A role for S1P and S1P1 in immature-B cell egress from mouse bone marrow

Abstract

B lymphocyte egress from secondary lymphoid organs requires sphingosine-1-phosphate (S1P) and S1P receptor-1 (S1P1). However, whether S1P contributes to immature-B cell egress from the bone marrow (BM) has not been fully assessed. Here we report that in S1P- and S1P1-conditionally deficient mice, the number of immature-B cells in the BM parenchyma increased, while it decreased in the blood. Moreover, a slower rate of bromodeoxyuridine incorporation suggested immature-B cells spent longer in the BM of mice in which S1P1-S1P signaling was genetically or pharmacologically impaired. Transgenic expression of S1P1 in developing B cells was sufficient to mobilize pro- and pre-B cells from the BM. We conclude that the S1P1-S1P pathway contributes to egress of immature-B cells from BM, and that this mechanism is partially redundant with other undefined pathways.

Figure 1. Progression into the Immature IgD^lo B cell stage coincides with increased egress efficiency.

(A) Expression of IgM and IgD in B220⁺ BM B-lineage cells. Numbers indicate percent of total B220⁺ cells. (B) Distribution of the indicated B-lineage populations in BM parenchyma and sinusoids. Numbers indicate percent of cells from each subset residing in sinusoids. (C) Expression of IgM and IgD in B220⁺ B-lineage cells in peripheral blood. Numbers indicate percent of total B220⁺ cells. (D) Enumeration of developing B cell subsets in BM parenchyma and sinusoids, and peripheral blood. In all panels, data shown are representative of more than 10 independent experiments. ** P<0.005 (unpaired, two-tailed Student's t-test).

Figure 2. Impaired BM egress of S1P1-deficient immature-B cells.

(A) Quantitative RT-PCR analysis of transcript abundance for the genes encoding S1P1 (Edg1), S1P2 (Edg5), S1P3 (Edg3), S1P4 (Edg6) and S1P5 (Edg8) in purified developing BM B cell subsets presented relative to Hprt1. Data shown are representative of 3 experiments. (B) Number of B cells in indicated subsets in the BM parenchyma and sinusoids, peripheral blood and spleens of Edg1^Fl/− Mb1^Cre/+ (gray bars) and Edg1^+/+ Mb1^Cre/+ (white bars) mice. Data are pooled from 4 independent experiments. (C) Analysis of B cell development in chimeras reconstituted with mixtures of Edg1^Fl/− Mb1^Cre/+ (gray bars, Ly5.2⁺) or Edg1^+/+ Mb1^Cre/+ (white bars, Ly5.2⁺) and wild-type (Ly5.1⁺) BM. Shown is the ratio of Ly5.2⁺ cells in the indicated developmental subsets with that of Pro-B cells in the same animal. Data are pooled from 3 independent experiments. (D) Number of developing B cells in total BM, blood and spleens of mice treated with FTY720 (1 mg/Kg, gray bars) or with saline (white bars) for 3 h. Data shown are representative of more than 3 independent experiments. * P<0.05; ** P<0.005 (unpaired, two-tailed Student's t-test).

Figure 3. S1P1 surface expression in BM parenchymal and sinusoidal cells and impaired egress of immature-B cells from the BM of S1P-deficient mice.

(A) S1P1 surface expression in BM parenchymal and sinusoidal naïve CD4⁺ T cells. Mice were treated with Ly5.2-PE antibodies intravenously for 2 minutes. T cells were gated as TCRβ⁺ CD4⁺ CD8⁻ L-sel⁺, and further gated as parenchymal (Ly5.2⁻, black) and sinusoidal (Ly5.2⁺, gray). Top panel shows cells stained with a control rabbit antiserum, and bottom panel shows cell staining with an anti-S1P1 rabbit antiserum. Data are representative of two experiments (n = 6). (B) Number of developing B cell subsets in the BM parenchyma, sinusoids, blood and spleen of Sphk-1 and -2-deficient and littermate control mice. Data shown were pooled from 4 independent experiments. * P<0.05; ** P<0.005 (unpaired, two-tailed Student's t-test).

Figure 4. Prolonged residence of immature-B cells in BM of S1P1 or S1P deficient mice.

(A) Analysis of BrdU incorporation for 48 h in developing B cell subsets in chimeras reconstituted with mixtures of Edg1^Fl/− Mb1^Cre/+ (gray bars, Ly5.2⁺) or Edg1^+/+ Mb1^Cre/+ (white bars, Ly5.2⁺) with wild-type (Ly5.1⁺) BM. Shown is the ratio of percent BrdU⁺ Ly5.2⁺ and BrdU⁺ Ly5.1⁺ cells in the indicated developmental subsets. Data are pooled from 3 independent experiments. (B) BrdU incorporation for 48 h in developing B cell subsets from Sphk-1 and -2-deficient (gray bars) and littermate controls (white bars). Data shown were pooled from 2 independent experiments. (C) BrdU incorporation for 48 h in developing B cell subsets from mice treated with saline (white bars) or FTY720 (1 mg/Kg; gray bars) for 3 days. Data shown are representative of 3 independent experiments. In all panels, bars indicate the mean, circles indicate individual mice. * P<0.05; ** P<0.005 (unpaired, two-tailed Student's t-test).

Figure 5. Premature egress of B cell precursors from the BM of S1P1-transgenic mice.

(A) Quantitative RT-PCR analysis of the expression of S1P1 encoding mRNA in purified developing BM B cell subsets from S1P1-transgenic (gray bars) and littermate control (white bars) mice, presented relative to Hprt1 mRNA (hypoxanthine guanine phosphoribosyl transferase). Data shown was pooled from 3 independent experiments. (B) Migration assays of developing B cells from S1P1-transgenic (gray bars) and littermate control (white bars) mice to S1P (100 nM) or SDF-1 (0.3 µg/mL). Data are representative of 4 experiments. (C) Enumeration of developing B cell subsets from S1P1-transgenic (gray bars) and littermate control (white bars) mice in the BM parenchyma, sinusoids, peripheral blood and spleen. In all panels, bars indicate the mean, circles indicate individual mice. * P<0.05; ** P<0.005 (unpaired, two-tailed Student's t-test).