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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

Reduced RANKL gene expression, in vitro, in BM stromal cell culture of SIRPα-mutant mice.The mRNA expression of rankl in wild-type (Wt–open bar) or SIRPα-mutant mice (Mut–black bar) BM stromal cells cultured for 12 days, was determined by RT-qPCR. Wild-type control was set to 100% and data of mRNA analyses are quantitative values normalized to the house keeping gene β-actin. The rankl mRNA expression was analyzed in three separate experiments and the data shown are the means±SEM of 4 samples/group in one representative experiment. **P<0.01, using Student’s t-test for unpaired analyses.
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pone.0134113.g005: Reduced RANKL gene expression, in vitro, in BM stromal cell culture of SIRPα-mutant mice.The mRNA expression of rankl in wild-type (Wt–open bar) or SIRPα-mutant mice (Mut–black bar) BM stromal cells cultured for 12 days, was determined by RT-qPCR. Wild-type control was set to 100% and data of mRNA analyses are quantitative values normalized to the house keeping gene β-actin. The rankl mRNA expression was analyzed in three separate experiments and the data shown are the means±SEM of 4 samples/group in one representative experiment. **P<0.01, using Student’s t-test for unpaired analyses.

Mentions: In contrast to that in lymphoid cells, SIRPα is expressed by both non-hematopoietic stromal cells [25] and hematopoietic cells [21], such as dendritic cells, which may all be involved in supporting maintenance and maturation of B cells in the BM and spleen [7,30]. To investigate how SIRPα signaling within the hematopoietic and non-hematopoietic compartments contributed to B cell maturation in the BM and spleen, we reconstituted either Ly5.2 wild-type or SIRPα-mutant mice with Ly5.1 wild-type BM cells, or Ly5.1 wild-type mice with Ly5.2 wild-type or mutant BM cells. We then followed the blood B cell numbers in these chimeric mice over time to find a blood B cell phenotype that was similar to the of naïve SIRPα-mutant mice. At 20 weeks after BM transfer, the number of CD19+ blood B cells was significantly reduced in SIRPα-mutants receiving wild-type BM, as compared with that in wild-type recipients of wild-type BM (Fig 4A). Using the gating strategy described in Fig 1A, we found that the number of immature B cells was normal whereas that of F-II and mature B cells were significantly reduced in SIRPα-mutant recipients of wild-type BM (P<0.001 and P<0.01, respectively) (Fig 4A). In contrast, all B cell subsets were normal in the blood of wild-type mice receiving either wild-type or mutant BM (Fig 4D). In the blood of SIRPα-mutant recipients of wild-type BM, the number of T cells was significantly reduced (P<0.05), while the numbers of neutrophils or monocytes were similar, as compared with that in wild-type recipients of wild-type BM (Fig 4B). However, in wild-type mice receiving either wild-type or mutant BM, we did not find any differences in the numbers of blood T cells, neutrophils or monocytes (Fig 4E). While the number of IgD-IgM+ immature B cells was not reduced in the BM of SIRPα-mutants reconstituted with wild-type BM, IgD+IgMhi F-II and IgD+IgMlo F-I B cells were significantly reduced, as compared with that in wild-types receiving wild-type BM (Fig 4C), a phenotype remarkably similar to that seen in naïve SIRPα-mutant mice (Fig 2C). In contrast, these B cell subsets were similar in the BM of wild-type recipients of either wild-type or mutant BM (Fig 4F). Thus, SIRPα-signaling within the non-hematopoietic compartment appeared to be important to maintain circulating B cells and normal B cell maturation in the BM. Although very little is known about factors regulating B cell maturation in the BM, and RANKL is unlikely to affect B cell maturation per se [9], we decided to investigate if SIRPα-mutant BM stromal cells showed a phenotype different from that of their wild-type counterparts by measuring stromal cell RANKL gene expression. Interestingly, the gene expression of RANKL was significantly reduced by more than 50% in SIRPα-mutant BM stromal cells cultured for 12 days, as compared to wild-type stromal cells (Fig 5). In marked contrast to that seen in the BM, and despite a drop in blood B cell numbers, SIRPα-mutant mice reconstituted with wild-type BM did not have a significantly reduced number of F-II or F-I B cells in the spleen (Fig 6A). However, while blood B cell numbers and B cell maturation in the BM appeared normal in wild-type recipients of mutant BM, splenic F-II or F-I B cell numbers were reduced to a similar extent as that seen in naïve SIRPα-mutant mice (Fig 6B).


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)

Reduced RANKL gene expression, in vitro, in BM stromal cell culture of SIRPα-mutant mice.The mRNA expression of rankl in wild-type (Wt–open bar) or SIRPα-mutant mice (Mut–black bar) BM stromal cells cultured for 12 days, was determined by RT-qPCR. Wild-type control was set to 100% and data of mRNA analyses are quantitative values normalized to the house keeping gene β-actin. The rankl mRNA expression was analyzed in three separate experiments and the data shown are the means±SEM of 4 samples/group in one representative experiment. **P<0.01, using Student’s t-test for unpaired analyses.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519279&req=5

pone.0134113.g005: Reduced RANKL gene expression, in vitro, in BM stromal cell culture of SIRPα-mutant mice.The mRNA expression of rankl in wild-type (Wt–open bar) or SIRPα-mutant mice (Mut–black bar) BM stromal cells cultured for 12 days, was determined by RT-qPCR. Wild-type control was set to 100% and data of mRNA analyses are quantitative values normalized to the house keeping gene β-actin. The rankl mRNA expression was analyzed in three separate experiments and the data shown are the means±SEM of 4 samples/group in one representative experiment. **P<0.01, using Student’s t-test for unpaired analyses.
Mentions: In contrast to that in lymphoid cells, SIRPα is expressed by both non-hematopoietic stromal cells [25] and hematopoietic cells [21], such as dendritic cells, which may all be involved in supporting maintenance and maturation of B cells in the BM and spleen [7,30]. To investigate how SIRPα signaling within the hematopoietic and non-hematopoietic compartments contributed to B cell maturation in the BM and spleen, we reconstituted either Ly5.2 wild-type or SIRPα-mutant mice with Ly5.1 wild-type BM cells, or Ly5.1 wild-type mice with Ly5.2 wild-type or mutant BM cells. We then followed the blood B cell numbers in these chimeric mice over time to find a blood B cell phenotype that was similar to the of naïve SIRPα-mutant mice. At 20 weeks after BM transfer, the number of CD19+ blood B cells was significantly reduced in SIRPα-mutants receiving wild-type BM, as compared with that in wild-type recipients of wild-type BM (Fig 4A). Using the gating strategy described in Fig 1A, we found that the number of immature B cells was normal whereas that of F-II and mature B cells were significantly reduced in SIRPα-mutant recipients of wild-type BM (P<0.001 and P<0.01, respectively) (Fig 4A). In contrast, all B cell subsets were normal in the blood of wild-type mice receiving either wild-type or mutant BM (Fig 4D). In the blood of SIRPα-mutant recipients of wild-type BM, the number of T cells was significantly reduced (P<0.05), while the numbers of neutrophils or monocytes were similar, as compared with that in wild-type recipients of wild-type BM (Fig 4B). However, in wild-type mice receiving either wild-type or mutant BM, we did not find any differences in the numbers of blood T cells, neutrophils or monocytes (Fig 4E). While the number of IgD-IgM+ immature B cells was not reduced in the BM of SIRPα-mutants reconstituted with wild-type BM, IgD+IgMhi F-II and IgD+IgMlo F-I B cells were significantly reduced, as compared with that in wild-types receiving wild-type BM (Fig 4C), a phenotype remarkably similar to that seen in naïve SIRPα-mutant mice (Fig 2C). In contrast, these B cell subsets were similar in the BM of wild-type recipients of either wild-type or mutant BM (Fig 4F). Thus, SIRPα-signaling within the non-hematopoietic compartment appeared to be important to maintain circulating B cells and normal B cell maturation in the BM. Although very little is known about factors regulating B cell maturation in the BM, and RANKL is unlikely to affect B cell maturation per se [9], we decided to investigate if SIRPα-mutant BM stromal cells showed a phenotype different from that of their wild-type counterparts by measuring stromal cell RANKL gene expression. Interestingly, the gene expression of RANKL was significantly reduced by more than 50% in SIRPα-mutant BM stromal cells cultured for 12 days, as compared to wild-type stromal cells (Fig 5). In marked contrast to that seen in the BM, and despite a drop in blood B cell numbers, SIRPα-mutant mice reconstituted with wild-type BM did not have a significantly reduced number of F-II or F-I B cells in the spleen (Fig 6A). However, while blood B cell numbers and B cell maturation in the BM appeared normal in wild-type recipients of mutant BM, splenic F-II or F-I B cell numbers were reduced to a similar extent as that seen in naïve SIRPα-mutant mice (Fig 6B).

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