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B cell development is arrested at the immature B cell stage in mice carrying a mutation in the cytoplasmic domain of immunoglobulin beta.

Reichlin A, Hu Y, Meffre E, Nagaoka H, Gong S, Kraus M, Rajewsky K, Nussenzweig MC - J. Exp. Med. (2001)

Bottom Line: The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)alpha and Ig beta, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM).However, Ig beta Delta C B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis.We conclude that the cytoplasmic domain Ig beta is required for B cell development beyond the immature B cell stage and that Ig alpha and Ig beta have distinct biologic activities in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA.

ABSTRACT
The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)alpha and Ig beta, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM). To determine the function of Ig beta, we produced mice that carry a deletion of the cytoplasmic domain of Ig beta (Ig beta Delta C mice) and compared them to mice that carry a similar mutation in Ig alpha (MB1 Delta C, herein referred to as Ig alpha Delta C mice). Ig beta Delta C mice differ from Ig alpha Delta C mice in that they show little impairment in early B cell development and they produce immature B cells that respond normally to BCR cross-linking as determined by Ca(2+) flux. However, Ig beta Delta C B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis. We conclude that the cytoplasmic domain Ig beta is required for B cell development beyond the immature B cell stage and that Ig alpha and Ig beta have distinct biologic activities in vivo.

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B cell development and death in bone marrow cultures. Bone marrow cells from IgβΔC and control mice were selected using CD19 MACS beads, sorted for B220+CD43−IgMlow immature B cells, and then cocultured with S17 stromal cells. (A) Dot plots show staining with anti-CD25 or anti-IgD and anti-IgM at the initiation of culture (left) and after 24 h (right). Numbers indicate percentages of B220+ cells in each quadrant. Wt, wild-type. (B) Bar graphs show the percentage of B220+ cells that were annexin V or propidium iodide. Results are the average of duplicate cultures performed on two independent mice. The variation between samples and mice was <5%.
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Figure 2: B cell development and death in bone marrow cultures. Bone marrow cells from IgβΔC and control mice were selected using CD19 MACS beads, sorted for B220+CD43−IgMlow immature B cells, and then cocultured with S17 stromal cells. (A) Dot plots show staining with anti-CD25 or anti-IgD and anti-IgM at the initiation of culture (left) and after 24 h (right). Numbers indicate percentages of B220+ cells in each quadrant. Wt, wild-type. (B) Bar graphs show the percentage of B220+ cells that were annexin V or propidium iodide. Results are the average of duplicate cultures performed on two independent mice. The variation between samples and mice was <5%.

Mentions: To determine whether arrest at the CD25+IgM+IgD− immature B cell stage is associated with increased cell death, we established in vitro bone marrow cultures 31. Immature B cells were purified by cell sorting using a Fab′ anti-IgM to avoid receptor cross-linking. Cell death was measured by propidium iodide exclusion and annexin V staining (Fig. 2). Annexin V staining varies between different stages of B cell development and is therefore unreliable when comparing B cells in different stages 39. However, annexin is a reliable marker for apoptosis when comparing cells at similar stages in development 39. Freshly isolated immature IgβΔC and control B cells were equally viable as measured by exclusion of propidium iodide. In culture, the control immature B cells developed into CD25−IgMhiIgD+ transitional B cells, whereas the IgβΔC B cells did not progress beyond the CD25+IgMloIgD− immature B cell stage. Instead, IgβΔC B cells became increasingly annexin V and propidium iodide positive (Fig. 2). Thus, IgβΔC B cells that reach the CD25+IgM+IgD− immature B cell stage fail to progress and die by apoptosis.


B cell development is arrested at the immature B cell stage in mice carrying a mutation in the cytoplasmic domain of immunoglobulin beta.

Reichlin A, Hu Y, Meffre E, Nagaoka H, Gong S, Kraus M, Rajewsky K, Nussenzweig MC - J. Exp. Med. (2001)

B cell development and death in bone marrow cultures. Bone marrow cells from IgβΔC and control mice were selected using CD19 MACS beads, sorted for B220+CD43−IgMlow immature B cells, and then cocultured with S17 stromal cells. (A) Dot plots show staining with anti-CD25 or anti-IgD and anti-IgM at the initiation of culture (left) and after 24 h (right). Numbers indicate percentages of B220+ cells in each quadrant. Wt, wild-type. (B) Bar graphs show the percentage of B220+ cells that were annexin V or propidium iodide. Results are the average of duplicate cultures performed on two independent mice. The variation between samples and mice was <5%.
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Related In: Results  -  Collection

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Figure 2: B cell development and death in bone marrow cultures. Bone marrow cells from IgβΔC and control mice were selected using CD19 MACS beads, sorted for B220+CD43−IgMlow immature B cells, and then cocultured with S17 stromal cells. (A) Dot plots show staining with anti-CD25 or anti-IgD and anti-IgM at the initiation of culture (left) and after 24 h (right). Numbers indicate percentages of B220+ cells in each quadrant. Wt, wild-type. (B) Bar graphs show the percentage of B220+ cells that were annexin V or propidium iodide. Results are the average of duplicate cultures performed on two independent mice. The variation between samples and mice was <5%.
Mentions: To determine whether arrest at the CD25+IgM+IgD− immature B cell stage is associated with increased cell death, we established in vitro bone marrow cultures 31. Immature B cells were purified by cell sorting using a Fab′ anti-IgM to avoid receptor cross-linking. Cell death was measured by propidium iodide exclusion and annexin V staining (Fig. 2). Annexin V staining varies between different stages of B cell development and is therefore unreliable when comparing B cells in different stages 39. However, annexin is a reliable marker for apoptosis when comparing cells at similar stages in development 39. Freshly isolated immature IgβΔC and control B cells were equally viable as measured by exclusion of propidium iodide. In culture, the control immature B cells developed into CD25−IgMhiIgD+ transitional B cells, whereas the IgβΔC B cells did not progress beyond the CD25+IgMloIgD− immature B cell stage. Instead, IgβΔC B cells became increasingly annexin V and propidium iodide positive (Fig. 2). Thus, IgβΔC B cells that reach the CD25+IgM+IgD− immature B cell stage fail to progress and die by apoptosis.

Bottom Line: The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)alpha and Ig beta, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM).However, Ig beta Delta C B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis.We conclude that the cytoplasmic domain Ig beta is required for B cell development beyond the immature B cell stage and that Ig alpha and Ig beta have distinct biologic activities in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA.

ABSTRACT
The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)alpha and Ig beta, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM). To determine the function of Ig beta, we produced mice that carry a deletion of the cytoplasmic domain of Ig beta (Ig beta Delta C mice) and compared them to mice that carry a similar mutation in Ig alpha (MB1 Delta C, herein referred to as Ig alpha Delta C mice). Ig beta Delta C mice differ from Ig alpha Delta C mice in that they show little impairment in early B cell development and they produce immature B cells that respond normally to BCR cross-linking as determined by Ca(2+) flux. However, Ig beta Delta C B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis. We conclude that the cytoplasmic domain Ig beta is required for B cell development beyond the immature B cell stage and that Ig alpha and Ig beta have distinct biologic activities in vivo.

Show MeSH
Related in: MedlinePlus