Sphingosine-1-phosphate-mediated mobilization of hematopoietic stem/progenitor cells during intravascular hemolysis requires attenuation of SDF-1-CXCR4 retention signaling in bone marrow

Abstract

Sphingosine-1-phosphate (S1P) is a crucial chemotactic factor in peripheral blood (PB) involved in the mobilization process and egress of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM). Since S1P is present at high levels in erythrocytes, one might assume that, by increasing the plasma S1P level, the hemolysis of red blood cells would induce mobilization of HSPCs. To test this assumption, we induced hemolysis in mice by employing phenylhydrazine (PHZ). We observed that doubling the S1P level in PB from damaged erythrocytes induced only a marginally increased level of mobilization. However, if mice were exposed to PHZ together with the CXCR4 blocking agent, AMD3100, a robust synergistic increase in the number of mobilized HSPCs occurred. We conclude that hemolysis, even if it significantly elevates the S1P level in PB, also requires attenuation of the CXCR4-SDF-1 axis-mediated retention in BM niches for HSPC mobilization to occur. Our data also further confirm that S1P is a major chemottractant present in plasma and chemoattracts HSPCs into PB under steady-state conditions. However, to egress from BM, HSPCs first have to be released from BM niches by blocking the SDF-1-CXCR4 retention signal.

Figure 1

PHZ induces an increase in the total level of S1P in PB. S1P was measured by employing mass spectrophotometry in PB samples harvested at the peak of the mobilization process from mice exposed to phenylhydrazine (PHZ) and from nonmobilized control animals. The data are combined from two independent experiments with 5 animals each. *P < 0.001.

Figure 2

Kinetic of effect of PHZ-induced hemolysis on the mobilization of SKL cells and CFU-GM clonogenic progenitors. C57Bl/6 mice (10 mice per group) were sacrificed 1, 6, and 24 h after injection of PHZ (40 mg/kg i.p.). Control animals were injected with saline (0.9%). (a) shows the number of Sca-1⁺Kit⁺Lin⁻ (SKL) HSPCs circulating in PB (*P < 0.01) and (b) shows the number of clonogenic CFU-GM progenitors circulating in PB (*P < 0.01).

Figure 3

PHZ-induced mobilization of HSPCs is significantly potentiated after administration of AMD3100. The numbers of circulating CFU-GM able to grow colonies in methylcellulose cultures isolated from control, PHZ-, AMD3100-, and PHZ + AMD3100-injected C57Bl/6 mice are shown. The data are combined from two different experiments with 10 animals each. *P < 0.001.