20 µl using an RNA PCR kit (R019A; Takara, Shiga, Japan) according to the manufacturer’s instructions.

The PCR reaction conditions were optimized for VH1–JH1 (J558 family), VH2–JH1 (Q52 family), VH11A–JH1 (CP3 family), VH11B–JH1 (CP3 family) and VH12–JH1 (CH27 family) primers as shown in Table 1. GAPDH DNA was also amplified as a standard to ensure that the cDNA concentrations in different reaction mixtures were approximately equal. The PCR products were electrophoresed in 1.2% agarose gel with ethidium bromide. The quantification of RNA was performed with an IPLab Gel densitometer (Signal Analytical, Vienna, VA). The results were expressed as the ratio of the OD value of the PCR products of VH1–JH1 to the OD value of the products of GAPDH according to the following formula: (OD of VH1–JH1/OD of GAPDH) x 100. The mRNA expression of IL-10 in B-1 cells from PEC of uninfected and T. gondii-infected BALB/c and B6 mice was detected as described above.


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Table 1. RT-PCR conditions (sequences of the oligonucleotide primers used for PCR amplification of VH gene families and IL-10 mRNA, number of PCR cycles and product size predicted)
 
Avidity assay
The avidity of IgG specific for rTgHSP70 and rmHSP70 was measured by protein-denaturing immunoassay (avidity ELISA). Briefly, 1/100, 1/200, 1/400, 1/800, 1/1600 and 1/3200 diluted sera of BALB/c and B6 mice 2 and 9 weeks after T. gondii infection were placed in microtiter wells coated with rTgHSP70 or rmHSP70. After withdrawal of the sera, the antibodies attached to the antigen were eluted with a protein denaturant (6 M urea) and the proportion of residual/total antigen-bound IgG was quantified immunoenzymatically (22,23). Absorbance was read at 405 nm. The avidity index was calculated by the following formula: percentage of urea-resistant IgG of total bound IgG (A405urea+/A405urea).

Quantitative competitive-PCR (QC-PCR)
NCR anti-TgHSP70 mAb (TgNCR C2; IgG2a) and CR anti-TgHSP70 mAb (TgCR 20; IgG3) were obtained as previously described (11). Age/sex-matched BALB/c and B6 mice were i.p. treated by injection of TgNCR C2 mAb or TgCR 20 mAb. Control groups were injected with PBS. Seven days after the treatment, the mice were p.o. infected with five T. gondii cysts of the Fukaya strain as previously described (21). The infected mice were sacrificed 6 weeks post-infection and the numbers of T. gondii in the brains of T. gondii-infected mice were measured by QC-PCR. Total genomic DNAs were purified as described previously (21,24) from ~1 mm3 of tissues. The resulting DNAs were tested for the presence of surface-specific antigen gene 1 (SAG1) by thermal amplification with specific primer pairs (21,25). Briefly, genomic DNA (1 µg) extracted from these organs was co-amplified with a constant concentration of truncated SAG1 DNA, which competitively binds the primers with wild-type SAG1. The amplified cDNAs were eletrophoretically separated on 1.2% agarose gel containing ethidium bromide and the ratio against competitor (T/C) SAG1 DNA subsequently amplified was measured by an ILPab Gel densitometer (Signal Analytical). The numbers of T. gondii were calculated as described previously (21,24).

Detection of intracellular IFN-{gamma}
Three days after transferring T. gondii-infected peritoneal B-1 cells (1 x106) to B6 mice, the mice were i.p. infected with 50 T. gondii cysts of the Fukaya strain. PEC were harvested 5 days after the infection. Then, the cells were fixed and permeabilized with a Cytofix/Cytoperm kit (PharMingen) and stained with 0.1 µg of FITC-conjugated rat anti-mouse IFN-{gamma} mAb (XMG1.2; PharMingen). Finally, the cells were washed with the permeabilization buffer and the IFN-{gamma} expression was analyzed by a FACScan (Becton Dickinson).

Statistics
Differences between mean values were analyzed by unpaired Student’s t-test. P values <0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Anti-mHSP70 autoantibodies were produced in PEC B-1 cells from T. gondii-infected susceptible B6 mice
The production of anti-TgHSP70 antibody and anti-mHSP70 autoantibody by B-1 cells in PEC of T. gondii-infected BALB/c and B6 mice was analyzed in vitro. The purity of B-1 cells (Fig. 1A) was 91.5%. High levels of anti-TgHSP70 antibody were produced by B-1 cells purified from PEC of T. gondii-infected BALB/c and B6 mice. In contrast, high levels of anti-mHSP70 autoantibody were observed in B-1 cells purified from PEC of T. gondii-infected B6 mice, while only low levels were observed in PEC B-1 cells of T. gondii-infected BALB/c mice (Fig. 1B).




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Fig. 1. Specific anti-mHSP70 autoantibody produced in B-1 cells. (A) Isolated peritoneal B-1 cells were tested for purity by flow cytometry as described in Methods. (B) Anti-TgHSP70 antibody and anti-mHSP70 autoantibody formation in B-1 cells from PEC of T. gondii-infected mice. B-1 cells were purified from PEC of T. gondii-infected BALB/c and B6 mice as described in Methods. After 3 days of incubation at 37°C, anti-TgHSP70 antibody and anti-mHSP70 autoantibody formation in the culture supernatants of B-1 cells from PEC of T. gondii-infected BALB/c and B6 mice was examined by ELISA. Data represent means ± SD of three to five mice in three separate experiments. *P > 0.05; **P < 0.01 compared with the culture supernatant from T. gondii-infected BALB/c mice. (C and D) Anti-mHSP70 autoantibody produced in PEC B-1 cells of T. gondii-infected B6 mice recognized cross-reactive antigenic determinants shared by rTgHSP70 and rmHSP70. B-1 cells were purified from PEC of uninfected (D) and T. gondii-infected (C) B6 mice. After 3 days of incubation at 37°C, the culture supernatants were diluted as described in Methods. The diluted culture supernatants of PEC B-1 cells from uninfected and T. gondii-infected B6 mice were adsorbed with either rTgHSP70, rmHSP70 or BSA on ice as described in Methods, and the reactivities of unadsorbed and preadsorbed culture supernatants with rmHSP70 were then tested by ELISA. Data represent means ± SD of three to five mice in three separate experiments. **P < 0.01.

 
To demonstrate the antigen specificity of anti-mHSP70 autoantibody produced by B-1 cells of T. gondii-infected mice, the production of this anti-mHSP70 autoantibody in PEC of uninfected and T. gondii-infected B6 mice was analyzed by adsorption of culture supernatant with rTgHSP70, rmHSP70 and BSA. The culture supernatant of B-1 cells from T. gondii-infected B6 mice adsorbed with rTgHSP70 and rmHSP70 did not react with rmHSP70. On the other hand, that preadsorbed with BSA reacted with rmHSP70, indicating that specific anti-mHSP70 autoantibody was produced by PEC B-1 cells of T. gondii-infected mice (Fig.1C). Anti-mHSP70 autoantibody was not detected in PEC B-1 cells from uninfected mice (Fig. 1D).

VH1–JH1 peritoneal exudate B-1 cells increased after T. gondii infection in B6 mice
As shown in Fig. 2(A), the mRNA expression of VH1–JH1 in B-1 cells from PEC of B6 mice significantly increased 1 week after T. gondii infection, whereas that from PEC of BALB/c mice showed only a slight increase. Moreover, the hybridoma lines producing anti-TgHSP70 antibody cross-reactive with mHSP70 (TgCR 16 and TgCR 20) expressed the VH1–JH1 gene. Furthermore, the B-1 cell hybridoma lines producing anti-TgHSP70 antibody cross-reactive with mHSP70 (TgCRB 21 and TgCRB 28) expressed the VH1–JH1 gene. One hybridoma line producing anti-TgHSP70 antibody non-cross-reactive with mHSP70 (TgNCR A5) expressed the VH11A–JH1 gene and another hybridoma line producing anti-TgHSP70 antibody non-cross-reactive with mHSP70 (TgNCR C2) expressed the VH12–JH1 gene (Fig. 2B). These data indicated that B-1 cells forming anti-mHSP70 autoantibody dominantly utilized the VH1–JH1 gene.




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Fig. 2. Expression of VH1–JH1. (A) VH1–JH1 expression in B-1 cells. B-1 cells from PEC of BALB/c and B6 mice were purified, as described in Methods, before infection and 1 week after T. gondii infection. The expression of VH1–JH1 in B-1 cells from PEC of uninfected and T. gondii-infected BALB/c and B6 mice was analyzed by RT-PCR as described in Methods. Data represent means ± SD of three to five mice in three separate experiments. *P < 0.05; ***P < 0.005 compared with uninfected B-1 cells. (B) Expression of VH families in hybridoma cell lines. Various hybridoma cell lines (TgCR 16, TgCR 20, TgNCR A5 and TgNCR C2) were established as previously described (11). The B-1 cell hybridoma lines (TgCRB 20 and TgCRB 28) were established as described in Methods. The expression of VH families including VH1–JH1, VH2–JH1, VH11A–JH1, VH11B–JH1 and VH12–JH1 in hybridoma cell lines was analyzed by RT-PCR. PCR amplification of the GAPDH gene was used as a control to ensure that the amounts of cDNA obtained from each sample were equivalent. Representative results from three independent experiments are shown.

 
Avidity maturation of anti-TgHSP70 IgG antibody and anti-mHSP70 IgG autoantibody in T. gondii-infected mice
Our previous studies demonstrated that anti-TgHSP70 antibody and anti-mHSP70 autoantibody were produced in both BALB/c and B6 mice after T. gondii infection. The avidity maturation of anti-TgHSP70 IgG antibody and anti-mHSP70 IgG autoantibody in the sera of BALB/c and B6 mice 2 and 9 weeks after T. gondii infection was examined. The avidity index of anti-TgHSP70 IgG antibody in sera of BALB/c mice 9 weeks after T. gondii infection was higher than that at 2 weeks post-infection (Fig. 3A), while that of anti-mHSP70 IgG autoantibody at 9 weeks was only slightly increased when compared with that at 2 weeks post-infection (Fig. 3B). In contrast, the avidity index of anti-TgHSP70 IgG antibody in sera of B6 mice 9 weeks after T. gondii infection was only slightly increased when compared with that at 2 weeks post-infection (Fig. 3C), whereas that of anti-mHSP70 IgG autoantibody reached a high level 9 weeks post-infection (Fig. 3D). The results imply that the avidity maturation of anti-HSP70 IgG autoantibody is correlated with the susceptibility of mice to T. gondii infection.



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Fig. 3. Avidity maturation of anti-TgHSP70 IgG antibody and anti-mHSP70 IgG autoantibody in T. gondii-infected mice. Effects of the concentration of IgG on the avidity index (solid lines: scale on the left) of anti-TgHSP70 IgG antibody (A) and anti-mHSP70 IgG autoantibody (B) in sera of BALB/c mice 2 (closed triangles) or 9 (closed circles) weeks after T. gondii infection were analyzed. Diluted sera were allowed to bind to rTgHSP70 or rmHSP70, then washed in PBS/Tween with or without 6 M urea. Bound IgG was quantitated immunoenzymatically. The avidity index was expressed as described in Methods. Avidity maturation of anti-TgHSP70 IgG antibody (C) and anti-mHSP70 IgG autoantibody (D) (solid lines: scale on the left) in sera of B6 mice 2 (closed triangles) or 9 (closed circles) weeks after T. gondii infection was examined. Dashed lines (scale on the right) show the levels of absorbance (A405urea). Open triangles represent anti-TgHSP70 IgG antibodies and anti-mHSP70 IgG autoantibodies in BALB/c and B6 mice 2 weeks after T. gondii infection; open circles represent anti-TgHSP70 IgG antibodies and anti-mHSP70 IgG autoantibodies in BALB/c and B6 mice 9 weeks after T. gondii infection.

 
B-1 cells producing anti-mHSP70 autoantibody and IL-10 down-regulate host defense in T. gondii-infected B6 mice
To demonstrate the biological significance of anti-mHSP70 autoantibody, the numbers of T. gondii in the brains of T. gondii-infected mice treated with TgNCR C2 mAb or TgCR 20 mAb were measured by QC-PCR. Those in B6 mice treated with TgCR 20 mAb were markedly higher than in those treated with TgNCR C2 mAb or PBS 6 weeks after the challenge infection. The numbers in B6 mice treated with TgNCR C2 mAb were similar to those in control mice. The numbers of T. gondii in the brains of T. gondii-infected BALB/c mice treated with TgNCR C2 mAb or TgCR 20 mAb were as low as those in control mice (Fig. 4A). Therefore, anti-HSP70 autoantibody produced by PEC B-1 cells deteriorated the host defense in T. gondii-infected mice.

To examine the roles of B-1 cells in the IL network, the mRNA expression of IL-10 in B-1 cells from PEC of T. gondii-infected BALB/c and B6 mice was analyzed. IL-10 mRNA expression was not detectable in B-1 cells from PEC of uninfected BALB/c and B6 mice. mRNA expression of IL-10 was significantly present in B-1 cells from PEC of B6 mice 7 days after T. gondii infection, but none was detected in infected BALB/c mice (Fig. 4B). The expression of IFN-{gamma} on PEC of B6 mice was observed after T. gondii infection (Fig. 4C) and injection of T. gondii-infected peritoneal B-1 cells then decreased this expression. These results suggested that B-1 cells producing anti-mHSP70 autoantibody and IL-10 down-regulated host defense in T. gondii-infected susceptible B6 mice.





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Fig. 4. (A) Biological significance of anti-mHSP70 autoantibody in the host defense to T. gondii infection. BALB/c and B6 mice treated with TgNCR C2 mAb or TgCR 20 mAb p.o. infected with the T. gondii Fukaya strain. Six weeks after infection, the mice were sacrificed and the numbers of T. gondii in the brain were measured by QC-PCR targeting the SAG1 gene. Control groups were injected with PBS. Data represent means ± SD of three to five mice in three separate experiments. *P > 0.05; **P < 0.01. (B) mRNA expression of IL-10 by PEC B-1 cells. B-1 cells were purified from PEC of uninfected mice and T. gondii-infected BALB/c and B6 mice 7 days after infection as described in Methods. mRNA expression of IL-10 in B-1 cells from PEC of uninfected and T. gondii-infected BALB/c and B6 mice was analyzed. Representative results from three independent experiments are shown. (C) Intracellular IFN-{gamma} expression on PEC from uninfected B6 mice (shaded histograms), T. gondii-infected B6 mice (solid line) and T. gondii-infected B6 mice transferred with peritoneal B-1 cells (dotted line). The PEC from uninfected B6 mice, T. gondii-infected B6 mice and T. gondii-infected B6 mice transferred with peritoneal B-1 cells were stained with FITC-labeled rat anti-mouse IFN-{gamma} mAb. Flow cytometric analysis was performed as described in Methods. Representative results from three independent experiments are shown.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Previous investigations of anti-mHSP70 autoantibody produced in T. gondii-infected mice have shown that the levels of anti-mHSP70 autoantibody in the sera of T. gondii-infected B6 mice (a susceptible strain) were higher than those in the sera of T. gondii-infected BALB/c mice (a resistant strain) and B-1 cells were responsible for anti-mHSP70 autoantibody formation in T. gondii-infected mice (11). However, whether anti-mHSP70 autoantibody produced by B-1 cells exerted effects at susceptibility/resistance of the mice to T. gondii infection has not been investigated. Many studies have suggested that autoantibody-producing cells are generated from B-1 cells in autoimmune-prone mice and in autoantibody-producing transgenic mice (2628). Several lines of evidence have also suggested that both phosphatidylcholine-reactive antibody and bromelain-treated mouse red blood cell-reactive antibody were highly produced in the normal B-1 cell population and predominantly used VH11 or VH12 genes with restricted complementarity-determining region 3 structure (2931). Moreover, previous studies have revealed that the B-1 cell IgH repertoire such as VH1, VH2 and VH8 was limited, and marked by characteristic specificities for self and particular bacterial antigens (3234). B-1 cells from PEC of T. gondii-infected B6 mice predominantly expressed the VH1–JH1 gene and produced high levels of anti-HSP70 autoantibody, whereas PEC B-1 cells of T. gondii-infected BALB/c mice expressed low levels of VH1–JH1 mRNA and produced low levels of anti-mHSP70 autoantibody. Moreover, the B-1 cell hybridoma lines producing anti-TgHSP70 antibody cross-reactive with mHSP70 expressed the VH1–JH1 gene. These results suggested that B-1 cells responsible for anti-mHSP70 autoantibody formation preferentially utilized the VH1–JH1 gene in T. gondii-infected mice and the higher level of VH1–JH1 gene expression in PEC B-1 cells may be correlated to the strain-dependent differences in susceptibility to T. gondii infection.

Lappalainen et al. have been reported that avidity maturation of anti-T. gondii IgG antibody occurs during the course of T. gondii infection in humans (23). In the present study, avidity maturation of anti-TgHSP70 IgG antibody in the sera of T. gondii-infected BALB/c mice (a resistant strain) and anti-mHSP70 IgG autoantibody in the sera of T. gondii-infected B6 mice (a susceptible strain) was observed. It was confirmed, by using an in vitro culture system as described previously (11), that the high-affinity anti-HSP70 autoantibody was shown to be produced by B-1 cells prepared from B6 mice infected with T. gondii 9 weeks ago (data not shown). The results imply that the avidity maturation of anti-HSP70 IgG autoantibody in susceptible B6 mice may be correlated with the susceptibility to T. gondii infection.

The main function of HSP70 molecules is known to be that of molecular chaperones under physiological and stress conditions (35,36). They play a role in antigen processing and presentation (810), and are detectable on the cell surface (37). We have reported that heat shock cognate protein 71 plays a potential role in antigen presentation and processing of T. gondii-infected melanoma cells to CD4+ CTL (38). Furthermore, Lakey et al. demonstrated that antibodies to HSP70 blocked the presentation of an antigenic peptide to a MHC class II-restricted T cell hybridoma (39). To address the functional significance of anti-HSP70 IgG autoantibody produced in T. gondii-infected mice, we analyzed T. gondii numbers in the brains of T. gondii-infected mice. Higher levels of T. gondii numbers in the brains of T. gondii-infected B6 mice treated with TgCR 20 mAb were observed, suggesting that anti-mHSP70 autoantibodies cause deterioration of the host defense to T. gondii infection by blocking HSP70 peptide–MHC class II complex formation. On the other hand, anti-TgHSP70 antibody did not influence the host defense to T. gondii infection.

While the present research on T. gondii infection has a focus on the humoral immune response, the protective immunity to T. gondii was characterized by the development of a cell-mediated immune response dominated by the production of IFN-{gamma} by T cells (a Th1-type response). The production of IFN-{gamma} by T. gondii-specific CD4+ T cells has been shown to up-regulate class II expression on target cells and promote their killing (4). Several other studies have indicated that IFN-{gamma} mediated anti-parasitic effects in the murine model primarily by up-regulating the expression of macrophage and microglial inducible NO synthase, and the production of NO that was believed not only to be directly toxoplasmacidal but also to promote tachyzoite to bradyzoite transformation by inhibition of mitochondrial respiration (4043). On the other hand, IL-10 enhances humoral immunity by inhibiting macrophage as well as Th1 cell activation and promoting the development of Th2 cytokine synthesis (4446). One of the characteristics of B-1 cells is their capacity to produce large amounts of IL-10 in response to lipopolysaccharide (47). Additionally, IL-10 is needed for the development of murine B-1 cells (48). Velupillai et al. reported that activation and expansion of IL-10- producing B-1 cells were governed via cross-regulatory cytokines (49). Although IL-10 probably directly antagonizes macrophage functions, particularly tumor necrosis factor-{alpha}, IL-1, IL-12 and NO production (45,50), Suzuki et al. recently reported that IL-10 was required for preventing the development of IFN-{gamma}-mediated pathology and mortality in T. gondii-infected mice (1). In the present study, the expression of IFN-{gamma} on PEC of susceptible B6 mice was decreased by injection of PEC B-1 cells from T. gondii-infected mice. The result supported the former interpretation regarding the role of IL-10 in the host defense to T. gondii infection.

Taken together, our results indicate that anti-HSP70 autoantibody and IL-10-forming B-1 cells and avidity maturation of anti-HSP70 IgG autoantibody regulate the susceptibility of mice to T. gondii infection.


    Acknowledgements
 
This work was supported in part by a Grant-in-Aid for Scientific Research of Health and Welfare, and a grant from the Ministry of Education, Science, Sports and Culture, Japan.


    Abbreviations
 
B6 —C57BL/6

CTL—cytotoxic T lymphocyte

GC—germinal center

HSP70—heat shock protein 70

m—mouse

NCR—non-cross-reactive

OD—optical density

PEC—peritoneal exudate cells

QC-PCR—quantitative competitive-PCR

r—recombinant

SAG1—surface-specific antigen gene 1

TgCR—T. gondii cross-reactive

TgNCR—T. gondii non-cross-reactive

TgHSP70—T. gondii HSP70


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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