S1P3 confers differential S1P-induced migration by autoreactive and non-autoreactive immature B cells and is required for normal B-cell development

Abstract

During B-cell development, immature B-cell fate is determined by whether the BCR is engaged in the bone marrow. Immature B cells that are non-autoreactive continue maturation and emigrate from the marrow, whereas autoreactive immature B cells remain and are tolerized. However, the microenvironment where these events occur and the chemoattractants responsible for immature B-cell trafficking within and out of the bone marrow remain largely undefined. Sphingosine 1-phosphate (S1P) is a chemoattractant that directs lymphocyte trafficking and thymocyte egress and in this study we investigated whether S1P contributes to B-cell development, egress and positioning within the bone marrow. Our findings show that immature B cells are chemotactic toward S1P but that this response is dependent on Ag receptor specificity: non-autoreactive, but not autoreactive, immature B cells migrate toward S1P and are shown to require S1P3 receptor for this response. Despite this response, S1P3 is shown not to facilitate immature B-cell egress but is required for normal B-cell development including the positioning of transitional B cells within bone marrow sinusoids. These data indicate that S1P3 signaling directs immature B cells to a bone marrow microenvironment important for both tolerance induction and maturation.

Figure 1

Immature B cells with non-autoreactive antigen receptors migrate towards S1P. A) Representative flow cytometric analysis of IgM and IgD expression by non-autoreactive immature B cells generated by IL-7 in vitro culture of bone marrow cells (left panel) and 3–83 idiotype (54.1) and IgM expression by ex vivo bone marrow immature B cells gated as B220⁺ IgD^low/− cells (right panel) from 3-83Igi, H-2^d mice. B) Chemotaxis of non-autoreactive immature B cells towards differing concentrations of S1P or media (−). Data are representative of at least 4 independent experiments and are shown as mean with SD for duplicate wells. C) Cumulative results as for the experiment in (B) at 10nM S1P, mean ± SEM of 18 independent experiments; *p<0.05 in a Student’s one-tailed unpaired t-test. Non-autoreactive immature B cells were generated from IL-7 bone marrow cultures using 3-83Igi, H-2^d mice (B, C). D) B220+ bone marrow cells were isolated from 3-83Igi, H-2b mice and migration to the indicated concentrations of S1P or 0.5μg/mL CXCL12 by autoreactive (IgM^low 3-83^low IgD⁻) and edited IgM⁺ 3-83⁻IgD⁻cells measured. Data are mean ± SEM of 4–8 independent experiments; *p<0.05 in a Student’s one-tailed unpaired t-test. CXCL12 migration is representative of 4 independent experiments. E) Non-autoreactive immature B cells from IL-7 bone marrow cultures were allowed to migrate towards media (−) or 10 nM S1P during an initial migration period (0–3 hr) after which non-responding cells were again assayed for migration towards media or S1P after a second 3 hour migration period (3–6 hr). Data are mean ± SEM of 3 independent experiments.

Figure 2

Immature B cell migration to S1P in vitro requires S1P3. A) S1P receptor mRNA expression relative to HPRT of non-autoreactive and autoreactive immature B cells was measured by quantitative PCR. Data are mean ± SEM of 4 independent experiments. B) Non-autoreactive immature B cells from IL-7 bone marrow cultures were tested for migration to 10 nM S1P, 0.5 μg/mL CXCL12 or media in the presence or absence of 100 μM of the CAY10444 S1P3 antagonist. Data are mean ± SEM of 3 independent experiments with *p=0.05 in a Student’s one-tailed unpaired t-test. C) Non-autoreactive immature B cells from IL-7 cultures were tested for migration to SEW2871, an S1P1 agonist, VPC24191, an S1P1 and S1P3 agonist, VPC23153, an S1P4 agonist, 10 nM S1P or media (−). Data are representative of 3 independent experiments. D) B220+ bone marrow cells were isolated from S1P3^+/− or S1P3^−/− mice and tested ex vivo for migration of immature B220⁺ IgM⁺ IgD⁻B cells to the indicated concentrations of S1P, 0.5 μg/mL CXCL12 or media. IgM⁺ IgD^high mature cells were excluded from analysis. Data are mean ± SEM of 4 independent experiments; *p<0.05 Student’s one-tailed unpaired t-test.

Figure 3

S1P3 deficient and sufficient immature B cells have similar bone marrow egress. A) Absolute (right panel) and relative (left panel) numbers of B220⁺ CD93⁺ (immature/transitional) and B220⁺ CD93⁺ (mature) B cells were determined in spleens of 11 week old S1P3^−/− (n = 6) and S1P3^+/− (n = 6) littermate mice. Data are mean ± SEM of 3 independent experiments; *p<0.05 Student’s one-tailed unpaired t-test. B) S1P3^+/+ (wt, n = 8) or S1P3^−/− (n = 8) mice were injected i.p. with 1 mg of BrdU every 24 hours for the duration of the experiment. On day four, bone marrow (BM), blood (Bld) and spleen (Spl) cells were harvested and the percentage of BrdU+ cells for each B cell subset was determined by flow cytometry. Data are mean ± SEM of 3 independent experiments. C) B cell frequencies were determined in chimeric mice (n = 17, total) reconstituted with a mixture of lineage depleted bone marrow cells from S1P3^−/− and B6.SJL wild type mice. S1P3^−/− B cells were identified as CD45.1- and wild type B cells were identified as CD45.1⁺. B cell subsets in the bone marrow were defined as B220⁺ IgM⁺ IgD⁻ (immature) and B220⁺ IgM⁺ IgD^mid (transitional) and in the blood and spleen as B220+ CD93^high (immature) and B220+ CD93^mid (transitional). Data are mean ±SEM of 3 independent experiments.

Figure 4

Abnormal B cell development in bone marrow of S1P3^−/− mice. A) Absolute numbers of B cell subsets were determined in bone marrow of 11 week old S1P3^−/− (n = 6) and S1P3^+/− (n = 6) littermate mice. B cell subsets in bone marrow were defined as B220⁺ CD2⁻IgM⁻ (pro), B220⁺ CD2⁺ IgM⁻ (pre), B220⁺ IgM⁺ IgD⁻ (immature), B220⁺ IgM⁺ IgD^mid (transitional) and B220⁺ IgM⁺ IgD^high (mature). Data are mean ±SEM of 3 independent experiments; *p<0.05 Student’s one-tailed unpaired t-test. B) Absolute and relative numbers of bone marrow sinusoidal B cells were determined in S1P3−/− (n = 8) and S1P3+/− or S1P3+/+ (n = 7) control mice injected i.v. with 1 μg anti-CD19-PE for 2 minutes. Sinusoidal (CD19-PE⁺) B cell subsets in bone marrow were defined as in (A). Data are mean ± SEM of 3 independent experiments; *p<0.05 Student’s one-tailed unpaired t-test. C) Percentages of bone marrow sinusoidal (CD19-PE⁺) B cells were determined in 3-83Igi, H-2^b (n = 6) and 3-83Igi, H-2^d (n = 6) mice injected i.v. with 1 μg anti-CD19-PE for 2 minutes. B cell subsets were defined as B220⁺ IgM^low 3-83^low IgD⁻ (immature autoreactive), B220⁺ IgM⁺ 3-83⁻IgD⁻ (immature receptor edited), B220⁺ IgM⁺ 3-83⁻IgD^mid (transitional receptor edited) in 3-83Igi, H-2^b (n = 6) mice and B220⁺ IgM⁺ 3-83⁺ IgD⁻ (immature non-autoreactive) and B220⁺ IgM⁺ 3-83⁺ IgD^mid (transitional non-autoreactive) in 3-83Igi, H-2^d (n = 6) mice. Data are mean ± SEM of 3 independent experiments; *p<0.05 Student’s one-tailed unpaired t-test. D) Percentage of Igλ+ B cells in total bone marrow in S1P3^−/− (n = 8) and S1P3^+/− or S1P3^+/+ (n = 7) control mice. B cells subsets were defined as described in (A). Data are mean ± SEM of 3 independent experiments; *p<0.05 Student’s one-tailed unpaired t-test.