Altered myelopoiesis in <i>Sgpl1^−/−</i>chimeras.

<p>(A) <i>Sgpl1^−/−</i>chimeras show increased CD11b⁺ (monocytes) and CD11b⁺Gr1⁺CD71⁻ neutrophil counts in the circulation (n = 10–11 per group). (B) In bone marrow the increase in neutrophils is already noticeable at the level of the CD11b⁺/Gr1⁺ precursors, while this is not the case for the CD14⁺/GR1⁺ monocytes (n = 10–11 per group). (C) <i>In vivo</i> proliferation of CD11b⁺ cells as measured by EdU incorporation is 3-fold increased in <i>Sgpl1^−/−</i> chimeras (n = 8 per group). (D) Growth-stimulation of bone marrow cells indicated an increased colonigenic capacity of <i>Sgpl1^−/−</i> cells in response to G-CSF and GM-CSF, while no effect for M-CSF was seen (n = 3 per group). (E) Bone marrow-derived macrophages of <i>Sgpl1^−/−</i>chimeras are unresponsive to S1P stimulus as assessed by Ca²⁺-flux measurement, while the Ca²⁺ response to ionomycin and MCP-1 remained unaltered (n = 3 per group). (F). Analysis of plasma and bone marrow-derived macrophage (BM-mφ) supernatants pointed to reduced MCP-1 generation in <i>Sgpl1^−/−</i>chimeras (plasma: n = 10–11 per group, BM-mφ: n = 5 per group). Reduced MCP1 expression by BM derived macrophages of <i>Sgpl1^−/−</i> chimeras was corroborated at an mRNA levels (G), while conversely expression of CCR2 was increased (H) in bone marrow-derived macrophages (n = 10 per group). However, in a model of mild peritonitis, MCP-1 triggered migration of CCR2⁺ inflammatory macrophages (F4/80, P<0.05) in <i>Sgpl1^+/+</i> transplanted mice but not <i>Sgpl1^−/−</i> chimeras (n = 4–5 per group) (I). White bars: wild-type (WT), black bars: <i>Sgpl1^−/−</i> (KO) chimeras. Data are represented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (WT versus KO).