Novel insight into stem cell mobilization-plasma sphingosine-1-phosphate is a major chemoattractant that directs the egress of hematopoietic stem progenitor cells from the bone marrow and its level in peripheral blood increases during mobilization due to activation of complement cascade/membrane attack complex - PubMed (original) (raw)

Novel insight into stem cell mobilization-plasma sphingosine-1-phosphate is a major chemoattractant that directs the egress of hematopoietic stem progenitor cells from the bone marrow and its level in peripheral blood increases during mobilization due to activation of complement cascade/membrane attack complex

M Z Ratajczak et al. Leukemia. 2010 May.

Abstract

The complement cascade (CC) becomes activated and its cleavage fragments play a crucial role in the mobilization of hematopoietic stem/progenitor cells (HSPCs). Here, we sought to determine which major chemoattractant present in peripheral blood (PB) is responsible for the egress of HSPCs from the bone marrow (BM). We noticed that normal and mobilized plasma strongly chemoattracts HSPCs in a stromal-derived factor-1 (SDF-1)-independent manner because (i) plasma SDF-1 level does not correlate with mobilization efficiency; (ii) the chemotactic plasma gradient is not affected in the presence of AMD3100 and (iii) it is resistant to denaturation by heat. Surprisingly, the observed loss of plasma chemotactic activity after charcoal stripping suggested the involvement of bioactive lipids and we focused on sphingosine-1-phosphate (S1P), a known chemoattracant of HSPCs. We found that S1P (i) creates in plasma a continuously present gradient for BM-residing HSPCs; (ii) is at physiologically relevant concentrations a chemoattractant several magnitudes stronger than SDF-1 and (iii) its plasma level increases during mobilization due to CC activation and interaction of the membrane attack complex (MAC) with erythrocytes that are a major reservoir of S1P. We conclude and propose a new paradigm that S1P is a crucial chemoattractant for BM-residing HSPCs and that CC through MAC induces the release of S1P from erythrocytes for optimal egress/mobilization of HSPCs.

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Figures

Figure 1

Figure 1. Plasma SDF-1 level does not correlate with mobilization efficiency of HSPCs

Panel A - SDF-1 concentrations are low (less than 1 ng/ml) and do not differ significantly in the plasma of patients who are poor (n=7 or good (n=9) G-CSF mobilizers Panel B - As in patients, SDF-1 concentration is murine plasma is also low (less than 2 ng/ml) and does not increase significantly during Zymosan- or G-CSF-induced mobilization. Panel C – SDF-1 at a physiological concentration does not show chemotactic activity against murine BM HSPCs. The data shown in Panels B and C represent the combined results from three independent experiments carried out in triplicate per group (n=9). * p<0.05, ** p<0.01

Figure 2

Figure 2. HSPCs egress from BM into PB in an SDF-1-independent manner

Panel A – Murine CFU-GM are much more efficiently chemoattracted by murine plasma than by a physiological concentration of SDF-1. Potential involvement of SDF-1 can be excluded because normal CFU-GM progenitor cells (black bar) responded to heat-inactivated plasma (HI-P) as well as the presence of 5 μM AMD3100 (white bar - NP). However, exposure to AMD3100-inhibited chemotaxis to SDF-1. Panel B - CFU-GM cells do not migrate to charcoal-stripped, lipid-free plasma; however they respond normally to the SDF-1 added (50 ng/ml) charcoal-stripped plasma. M, media-alone control; NP, normal murine plasma (2.5%); HI-P, heat-inactivated murine plasma (2.5%); SDF-1 (ng/ml); FP, filtered normal plasma filtered with 0.22 μm membrane; FP-LF, charcoal-stripped and then filtered plasma. * p<0.01. The data shown are the combined results from three independent experiments carried out in triplicate per group (n=9).

Figure 3

Figure 3. S1P is a major chemotactic component of plasma

Panel A - Murine plasma S1P levels increase during mobilization (10 min after zymosan-, 1 h after AMD3100-, and 6 h at day 6 after the last G-CSF-treatment). Panel B and C - However, S1P does not chemoattract differentiated/mature BMNCs; (B), it strongly chemoattracts BM-residing naïve HSPCs (C). Panel D – S1P has much lower chemotactic activity against G-CSF-mobilized PB circulating HSPCs. Ctl, normal mouse plasma; M, media only control; SDF-1 (ng/ml). * p<0.01, ** p<0.001. The data shown represent the combined results from three independent experiments carried out in triplicate per group (n=9).

Figure 4

Figure 4. S1P exposed- or plasma pre-incubated BM-HSPCs do not respond to S1P

Pre-incubation of BM-HSPCs with plasma from the same donor (Panel A, striped bar) or with S1P (Panel B), gray bar-100 nM, white bar-500 nM) for 1 h abrogated migration of BM-naïve CFU-GM progenitors to an S1P gradient. M, media alone control. * p<0.001. The data shown represent the combined results from three independent experiments carried out in triplicate per group (n=9).

Figure 5

Figure 5. The role of MAC in mobilization

Panel A – Free hemoglobin level in plasma correlates with strength of CC activation. Panel B – MAC deposits are detectable in BM of G-CSF-mobilized wt but not C5−/− mice. Diffuse MAC deposition is visible in endothelial cells (arrow), osteoblasts (arrow head), and interstitium. Original magnification ×200. Panel C – Chemotactic response of BM CFU-GM to erythrocyte lysates prepared from plasma-washed erythrocytes followed by repeated freezing and thawing. Ctl, normal mouse plasma; M, media only control. * p<0.01, ** p<0.001. The data shown represent the combined results from three independent experiments carried out in triplicate per group (n=9).

Figure 6

Figure 6. Impaired responsiveness of HSPCs from DOP- treated mice to S1P

Panel A - BM HSPCs harvested from DOP- treated mice (white bar) do not migrate to a S1P gradient. Panel B - D - DOP treated mice are poor AMD3100 mobilizers *p<0.0001. The data shown represent the combined results from three independent experiments carried out in triplicate per group (n=9) and the combined results of three independent mobilization experiments carried out with 5 mice per group (n=15).

Figure 7

Figure 7. Novel mechanistic insight into BM HSPC mobilization

HSPCs are actively retained in BM and retention signals in the BM niches counteract a S1P-mediated chemotactic plasma gradient. Mobilizing agents disrupt major HSPC-anchoring signals (i.e., CXCR4-SDF-1 and VLA4-VCAM-1 interactions) and release HSPCs from their niches (Step I). First, as reported previously,, activated granulocytes egress from BM into sinusoids in response to C5 cleavage fragments and thus pave the way for HSPC (Step II). Next, MAC generated in the final step of CC activation enhances release of S1P from erythrocytes into the plasma, and the plasma S1P level directs egress of HSPCs into PB (Step III).

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