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B cell activating factor (BAFF) and T cells cooperate to breach B cell tolerance in lupus-prone New Zealand Black (NZB) mice.

Chang NH, Cheung YH, Loh C, Pau E, Roy V, Cai YC, Wither J - PLoS ONE (2010)

Bottom Line: Treatment of NZB sHEL recipient mice with TACI-Ig reduced NZB dTg B cell survival following adoptive transfer, confirming the role of BAFF in this process.In contrast, T cell blockade had a minimal effect on B cell survival, but inhibited anti-HEL antibody production.The findings suggest that the modest BAFF elevations in NZB mice are sufficient to perturb B cell tolerance, particularly when acting in concert with B cell functional abnormalities and T cell help.

View Article: PubMed Central - PubMed

Affiliation: Arthritis Centre of Excellence, Toronto Western Research Institute, Toronto, Ontario, Canada.

ABSTRACT
The presence of autoantibodies in New Zealand Black (NZB) mice suggests a B cell tolerance defect however the nature of this defect is unknown. To determine whether defects in B cell anergy contribute to the autoimmune phenotype in NZB mice, soluble hen egg lysozyme (sHEL) and anti-HEL Ig transgenes were bred onto the NZB background to generate double transgenic (dTg) mice. NZB dTg mice had elevated levels of anti-HEL antibodies, despite apparently normal B cell functional anergy in-vitro. NZB dTg B cells also demonstrated increased survival and abnormal entry into the follicular compartment following transfer into sHEL mice. Since this process is dependent on BAFF, BAFF serum and mRNA levels were assessed and were found to be significantly elevated in NZB dTg mice. Treatment of NZB sHEL recipient mice with TACI-Ig reduced NZB dTg B cell survival following adoptive transfer, confirming the role of BAFF in this process. Although NZB mice had modestly elevated BAFF, the enhanced NZB B cell survival response appeared to result from an altered response to BAFF. In contrast, T cell blockade had a minimal effect on B cell survival, but inhibited anti-HEL antibody production. The findings suggest that the modest BAFF elevations in NZB mice are sufficient to perturb B cell tolerance, particularly when acting in concert with B cell functional abnormalities and T cell help.

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Heightened survival response of NZB dTg B cells to BAFF in-vitro.(A) Up-regulation of Bcl-2 in NZB IgTg and dTg B cells. B cells isolated from B6 (open bars) or NZB (filled bars) IgTg or dTg mice were incubated with media alone or media containing HEL (100 ng/ml), BAFF (40 ng/ml), or a combination of both, for 20 or 96 h at 37°C. The percent Bcl-2 positive cells was determined by flow cytometry gating on the B220+ population. Results shown are for a single mouse from each strain and are representative experiment of four independent experiments in which a total of 2 nTg, 5 IgTg, 7 dTg B6 mice, and 3 nTg, 6 IgTg, 4 dTg NZB mice were examined. (B) Enhanced expression of Bcl-2 in all B cell subsets of NZB IgTg or dTg B cells after incubation with HEL and BAFF. Cells were isolated and incubated for 96 h at 37°C, as outlined in (A). The percent Bcl-2 positive cells was determined by flow cytometry gating on the B220+ population and using CD21 and CD24 antibodies to subset B cells, as shown in Table 1. (C) Scatterplots showing the proportion of BAFF-R+ cells in various B cell subsets in B6 (open circles) and NZB (closed circles), IgTg and dTg mice, gated as in (B). Background staining with a relevant isotype control was extremely low, and similar in B6 and NZB mice. Asterisks indicate the significance level for comparison between B6 and NZB mice as determined by the Mann-Whitney test: * p<0.05, ** p<0.005, *** p<0.0005.
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pone-0011691-g005: Heightened survival response of NZB dTg B cells to BAFF in-vitro.(A) Up-regulation of Bcl-2 in NZB IgTg and dTg B cells. B cells isolated from B6 (open bars) or NZB (filled bars) IgTg or dTg mice were incubated with media alone or media containing HEL (100 ng/ml), BAFF (40 ng/ml), or a combination of both, for 20 or 96 h at 37°C. The percent Bcl-2 positive cells was determined by flow cytometry gating on the B220+ population. Results shown are for a single mouse from each strain and are representative experiment of four independent experiments in which a total of 2 nTg, 5 IgTg, 7 dTg B6 mice, and 3 nTg, 6 IgTg, 4 dTg NZB mice were examined. (B) Enhanced expression of Bcl-2 in all B cell subsets of NZB IgTg or dTg B cells after incubation with HEL and BAFF. Cells were isolated and incubated for 96 h at 37°C, as outlined in (A). The percent Bcl-2 positive cells was determined by flow cytometry gating on the B220+ population and using CD21 and CD24 antibodies to subset B cells, as shown in Table 1. (C) Scatterplots showing the proportion of BAFF-R+ cells in various B cell subsets in B6 (open circles) and NZB (closed circles), IgTg and dTg mice, gated as in (B). Background staining with a relevant isotype control was extremely low, and similar in B6 and NZB mice. Asterisks indicate the significance level for comparison between B6 and NZB mice as determined by the Mann-Whitney test: * p<0.05, ** p<0.005, *** p<0.0005.

Mentions: The increased survival of NZB dTg B cells following transfer into sHEL recipients was not solely due to increased levels of BAFF in the NZB environment, because NZB dTg B cells also demonstrated enhanced survival following transfer into sHEL (NZB x B6)F1 recipients (see Figure 3A). This finding raised the possibility that NZB dTg B cells have a heightened response to BAFF leading to their increased survival. Since BAFF has been shown to enhance B cell survival by at least two mechanisms: down-regulation of the pro-apoptotic molecule Bim [32], [33] and up-regulation of anti-apoptotic molecules such as Bcl-2 [15], [34], [35], we hypothesized that the increased survival of NZB dTg B cells results from altered expression of these molecules. To assess this possibility, B cells from B6 and NZB non-Tg, IgTg or dTg mice were stimulated with HEL in the presence or absence of BAFF for 20 hr and expression of Bim or Bcl-2 assessed using flow cytometry. Bim expression was unaffected by the presence or absence of BAFF or HEL for both B6 and NZB B cells at 20 hr (data not shown). Although incubation of NZB IgTg B cells with BAFF also did not result in significant changes in Bcl-2 expression at 20 hr, Bcl-2 expression was induced by incubation with HEL (Figure 5A). At 96 hr, Bcl-2 expression was significantly increased in IgTg B cells incubated with BAFF in the presence or absence of HEL (Figure 5A). Notably, NZB dTg B cells responded similarly to IgTg B cells with increased expression of Bcl-2 in response to HEL at 20 hr and increased expression of Bcl-2 in response to BAFF and HEL at 96 hr. Incubation of B6 dTg B cells with HEL and/or BAFF resulted in minimal changes in the expression of Bcl-2 at 20 or 96 hr. This was not due to the altered proportions of B cell subsets in NZB IgTg and dTg mice, because increased expression of Bcl-2 was seen in all peripheral B cell subsets (T1, T2, MZP and Fo) of these mice (Figure 5B). These findings suggest that the increased survival response of NZB dTg B cells results from altered expression of Bcl-2, but not Bim. Notably, there was a trend to increased expression of BAFF-R on all peripheral B cell populations in NZB IgTg and dTg mice, as compared to their B6 counterparts. This appeared to reflect an increased proportion of cells expressing BAFF-R rather than a shift in overall staining within these populations and did not arise from differences in BAFF binding between these mice, as staining with the anti-BAFF-R antibody is not affected by binding to BAFF. Thus, it is likely that the altered BAFF response in NZB dTg mice arises at least in part from increased BAFF-R expression.


B cell activating factor (BAFF) and T cells cooperate to breach B cell tolerance in lupus-prone New Zealand Black (NZB) mice.

Chang NH, Cheung YH, Loh C, Pau E, Roy V, Cai YC, Wither J - PLoS ONE (2010)

Heightened survival response of NZB dTg B cells to BAFF in-vitro.(A) Up-regulation of Bcl-2 in NZB IgTg and dTg B cells. B cells isolated from B6 (open bars) or NZB (filled bars) IgTg or dTg mice were incubated with media alone or media containing HEL (100 ng/ml), BAFF (40 ng/ml), or a combination of both, for 20 or 96 h at 37°C. The percent Bcl-2 positive cells was determined by flow cytometry gating on the B220+ population. Results shown are for a single mouse from each strain and are representative experiment of four independent experiments in which a total of 2 nTg, 5 IgTg, 7 dTg B6 mice, and 3 nTg, 6 IgTg, 4 dTg NZB mice were examined. (B) Enhanced expression of Bcl-2 in all B cell subsets of NZB IgTg or dTg B cells after incubation with HEL and BAFF. Cells were isolated and incubated for 96 h at 37°C, as outlined in (A). The percent Bcl-2 positive cells was determined by flow cytometry gating on the B220+ population and using CD21 and CD24 antibodies to subset B cells, as shown in Table 1. (C) Scatterplots showing the proportion of BAFF-R+ cells in various B cell subsets in B6 (open circles) and NZB (closed circles), IgTg and dTg mice, gated as in (B). Background staining with a relevant isotype control was extremely low, and similar in B6 and NZB mice. Asterisks indicate the significance level for comparison between B6 and NZB mice as determined by the Mann-Whitney test: * p<0.05, ** p<0.005, *** p<0.0005.
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Related In: Results  -  Collection

Show All Figures
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pone-0011691-g005: Heightened survival response of NZB dTg B cells to BAFF in-vitro.(A) Up-regulation of Bcl-2 in NZB IgTg and dTg B cells. B cells isolated from B6 (open bars) or NZB (filled bars) IgTg or dTg mice were incubated with media alone or media containing HEL (100 ng/ml), BAFF (40 ng/ml), or a combination of both, for 20 or 96 h at 37°C. The percent Bcl-2 positive cells was determined by flow cytometry gating on the B220+ population. Results shown are for a single mouse from each strain and are representative experiment of four independent experiments in which a total of 2 nTg, 5 IgTg, 7 dTg B6 mice, and 3 nTg, 6 IgTg, 4 dTg NZB mice were examined. (B) Enhanced expression of Bcl-2 in all B cell subsets of NZB IgTg or dTg B cells after incubation with HEL and BAFF. Cells were isolated and incubated for 96 h at 37°C, as outlined in (A). The percent Bcl-2 positive cells was determined by flow cytometry gating on the B220+ population and using CD21 and CD24 antibodies to subset B cells, as shown in Table 1. (C) Scatterplots showing the proportion of BAFF-R+ cells in various B cell subsets in B6 (open circles) and NZB (closed circles), IgTg and dTg mice, gated as in (B). Background staining with a relevant isotype control was extremely low, and similar in B6 and NZB mice. Asterisks indicate the significance level for comparison between B6 and NZB mice as determined by the Mann-Whitney test: * p<0.05, ** p<0.005, *** p<0.0005.
Mentions: The increased survival of NZB dTg B cells following transfer into sHEL recipients was not solely due to increased levels of BAFF in the NZB environment, because NZB dTg B cells also demonstrated enhanced survival following transfer into sHEL (NZB x B6)F1 recipients (see Figure 3A). This finding raised the possibility that NZB dTg B cells have a heightened response to BAFF leading to their increased survival. Since BAFF has been shown to enhance B cell survival by at least two mechanisms: down-regulation of the pro-apoptotic molecule Bim [32], [33] and up-regulation of anti-apoptotic molecules such as Bcl-2 [15], [34], [35], we hypothesized that the increased survival of NZB dTg B cells results from altered expression of these molecules. To assess this possibility, B cells from B6 and NZB non-Tg, IgTg or dTg mice were stimulated with HEL in the presence or absence of BAFF for 20 hr and expression of Bim or Bcl-2 assessed using flow cytometry. Bim expression was unaffected by the presence or absence of BAFF or HEL for both B6 and NZB B cells at 20 hr (data not shown). Although incubation of NZB IgTg B cells with BAFF also did not result in significant changes in Bcl-2 expression at 20 hr, Bcl-2 expression was induced by incubation with HEL (Figure 5A). At 96 hr, Bcl-2 expression was significantly increased in IgTg B cells incubated with BAFF in the presence or absence of HEL (Figure 5A). Notably, NZB dTg B cells responded similarly to IgTg B cells with increased expression of Bcl-2 in response to HEL at 20 hr and increased expression of Bcl-2 in response to BAFF and HEL at 96 hr. Incubation of B6 dTg B cells with HEL and/or BAFF resulted in minimal changes in the expression of Bcl-2 at 20 or 96 hr. This was not due to the altered proportions of B cell subsets in NZB IgTg and dTg mice, because increased expression of Bcl-2 was seen in all peripheral B cell subsets (T1, T2, MZP and Fo) of these mice (Figure 5B). These findings suggest that the increased survival response of NZB dTg B cells results from altered expression of Bcl-2, but not Bim. Notably, there was a trend to increased expression of BAFF-R on all peripheral B cell populations in NZB IgTg and dTg mice, as compared to their B6 counterparts. This appeared to reflect an increased proportion of cells expressing BAFF-R rather than a shift in overall staining within these populations and did not arise from differences in BAFF binding between these mice, as staining with the anti-BAFF-R antibody is not affected by binding to BAFF. Thus, it is likely that the altered BAFF response in NZB dTg mice arises at least in part from increased BAFF-R expression.

Bottom Line: Treatment of NZB sHEL recipient mice with TACI-Ig reduced NZB dTg B cell survival following adoptive transfer, confirming the role of BAFF in this process.In contrast, T cell blockade had a minimal effect on B cell survival, but inhibited anti-HEL antibody production.The findings suggest that the modest BAFF elevations in NZB mice are sufficient to perturb B cell tolerance, particularly when acting in concert with B cell functional abnormalities and T cell help.

View Article: PubMed Central - PubMed

Affiliation: Arthritis Centre of Excellence, Toronto Western Research Institute, Toronto, Ontario, Canada.

ABSTRACT
The presence of autoantibodies in New Zealand Black (NZB) mice suggests a B cell tolerance defect however the nature of this defect is unknown. To determine whether defects in B cell anergy contribute to the autoimmune phenotype in NZB mice, soluble hen egg lysozyme (sHEL) and anti-HEL Ig transgenes were bred onto the NZB background to generate double transgenic (dTg) mice. NZB dTg mice had elevated levels of anti-HEL antibodies, despite apparently normal B cell functional anergy in-vitro. NZB dTg B cells also demonstrated increased survival and abnormal entry into the follicular compartment following transfer into sHEL mice. Since this process is dependent on BAFF, BAFF serum and mRNA levels were assessed and were found to be significantly elevated in NZB dTg mice. Treatment of NZB sHEL recipient mice with TACI-Ig reduced NZB dTg B cell survival following adoptive transfer, confirming the role of BAFF in this process. Although NZB mice had modestly elevated BAFF, the enhanced NZB B cell survival response appeared to result from an altered response to BAFF. In contrast, T cell blockade had a minimal effect on B cell survival, but inhibited anti-HEL antibody production. The findings suggest that the modest BAFF elevations in NZB mice are sufficient to perturb B cell tolerance, particularly when acting in concert with B cell functional abnormalities and T cell help.

Show MeSH
Related in: MedlinePlus