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Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T-B interface of the splenic follicle.

Mandik-Nayak L, Bui A, Noorchashm H, Eaton A, Erikson J - J. Exp. Med. (1997)

Bottom Line: Because the VH3H9 H chain can pair with endogenous L chains to generate anti-single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire.We have identified anti-dsDNA B cells that are located at the T-B interface in the splenic follicle where they have an increased in vivo turnover rate.These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.

View Article: PubMed Central - PubMed

Affiliation: The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Systemic lupus erythematosus (SLE) and the MRL-lpr/lpr murine model for SLE are characterized by the presence of serum anti-double-stranded (ds)DNA antibodies (Abs), whereas nonautoimmune individuals have negligible levels of these Abs. To increase the frequency of anti-DNA B cells and identify the mechanisms involved in their regulation in nonautoimmune mice, we have used Ig transgenes (tgs). In the present study, we used the VH3H9 heavy (H) chain tg which expresses an H chain that was repeatedly isolated from anti-dsDNA Abs from MRL-lpr/lpr mice. Because the VH3H9 H chain can pair with endogenous L chains to generate anti-single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire. We have identified anti-dsDNA B cells that are located at the T-B interface in the splenic follicle where they have an increased in vivo turnover rate. These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.

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VH3H9/λ B cells have an increased in vivo turnover rate.  Tg(−) (top) and VH3H9 (bottom) mice were continuously labeled with  BrdU for 8 d. Spleen cells were then stained with anti-λ-biotin/streptavidin-Red670 and anti-B220-PE or anti–IgM + IgD–PE (Ig), fixed, permeabilized, and then incorporated BrdU was detected with anti-BrdU  Ab. (A) Dot plots show B220 versus λ (left) and histograms show BrdU  label within the B220+λ+ gate (right). Percentages are given for the  BrdU+ cells. (B) Dot plots show B220 versus Ig (left) and histograms show  BrdU label for the indicated B220+Ighigh and B220+Iglow gates (right).  These are representative plots for n = 4 tg(−) and n = 3 VH3H9 tg mice.
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Figure 3: VH3H9/λ B cells have an increased in vivo turnover rate. Tg(−) (top) and VH3H9 (bottom) mice were continuously labeled with BrdU for 8 d. Spleen cells were then stained with anti-λ-biotin/streptavidin-Red670 and anti-B220-PE or anti–IgM + IgD–PE (Ig), fixed, permeabilized, and then incorporated BrdU was detected with anti-BrdU Ab. (A) Dot plots show B220 versus λ (left) and histograms show BrdU label within the B220+λ+ gate (right). Percentages are given for the BrdU+ cells. (B) Dot plots show B220 versus Ig (left) and histograms show BrdU label for the indicated B220+Ighigh and B220+Iglow gates (right). These are representative plots for n = 4 tg(−) and n = 3 VH3H9 tg mice.

Mentions: The in vivo lifespan of a B cell can be estimated by continuously labeling mice with the thymidine analogue BrdU, and then measuring the incorporation of BrdU-labeled cells into the splenic population (4, 52). A population that is rapidly turning over will be replaced more quickly with labeled cells from the bone marrow. Studies of BALB/c splenic B cell turnover rates have estimated that B cells have an average lifespan of 3–4 wk (4). To estimate the lifespan of anti-dsDNA B cells, tg(−) and VH3H9 tg mice were labeled with BrdU for 8 d and then the incorporation of BrdU was measured using flow cytometry. We chose to analyze the 8-d time point since this is when relatively few B cells will be labeled, and cells with an increased turnover rate will be readily apparent (4, 26). To assess the lifespan of the anti-dsDNA population of cells, we examined BrdU incorporation in the λ+ subset. Fig. 3 A demonstrates that among the λ+ cells, the frequency of BrdU-labeled cells is significantly higher in VH3H9 tg mice than in tg(−) mice (63.1 ± 5.6% versus 9.5 ± 0.4%), suggesting that the anti-dsDNA B cells have a decreased in vivo lifespan. Alternatively, the increased BrdU uptake in this population could be due to the active proliferation of the λ+ B cells in vivo. To address this, we pulsed VH3H9 tg and tg(−) mice with BrdU for 2 h and then measured the uptake of BrdU in the splenic B cell populations. This is a time point when actively proliferating cells will take up BrdU, but is not a long enough period for replacement of cells from the bone marrow pool (52). We were unable to detect BrdU label in the λ+ B cells in VH3H9 tg mice spleens. Labeling was analyzed and detected in the rapidly dividing populations of the bone marrow, thus ensuring that the mice did receive BrdU (data not shown). Together, these data suggest that the increased BrdU labeling of the VH3H9/λ B cells is due to their decreased in vivo lifespan.


Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T-B interface of the splenic follicle.

Mandik-Nayak L, Bui A, Noorchashm H, Eaton A, Erikson J - J. Exp. Med. (1997)

VH3H9/λ B cells have an increased in vivo turnover rate.  Tg(−) (top) and VH3H9 (bottom) mice were continuously labeled with  BrdU for 8 d. Spleen cells were then stained with anti-λ-biotin/streptavidin-Red670 and anti-B220-PE or anti–IgM + IgD–PE (Ig), fixed, permeabilized, and then incorporated BrdU was detected with anti-BrdU  Ab. (A) Dot plots show B220 versus λ (left) and histograms show BrdU  label within the B220+λ+ gate (right). Percentages are given for the  BrdU+ cells. (B) Dot plots show B220 versus Ig (left) and histograms show  BrdU label for the indicated B220+Ighigh and B220+Iglow gates (right).  These are representative plots for n = 4 tg(−) and n = 3 VH3H9 tg mice.
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Figure 3: VH3H9/λ B cells have an increased in vivo turnover rate. Tg(−) (top) and VH3H9 (bottom) mice were continuously labeled with BrdU for 8 d. Spleen cells were then stained with anti-λ-biotin/streptavidin-Red670 and anti-B220-PE or anti–IgM + IgD–PE (Ig), fixed, permeabilized, and then incorporated BrdU was detected with anti-BrdU Ab. (A) Dot plots show B220 versus λ (left) and histograms show BrdU label within the B220+λ+ gate (right). Percentages are given for the BrdU+ cells. (B) Dot plots show B220 versus Ig (left) and histograms show BrdU label for the indicated B220+Ighigh and B220+Iglow gates (right). These are representative plots for n = 4 tg(−) and n = 3 VH3H9 tg mice.
Mentions: The in vivo lifespan of a B cell can be estimated by continuously labeling mice with the thymidine analogue BrdU, and then measuring the incorporation of BrdU-labeled cells into the splenic population (4, 52). A population that is rapidly turning over will be replaced more quickly with labeled cells from the bone marrow. Studies of BALB/c splenic B cell turnover rates have estimated that B cells have an average lifespan of 3–4 wk (4). To estimate the lifespan of anti-dsDNA B cells, tg(−) and VH3H9 tg mice were labeled with BrdU for 8 d and then the incorporation of BrdU was measured using flow cytometry. We chose to analyze the 8-d time point since this is when relatively few B cells will be labeled, and cells with an increased turnover rate will be readily apparent (4, 26). To assess the lifespan of the anti-dsDNA population of cells, we examined BrdU incorporation in the λ+ subset. Fig. 3 A demonstrates that among the λ+ cells, the frequency of BrdU-labeled cells is significantly higher in VH3H9 tg mice than in tg(−) mice (63.1 ± 5.6% versus 9.5 ± 0.4%), suggesting that the anti-dsDNA B cells have a decreased in vivo lifespan. Alternatively, the increased BrdU uptake in this population could be due to the active proliferation of the λ+ B cells in vivo. To address this, we pulsed VH3H9 tg and tg(−) mice with BrdU for 2 h and then measured the uptake of BrdU in the splenic B cell populations. This is a time point when actively proliferating cells will take up BrdU, but is not a long enough period for replacement of cells from the bone marrow pool (52). We were unable to detect BrdU label in the λ+ B cells in VH3H9 tg mice spleens. Labeling was analyzed and detected in the rapidly dividing populations of the bone marrow, thus ensuring that the mice did receive BrdU (data not shown). Together, these data suggest that the increased BrdU labeling of the VH3H9/λ B cells is due to their decreased in vivo lifespan.

Bottom Line: Because the VH3H9 H chain can pair with endogenous L chains to generate anti-single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire.We have identified anti-dsDNA B cells that are located at the T-B interface in the splenic follicle where they have an increased in vivo turnover rate.These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.

View Article: PubMed Central - PubMed

Affiliation: The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Systemic lupus erythematosus (SLE) and the MRL-lpr/lpr murine model for SLE are characterized by the presence of serum anti-double-stranded (ds)DNA antibodies (Abs), whereas nonautoimmune individuals have negligible levels of these Abs. To increase the frequency of anti-DNA B cells and identify the mechanisms involved in their regulation in nonautoimmune mice, we have used Ig transgenes (tgs). In the present study, we used the VH3H9 heavy (H) chain tg which expresses an H chain that was repeatedly isolated from anti-dsDNA Abs from MRL-lpr/lpr mice. Because the VH3H9 H chain can pair with endogenous L chains to generate anti-single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire. We have identified anti-dsDNA B cells that are located at the T-B interface in the splenic follicle where they have an increased in vivo turnover rate. These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.

Show MeSH
Related in: MedlinePlus