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Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool.

Turner M, Gulbranson-Judge A, Quinn ME, Walters AE, MacLennan IC, Tybulewicz VL - J. Exp. Med. (1997)

Bottom Line: Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells.Normally only a proportion of immature B cells is recruited into the recirculating pool.Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Medical Research, Mill Hill, London, NW7 1AA, United Kingdom.

ABSTRACT
The tyrosine kinase Syk has been implicated as a key signal transducer from the B cell antigen receptor (BCR). We show here that mutation of the Syk gene completely blocks the maturation of immature B cells into recirculating cells and stops their entry into B cell follicles. Furthermore, using radiation chimeras we demonstrate that this developmental block is due to the absence of Syk in the B cells themselves. Syk-deficient B cells are shown to have the life span of normal immature B cells. If this is extended by over-expression of Bcl-2, they accumulate in the T zone and red pulp of the spleen in increased numbers, but still fail to mature to become recirculating follicular B cells. Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells. Normally only a proportion of immature B cells is recruited into the recirculating pool. Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.

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Immature Syk−/− B cells migrate to the spleen but do not mature into follicular B cells. Immunohistochemistry of peripheral B cells in radiation chimeras whose nature is shown to the left above each micrograph; i.e., whether the reconstituting livers were Syk+/− or Syk−/− and if they contained the 3-83 or Bcl-2 transgenes (29, 33). All fetal liver donors were of the IgHb immunoglobulin allotype; all hosts were IgHa. 20 Syk−/− nontransgenic  chimeras and at least three chimeras from each group of transgenic donors were analyzed. The specificity of the antibodies used for staining the sections is  shown to the right above each micrograph; the molecule stained gold by immunoperoxidase is shown first, the one stained blue by immunoalkaline phosphatase is shown second and, where used, BrdU staining is in red. Cells double positive for gold and blue appear dark brown. Scale: black bar represents  100 μm. (a) Donor IgMb+ Syk−/− immature B cells (small arrows) are present in the T zone (T) and red pulp (R) of the spleen, but not in the partially filled  follicles (F), which contain mature IgD+ (gold) cells of host origin. Donor IgMb+Syk−/− plasma cells in the red pulp are indicated by large arrows. (b) Section through a lymph node from a similar chimera to that in a. Well-developed primary follicles are present but no donor IgMb+Syk−/− B cells were detected in this or in any lymph node from similar chimeras. (c) Spleen section from a Syk+/−/3-83 chimera that, like the ones shown in d and e, had been  given BrdU for 12 h, 8 d before the spleen was taken. The follicular mantles are filled with mature 3-83+IgD+ (dark brown) donor B cells; occasional cells  (arrowed) have a red-stained nucleus, indicating that they had taken up BrdU during the 12-h pulse 8 d previously. Some 3-83+IgD− (blue) donor B cells can be  seen in the outer T zone and red pulp; some of these may be immature B cells, but none are BrdU+. The germinal center (G) is filled with donor-derived  3-83−IgHb+ B cells (data not shown) that arise from occasional rearrangement of endogenous Ig genes in the 3-83 transgenics. (d) Spleen section from a  Syk−/−/3-83 chimera. Small numbers of 3-83+IgD− donor B cells (blue and arrowed) can be seen in the outer T zone and red pulp; none of these are  BrdU+. As in a the follicles exclusively contain host IgD+ cells (gold) but no donor B cells. (e) Spleen section from a Syk−/−/3-83/Bcl-2Tg chimera. More  donor B cells are present in the T zone and red pulp than in the chimera in d not carrying Bcl-2Tg. Nevertheless, there were no donor B cells in follicles.  Some of the donor 3-83+ B cells had BrdU in their nuclei (arrowed and red). These must have been in cell cycle as pre-B cells 8 d previously, indicating  an extended life span for these immature B cells.
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Figure 2: Immature Syk−/− B cells migrate to the spleen but do not mature into follicular B cells. Immunohistochemistry of peripheral B cells in radiation chimeras whose nature is shown to the left above each micrograph; i.e., whether the reconstituting livers were Syk+/− or Syk−/− and if they contained the 3-83 or Bcl-2 transgenes (29, 33). All fetal liver donors were of the IgHb immunoglobulin allotype; all hosts were IgHa. 20 Syk−/− nontransgenic chimeras and at least three chimeras from each group of transgenic donors were analyzed. The specificity of the antibodies used for staining the sections is shown to the right above each micrograph; the molecule stained gold by immunoperoxidase is shown first, the one stained blue by immunoalkaline phosphatase is shown second and, where used, BrdU staining is in red. Cells double positive for gold and blue appear dark brown. Scale: black bar represents 100 μm. (a) Donor IgMb+ Syk−/− immature B cells (small arrows) are present in the T zone (T) and red pulp (R) of the spleen, but not in the partially filled follicles (F), which contain mature IgD+ (gold) cells of host origin. Donor IgMb+Syk−/− plasma cells in the red pulp are indicated by large arrows. (b) Section through a lymph node from a similar chimera to that in a. Well-developed primary follicles are present but no donor IgMb+Syk−/− B cells were detected in this or in any lymph node from similar chimeras. (c) Spleen section from a Syk+/−/3-83 chimera that, like the ones shown in d and e, had been given BrdU for 12 h, 8 d before the spleen was taken. The follicular mantles are filled with mature 3-83+IgD+ (dark brown) donor B cells; occasional cells (arrowed) have a red-stained nucleus, indicating that they had taken up BrdU during the 12-h pulse 8 d previously. Some 3-83+IgD− (blue) donor B cells can be seen in the outer T zone and red pulp; some of these may be immature B cells, but none are BrdU+. The germinal center (G) is filled with donor-derived 3-83−IgHb+ B cells (data not shown) that arise from occasional rearrangement of endogenous Ig genes in the 3-83 transgenics. (d) Spleen section from a Syk−/−/3-83 chimera. Small numbers of 3-83+IgD− donor B cells (blue and arrowed) can be seen in the outer T zone and red pulp; none of these are BrdU+. As in a the follicles exclusively contain host IgD+ cells (gold) but no donor B cells. (e) Spleen section from a Syk−/−/3-83/Bcl-2Tg chimera. More donor B cells are present in the T zone and red pulp than in the chimera in d not carrying Bcl-2Tg. Nevertheless, there were no donor B cells in follicles. Some of the donor 3-83+ B cells had BrdU in their nuclei (arrowed and red). These must have been in cell cycle as pre-B cells 8 d previously, indicating an extended life span for these immature B cells.

Mentions: It was unclear from our preliminary study if this developmental block is due to a malfunction within B cells, or some other cell type required for the formation of follicles, or both. To address this issue, chimeras were constructed by transferring Syk−/− fetal liver into irradiated wild-type mice which differed in their immunoglobulin heavy chain allotype; this allotype difference allowed B cells of host and donor origin to be distinguished. Flow cytometric analysis of these mice confirmed that they contained Syk−/− immature B cells in the marrow, but none were detectable in peripheral lymphoid tissue (data not shown). The spleens from 20 of these chimeras were examined by immunohistology, and all were found to have small numbers of donor Syk−/− immature B cells (IgMb+IgD−) in the T zones and red pulp, but no Syk-deficient B cells in the follicles (Fig. 2 a); the B cells in these follicles were exclusively host (IgMa+) cells (Fig. 2 a and data not shown). Furthermore, analysis of lymph nodes from the same chimeras failed to find any donor Syk-deficient B cells, although again host IgMa+IgD+ cells were found in the follicles (Fig. 2 b). In contrast, chimeras made with Syk+/+ or Syk+/− fetal liver had normal follicles filled with donor IgMb+IgD+ B cells (data not shown). Effective reconstitution of the irradiated mice with Syk−/− donor hematopoietic cells could be clearly documented in the T lineage (data not shown); however, there was always a small, although variable, level of residual host lymphopoiesis. The presence of significant numbers of host (Syk+/+) follicular IgMa+IgD+ B cells, particularly in lymph nodes, demonstrates that wild-type B cells can mature in these Syk−/− chimeras; consequently the maturational arrest of the Syk−/− immature B cells appears to be due to the absence of Syk within the B cells themselves.


Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool.

Turner M, Gulbranson-Judge A, Quinn ME, Walters AE, MacLennan IC, Tybulewicz VL - J. Exp. Med. (1997)

Immature Syk−/− B cells migrate to the spleen but do not mature into follicular B cells. Immunohistochemistry of peripheral B cells in radiation chimeras whose nature is shown to the left above each micrograph; i.e., whether the reconstituting livers were Syk+/− or Syk−/− and if they contained the 3-83 or Bcl-2 transgenes (29, 33). All fetal liver donors were of the IgHb immunoglobulin allotype; all hosts were IgHa. 20 Syk−/− nontransgenic  chimeras and at least three chimeras from each group of transgenic donors were analyzed. The specificity of the antibodies used for staining the sections is  shown to the right above each micrograph; the molecule stained gold by immunoperoxidase is shown first, the one stained blue by immunoalkaline phosphatase is shown second and, where used, BrdU staining is in red. Cells double positive for gold and blue appear dark brown. Scale: black bar represents  100 μm. (a) Donor IgMb+ Syk−/− immature B cells (small arrows) are present in the T zone (T) and red pulp (R) of the spleen, but not in the partially filled  follicles (F), which contain mature IgD+ (gold) cells of host origin. Donor IgMb+Syk−/− plasma cells in the red pulp are indicated by large arrows. (b) Section through a lymph node from a similar chimera to that in a. Well-developed primary follicles are present but no donor IgMb+Syk−/− B cells were detected in this or in any lymph node from similar chimeras. (c) Spleen section from a Syk+/−/3-83 chimera that, like the ones shown in d and e, had been  given BrdU for 12 h, 8 d before the spleen was taken. The follicular mantles are filled with mature 3-83+IgD+ (dark brown) donor B cells; occasional cells  (arrowed) have a red-stained nucleus, indicating that they had taken up BrdU during the 12-h pulse 8 d previously. Some 3-83+IgD− (blue) donor B cells can be  seen in the outer T zone and red pulp; some of these may be immature B cells, but none are BrdU+. The germinal center (G) is filled with donor-derived  3-83−IgHb+ B cells (data not shown) that arise from occasional rearrangement of endogenous Ig genes in the 3-83 transgenics. (d) Spleen section from a  Syk−/−/3-83 chimera. Small numbers of 3-83+IgD− donor B cells (blue and arrowed) can be seen in the outer T zone and red pulp; none of these are  BrdU+. As in a the follicles exclusively contain host IgD+ cells (gold) but no donor B cells. (e) Spleen section from a Syk−/−/3-83/Bcl-2Tg chimera. More  donor B cells are present in the T zone and red pulp than in the chimera in d not carrying Bcl-2Tg. Nevertheless, there were no donor B cells in follicles.  Some of the donor 3-83+ B cells had BrdU in their nuclei (arrowed and red). These must have been in cell cycle as pre-B cells 8 d previously, indicating  an extended life span for these immature B cells.
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Related In: Results  -  Collection

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Figure 2: Immature Syk−/− B cells migrate to the spleen but do not mature into follicular B cells. Immunohistochemistry of peripheral B cells in radiation chimeras whose nature is shown to the left above each micrograph; i.e., whether the reconstituting livers were Syk+/− or Syk−/− and if they contained the 3-83 or Bcl-2 transgenes (29, 33). All fetal liver donors were of the IgHb immunoglobulin allotype; all hosts were IgHa. 20 Syk−/− nontransgenic chimeras and at least three chimeras from each group of transgenic donors were analyzed. The specificity of the antibodies used for staining the sections is shown to the right above each micrograph; the molecule stained gold by immunoperoxidase is shown first, the one stained blue by immunoalkaline phosphatase is shown second and, where used, BrdU staining is in red. Cells double positive for gold and blue appear dark brown. Scale: black bar represents 100 μm. (a) Donor IgMb+ Syk−/− immature B cells (small arrows) are present in the T zone (T) and red pulp (R) of the spleen, but not in the partially filled follicles (F), which contain mature IgD+ (gold) cells of host origin. Donor IgMb+Syk−/− plasma cells in the red pulp are indicated by large arrows. (b) Section through a lymph node from a similar chimera to that in a. Well-developed primary follicles are present but no donor IgMb+Syk−/− B cells were detected in this or in any lymph node from similar chimeras. (c) Spleen section from a Syk+/−/3-83 chimera that, like the ones shown in d and e, had been given BrdU for 12 h, 8 d before the spleen was taken. The follicular mantles are filled with mature 3-83+IgD+ (dark brown) donor B cells; occasional cells (arrowed) have a red-stained nucleus, indicating that they had taken up BrdU during the 12-h pulse 8 d previously. Some 3-83+IgD− (blue) donor B cells can be seen in the outer T zone and red pulp; some of these may be immature B cells, but none are BrdU+. The germinal center (G) is filled with donor-derived 3-83−IgHb+ B cells (data not shown) that arise from occasional rearrangement of endogenous Ig genes in the 3-83 transgenics. (d) Spleen section from a Syk−/−/3-83 chimera. Small numbers of 3-83+IgD− donor B cells (blue and arrowed) can be seen in the outer T zone and red pulp; none of these are BrdU+. As in a the follicles exclusively contain host IgD+ cells (gold) but no donor B cells. (e) Spleen section from a Syk−/−/3-83/Bcl-2Tg chimera. More donor B cells are present in the T zone and red pulp than in the chimera in d not carrying Bcl-2Tg. Nevertheless, there were no donor B cells in follicles. Some of the donor 3-83+ B cells had BrdU in their nuclei (arrowed and red). These must have been in cell cycle as pre-B cells 8 d previously, indicating an extended life span for these immature B cells.
Mentions: It was unclear from our preliminary study if this developmental block is due to a malfunction within B cells, or some other cell type required for the formation of follicles, or both. To address this issue, chimeras were constructed by transferring Syk−/− fetal liver into irradiated wild-type mice which differed in their immunoglobulin heavy chain allotype; this allotype difference allowed B cells of host and donor origin to be distinguished. Flow cytometric analysis of these mice confirmed that they contained Syk−/− immature B cells in the marrow, but none were detectable in peripheral lymphoid tissue (data not shown). The spleens from 20 of these chimeras were examined by immunohistology, and all were found to have small numbers of donor Syk−/− immature B cells (IgMb+IgD−) in the T zones and red pulp, but no Syk-deficient B cells in the follicles (Fig. 2 a); the B cells in these follicles were exclusively host (IgMa+) cells (Fig. 2 a and data not shown). Furthermore, analysis of lymph nodes from the same chimeras failed to find any donor Syk-deficient B cells, although again host IgMa+IgD+ cells were found in the follicles (Fig. 2 b). In contrast, chimeras made with Syk+/+ or Syk+/− fetal liver had normal follicles filled with donor IgMb+IgD+ B cells (data not shown). Effective reconstitution of the irradiated mice with Syk−/− donor hematopoietic cells could be clearly documented in the T lineage (data not shown); however, there was always a small, although variable, level of residual host lymphopoiesis. The presence of significant numbers of host (Syk+/+) follicular IgMa+IgD+ B cells, particularly in lymph nodes, demonstrates that wild-type B cells can mature in these Syk−/− chimeras; consequently the maturational arrest of the Syk−/− immature B cells appears to be due to the absence of Syk within the B cells themselves.

Bottom Line: Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells.Normally only a proportion of immature B cells is recruited into the recirculating pool.Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Medical Research, Mill Hill, London, NW7 1AA, United Kingdom.

ABSTRACT
The tyrosine kinase Syk has been implicated as a key signal transducer from the B cell antigen receptor (BCR). We show here that mutation of the Syk gene completely blocks the maturation of immature B cells into recirculating cells and stops their entry into B cell follicles. Furthermore, using radiation chimeras we demonstrate that this developmental block is due to the absence of Syk in the B cells themselves. Syk-deficient B cells are shown to have the life span of normal immature B cells. If this is extended by over-expression of Bcl-2, they accumulate in the T zone and red pulp of the spleen in increased numbers, but still fail to mature to become recirculating follicular B cells. Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells. Normally only a proportion of immature B cells is recruited into the recirculating pool. Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.

Show MeSH
Related in: MedlinePlus