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Non-Hematopoietic and Hematopoietic SIRPα Signaling Differently Regulates Murine B Cell Maturation in Bone Marrow and Spleen.

Kolan SS, Lejon K, Koskinen Holm C, Sulniute R, Lundberg P, Matozaki T, Oldenborg PA - PLoS ONE (2015)

Bottom Line: Here we provide evidence that signal regulatory protein α (SIRPα), an Ig-superfamily ITIM-receptor expressed by myeloid but not by lymphoid cells, is involved in regulating B cell maturation.Lack of SIRPα signaling in adult SIRPα-mutant mice resulted in a reduced maturation of B cells in the bone marrow, evident by reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells, as well as reduced blood B cell numbers.In addition, lack of SIRPα signaling also impaired follicular B cell maturation in the spleen.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Medical Biology, Umeå University, Umeå, Sweden.

ABSTRACT
B lymphocyte development occurs in the bone marrow, while final differentiation and maturation can occur in both the bone marrow and the spleen. Here we provide evidence that signal regulatory protein α (SIRPα), an Ig-superfamily ITIM-receptor expressed by myeloid but not by lymphoid cells, is involved in regulating B cell maturation. Lack of SIRPα signaling in adult SIRPα-mutant mice resulted in a reduced maturation of B cells in the bone marrow, evident by reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells, as well as reduced blood B cell numbers. In addition, lack of SIRPα signaling also impaired follicular B cell maturation in the spleen. Maturing BM or splenic B cells of SIRPα-mutant mice were found to express higher levels of the pro-apoptotic protein BIM and apoptosis was increased among these B cells. Bone marrow reconstitution experiments revealed that the B cell maturation defect in bone marrow and blood was due to lack of SIRPα signaling in non-hematopoietic cells, while hematopoietic SIRPα signaling was important for follicular B cell maturation in the spleen. Adding on to our previous findings of a stromal cell defect in SIRPα-mutant mice was the finding that gene expression of receptor activator of nuclear factor-ĸB ligand (RANKL) was significantly lower in cultured bone marrow stromal cells of SIRPα mutant mice. These data suggest a novel and opposite contribution of SIRPα signaling within non-hematopoietic and hematopoietic cells, respectively, to maintain B cell maturation and to prevent apoptosis in the bone marrow and spleen of adult mice.

No MeSH data available.


Related in: MedlinePlus

Impaired B cell maturation in the bone marrow of SIRPα-mutant mice.(A) Absolute number of B220+ B cells in BM of 12 weeks old wild-type (open bar) or SIRPα-mutant mice (black bar). Data are means±SEM for 9 mice/group. (B) Representative flow cytometry analyses of BM B cell maturation based on expression of IgD and IgM in B220+ BM cells of wild-type or SIRPα-mutant mice. (C) Absolute numbers, or (D) relative numbers, of IgD-IgM+ immature (Imm B), IgDloIgMhi transitional 1 (T1), IgD+IgMhi follicular type-II (F-II) and IgD+IgMlo follicular type-I (F-I) B cells in BM of 12 weeks old wild-type (open bars) or SIRPα-mutant mice (black bars). Data are means±SEM for 9 mice/group. (E) The relative expression of BIM protein, and (F) the relative increase in annexin V+ apoptotic cells, were quantified among BM B cell subsets defined as described in panels C-D. Data are means±SEM for 5 mice/group. *P<0.05, **P<0.01 and ***P<0.001, using Student’s t-test for unpaired analyses.
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pone.0134113.g002: Impaired B cell maturation in the bone marrow of SIRPα-mutant mice.(A) Absolute number of B220+ B cells in BM of 12 weeks old wild-type (open bar) or SIRPα-mutant mice (black bar). Data are means±SEM for 9 mice/group. (B) Representative flow cytometry analyses of BM B cell maturation based on expression of IgD and IgM in B220+ BM cells of wild-type or SIRPα-mutant mice. (C) Absolute numbers, or (D) relative numbers, of IgD-IgM+ immature (Imm B), IgDloIgMhi transitional 1 (T1), IgD+IgMhi follicular type-II (F-II) and IgD+IgMlo follicular type-I (F-I) B cells in BM of 12 weeks old wild-type (open bars) or SIRPα-mutant mice (black bars). Data are means±SEM for 9 mice/group. (E) The relative expression of BIM protein, and (F) the relative increase in annexin V+ apoptotic cells, were quantified among BM B cell subsets defined as described in panels C-D. Data are means±SEM for 5 mice/group. *P<0.05, **P<0.01 and ***P<0.001, using Student’s t-test for unpaired analyses.

Mentions: It has been suggested that approximately two thirds of the sIgM+ immature B cells generated in the BM will be released into the blood and migrate to the spleen for further maturation, while the remaining immature B cells mature within the BM itself [5,27]. Therefore, we next investigated if the maturation of B cells in the BM was affected in SIRPα-mutant mice. While the total number of BM cells in 13 weeks old SIRPα-mutant mice was similar to that in wild-type mice (S1A Fig), mutants had significantly less B220+ B cells (P<0.05, Fig 2A). We found no difference in the absolute numbers of early B lineage Hardy fractions A (B220+ IgM- CD43hi CD19-), B-C (B220+ IgM- CD43hi CD19+), or D (B220+ IgM- CD43lo CD19+) cells in the BM when comparing wild-type and SIRPα-mutant mice (S1B Fig). Further analysis of BM B cell subsets ranging from immature to mature B cells can be made by following the expression levels of IgD and IgM. IgD-IgM+ immature B cells that mature within the BM first become IgDloIgMhi transitional 1 (T1) B cells, followed by becoming semi-mature IgD+IgMhi B cells (corresponding to splenic follicular type-II cells; F-II) and then fully mature IgD+IgMlo B cells (corresponding to splenic follicular type-I cells; F-I), (Fig 2B) [4–6]. Using this approach, we found that the reduced amount of IgD-IgM+ immature B cells found in SIRPα-mutant mice did not reach a higher level of statistical significance (P = 0.075, Fig 2C). However, the numbers of the T1, F-II and F-I subsets were all significantly reduced in the BM of SIRPα-mutant mice (P<0.01, P<0.001 and P<0.001, respectively) (Fig 2C). We also investigated the relative frequency as fractions of the total number of B220+ BM cells. This analysis revealed that the fractions of IgD- IgM+ immature B cells and IgDloIgMhi T1 B cells were not significantly different from that in wild-type mice (Fig 2D), whereas the fraction of F-II cells was reduced by 40% and that of F-I cells was reduced by 55% (P<0.05 and P<0.001, respectively) (Fig 2D). Since the majority of immature B cells mature in the spleen [5,27], we further investigated the numbers of B220+CD21intIgDhiIgMhi F-II and B220+CD21intIgDhiIgMlo F-I B cells in the spleens of SIRPα-mutant mice. This analysis first revealed that 13 weeks old mutant mice had a significantly reduced number of B220+ B cells (P<0.05, Fig 3A). Second, both the F-II and F-I B cell subsets were significantly reduced in SIRPα-mutant spleens, as compared with that in wild-type spleens (P<0.01, Fig 3B). Supporting our previous finding that B cells lack SIRPα expression in secondary lymphoid organs [24], we found that SIRPα was neither expressed by B220+ IgM+ CD43- B cells in the BM (S2A Fig) nor by splenic FoB cells (S2B Fig). Thus, SIRPα signaling outside the B cell compartment appeared to be required to maintain normal levels of immature B cells in the BM and to support B cell maturation in both the BM and the spleen. B cell survival is regulated by pro-survival proteins (e.g. Bcl-2 and Bcl-XL) and pro-apoptotic proteins (e.g. BIM) [28]. To test the hypothesis that there was a reduced survival of mature follicular B cells in mutant spleens, we quantified the mRNA expression levels of Bcl-2 and BIM in FACS-sorted CD19+CD21intCD23hi mature splenic FoB cells. This analysis showed that there were no differences in gene expression of these two proteins when comparing wild-type and mutant FoB cells (Fig 3C). However, it has been recognized that in lymphocytes, the pro-apoptotic activity of BIM can also be regulated at the posttranslational level [29]. Therefore, we next investigated BIM protein levels in BM or splenic B cells of wild-type or SIRPα-mutant mice. In the BM, we found an increased level of BIM protein in immature and T1 B cells (P = 0.08 and P<0.01, respectively; Fig 2E). In addition, both splenic F-II and F-I B cells showed an increased level of BIM protein (P<0.01 and P<0.05, respectively; Fig 3D). To further investigate the possibility of an increased rate of B cell apoptosis in SIRPα-mutant mice, we quantified the amount of annexin V-positive cells. We found a significantly increased amount of apoptotic immature, T1 and F-II B cells in the BM of mutant mice, as compared with that in wild-type mice (P<0.001, P<0.05 and P<0.05, respectively; Fig 2F). In the spleen, the fraction of apoptotic F-II B cells was significantly increased in SIRPα-mutant mice, as compared with that in wild-type mice (P<0.01; Fig 3E). In contrast, we did not find any differences in cell cycle activity among these B cell subsets in the spleen or BM when comparing wild-type and SIRPα-mutant mice (S3 Fig). Thus, a reduced number of maturing B cells in the BM and spleen of SIRPα-mutant mice appeared to be associated with an increased rate of B cell apoptosis.


Non-Hematopoietic and Hematopoietic SIRPα Signaling Differently Regulates Murine B Cell Maturation in Bone Marrow and Spleen.

Kolan SS, Lejon K, Koskinen Holm C, Sulniute R, Lundberg P, Matozaki T, Oldenborg PA - PLoS ONE (2015)

Impaired B cell maturation in the bone marrow of SIRPα-mutant mice.(A) Absolute number of B220+ B cells in BM of 12 weeks old wild-type (open bar) or SIRPα-mutant mice (black bar). Data are means±SEM for 9 mice/group. (B) Representative flow cytometry analyses of BM B cell maturation based on expression of IgD and IgM in B220+ BM cells of wild-type or SIRPα-mutant mice. (C) Absolute numbers, or (D) relative numbers, of IgD-IgM+ immature (Imm B), IgDloIgMhi transitional 1 (T1), IgD+IgMhi follicular type-II (F-II) and IgD+IgMlo follicular type-I (F-I) B cells in BM of 12 weeks old wild-type (open bars) or SIRPα-mutant mice (black bars). Data are means±SEM for 9 mice/group. (E) The relative expression of BIM protein, and (F) the relative increase in annexin V+ apoptotic cells, were quantified among BM B cell subsets defined as described in panels C-D. Data are means±SEM for 5 mice/group. *P<0.05, **P<0.01 and ***P<0.001, using Student’s t-test for unpaired analyses.
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pone.0134113.g002: Impaired B cell maturation in the bone marrow of SIRPα-mutant mice.(A) Absolute number of B220+ B cells in BM of 12 weeks old wild-type (open bar) or SIRPα-mutant mice (black bar). Data are means±SEM for 9 mice/group. (B) Representative flow cytometry analyses of BM B cell maturation based on expression of IgD and IgM in B220+ BM cells of wild-type or SIRPα-mutant mice. (C) Absolute numbers, or (D) relative numbers, of IgD-IgM+ immature (Imm B), IgDloIgMhi transitional 1 (T1), IgD+IgMhi follicular type-II (F-II) and IgD+IgMlo follicular type-I (F-I) B cells in BM of 12 weeks old wild-type (open bars) or SIRPα-mutant mice (black bars). Data are means±SEM for 9 mice/group. (E) The relative expression of BIM protein, and (F) the relative increase in annexin V+ apoptotic cells, were quantified among BM B cell subsets defined as described in panels C-D. Data are means±SEM for 5 mice/group. *P<0.05, **P<0.01 and ***P<0.001, using Student’s t-test for unpaired analyses.
Mentions: It has been suggested that approximately two thirds of the sIgM+ immature B cells generated in the BM will be released into the blood and migrate to the spleen for further maturation, while the remaining immature B cells mature within the BM itself [5,27]. Therefore, we next investigated if the maturation of B cells in the BM was affected in SIRPα-mutant mice. While the total number of BM cells in 13 weeks old SIRPα-mutant mice was similar to that in wild-type mice (S1A Fig), mutants had significantly less B220+ B cells (P<0.05, Fig 2A). We found no difference in the absolute numbers of early B lineage Hardy fractions A (B220+ IgM- CD43hi CD19-), B-C (B220+ IgM- CD43hi CD19+), or D (B220+ IgM- CD43lo CD19+) cells in the BM when comparing wild-type and SIRPα-mutant mice (S1B Fig). Further analysis of BM B cell subsets ranging from immature to mature B cells can be made by following the expression levels of IgD and IgM. IgD-IgM+ immature B cells that mature within the BM first become IgDloIgMhi transitional 1 (T1) B cells, followed by becoming semi-mature IgD+IgMhi B cells (corresponding to splenic follicular type-II cells; F-II) and then fully mature IgD+IgMlo B cells (corresponding to splenic follicular type-I cells; F-I), (Fig 2B) [4–6]. Using this approach, we found that the reduced amount of IgD-IgM+ immature B cells found in SIRPα-mutant mice did not reach a higher level of statistical significance (P = 0.075, Fig 2C). However, the numbers of the T1, F-II and F-I subsets were all significantly reduced in the BM of SIRPα-mutant mice (P<0.01, P<0.001 and P<0.001, respectively) (Fig 2C). We also investigated the relative frequency as fractions of the total number of B220+ BM cells. This analysis revealed that the fractions of IgD- IgM+ immature B cells and IgDloIgMhi T1 B cells were not significantly different from that in wild-type mice (Fig 2D), whereas the fraction of F-II cells was reduced by 40% and that of F-I cells was reduced by 55% (P<0.05 and P<0.001, respectively) (Fig 2D). Since the majority of immature B cells mature in the spleen [5,27], we further investigated the numbers of B220+CD21intIgDhiIgMhi F-II and B220+CD21intIgDhiIgMlo F-I B cells in the spleens of SIRPα-mutant mice. This analysis first revealed that 13 weeks old mutant mice had a significantly reduced number of B220+ B cells (P<0.05, Fig 3A). Second, both the F-II and F-I B cell subsets were significantly reduced in SIRPα-mutant spleens, as compared with that in wild-type spleens (P<0.01, Fig 3B). Supporting our previous finding that B cells lack SIRPα expression in secondary lymphoid organs [24], we found that SIRPα was neither expressed by B220+ IgM+ CD43- B cells in the BM (S2A Fig) nor by splenic FoB cells (S2B Fig). Thus, SIRPα signaling outside the B cell compartment appeared to be required to maintain normal levels of immature B cells in the BM and to support B cell maturation in both the BM and the spleen. B cell survival is regulated by pro-survival proteins (e.g. Bcl-2 and Bcl-XL) and pro-apoptotic proteins (e.g. BIM) [28]. To test the hypothesis that there was a reduced survival of mature follicular B cells in mutant spleens, we quantified the mRNA expression levels of Bcl-2 and BIM in FACS-sorted CD19+CD21intCD23hi mature splenic FoB cells. This analysis showed that there were no differences in gene expression of these two proteins when comparing wild-type and mutant FoB cells (Fig 3C). However, it has been recognized that in lymphocytes, the pro-apoptotic activity of BIM can also be regulated at the posttranslational level [29]. Therefore, we next investigated BIM protein levels in BM or splenic B cells of wild-type or SIRPα-mutant mice. In the BM, we found an increased level of BIM protein in immature and T1 B cells (P = 0.08 and P<0.01, respectively; Fig 2E). In addition, both splenic F-II and F-I B cells showed an increased level of BIM protein (P<0.01 and P<0.05, respectively; Fig 3D). To further investigate the possibility of an increased rate of B cell apoptosis in SIRPα-mutant mice, we quantified the amount of annexin V-positive cells. We found a significantly increased amount of apoptotic immature, T1 and F-II B cells in the BM of mutant mice, as compared with that in wild-type mice (P<0.001, P<0.05 and P<0.05, respectively; Fig 2F). In the spleen, the fraction of apoptotic F-II B cells was significantly increased in SIRPα-mutant mice, as compared with that in wild-type mice (P<0.01; Fig 3E). In contrast, we did not find any differences in cell cycle activity among these B cell subsets in the spleen or BM when comparing wild-type and SIRPα-mutant mice (S3 Fig). Thus, a reduced number of maturing B cells in the BM and spleen of SIRPα-mutant mice appeared to be associated with an increased rate of B cell apoptosis.

Bottom Line: Here we provide evidence that signal regulatory protein α (SIRPα), an Ig-superfamily ITIM-receptor expressed by myeloid but not by lymphoid cells, is involved in regulating B cell maturation.Lack of SIRPα signaling in adult SIRPα-mutant mice resulted in a reduced maturation of B cells in the bone marrow, evident by reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells, as well as reduced blood B cell numbers.In addition, lack of SIRPα signaling also impaired follicular B cell maturation in the spleen.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Medical Biology, Umeå University, Umeå, Sweden.

ABSTRACT
B lymphocyte development occurs in the bone marrow, while final differentiation and maturation can occur in both the bone marrow and the spleen. Here we provide evidence that signal regulatory protein α (SIRPα), an Ig-superfamily ITIM-receptor expressed by myeloid but not by lymphoid cells, is involved in regulating B cell maturation. Lack of SIRPα signaling in adult SIRPα-mutant mice resulted in a reduced maturation of B cells in the bone marrow, evident by reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells, as well as reduced blood B cell numbers. In addition, lack of SIRPα signaling also impaired follicular B cell maturation in the spleen. Maturing BM or splenic B cells of SIRPα-mutant mice were found to express higher levels of the pro-apoptotic protein BIM and apoptosis was increased among these B cells. Bone marrow reconstitution experiments revealed that the B cell maturation defect in bone marrow and blood was due to lack of SIRPα signaling in non-hematopoietic cells, while hematopoietic SIRPα signaling was important for follicular B cell maturation in the spleen. Adding on to our previous findings of a stromal cell defect in SIRPα-mutant mice was the finding that gene expression of receptor activator of nuclear factor-ĸB ligand (RANKL) was significantly lower in cultured bone marrow stromal cells of SIRPα mutant mice. These data suggest a novel and opposite contribution of SIRPα signaling within non-hematopoietic and hematopoietic cells, respectively, to maintain B cell maturation and to prevent apoptosis in the bone marrow and spleen of adult mice.

No MeSH data available.


Related in: MedlinePlus