Elevation of Plasma Leukotriene B4 on G-CSF-Induced Hematopoietic Stem Cells Mobilization In Normal Individuals

Previously, we had reported that mobilization kinetics of CD34+ cells association of CD44 and CD31 expression during continuous intravenous administration of G-CSF in normal donors(Stem Cells 18:281-286, 2000, BMT 36:1027-1032, 2005). Meanwhile, a number of studies have reported the mechanisms of G-CSF-induced HSCs mobilization, but the underlying mechanisms are not clear yet. Some chemokines, macrophage inflammatory protein-1α (MIP-1α/CCL3), stem cell-derived factor 1α (SDF-1α/CCL12), interleukin 8 (IL-8/CXCL8), and GROβ/CXCL2, can mobilize HSCs, and we also reported that LTB4, which has considerable functional overlap with the chemokine family of chemoattractant peptides, had mobilized HSCs in murine model. In this study, we have investigated whether G-CSF may affect plasma LTB4 level during HSCs mobilization and G-CSF-induced HSCs mobilization may be modulated by LTB4.

To investigate that G-CSF may modulate plasma LTB4 level, G-CSF (Filgrastim, Kirin Brewery Co. Tokyo, Japan; 10 microgram/kg/day) was administered subcutaneously into 4 healthy donors for 5 days and then, apheresis were performed on day 4 and 5, and were collected for their serum plasma at base line, 24hours, 48hours and 72hours after 1^st dose of G-CSF. LTB4 concentration was measured by enzyme-linked immunosorbent assay (Parameter™ LTB4 Assay; R&D Systems, Mineapolis, MN, USA). Meanwhile, to evaluate the effect of LTB4 inhibition on G-CSF-induced HSCs mobilization, LTB4APA and U75302, which are LTB4 receptor antagonists were given to C57BL/6 mice followed by G-CSF 5μ g or LTB4 1μ g administration intravenously 2 hours later. 24 hours after the G-CSF injection or 4 hours after LTB4 injection, peripheral blood samples were obtained and analyzed for HSCs mobilization by flow cytometry using Sca-1, CD45R(B220), CD116, Gr-1 and TER119.

Plasma LTB4 levels of healthy donors demonstrated increases in 24hrs after G-CSF administration; 415.3±112.1 pg/ml before treatment of G-CSF and 706.4±154.7 pg/ml 24hours after 1^st dose of G-CSF (p=0.005), and then plasma LTB4 levels were decreased continuously after peak level on 24hours. Meanwhile, when LTB4 receptor antagonists were given, the number of HSCs were decreased in the G-CSF mobilized mice; 4.69×10³ cells/ml blood before treatment of G-CSF, 80.08×10³ cells/ml blood after treatment G-CSF without LTB4 receptor antagonist, 9.24×10³ cells/ml blood after treatment of G-CSF with LTB4APA 1μ g respectively(p<0.05, compared with the data of G-CSF alone), 22.86×10³ cells/ml blood after treatment of G-CSF with U75302 1μ g (p<0.05, compared with the data of G-CSF alone). The blocking effects on mobilization of HSCs by LTB4 receptor antagonists were also demonstrated in the LTB4 mobilized mice (data not shown).

We observed that G-CSF increases plasma LTB4 levels during HSCs mobilization in healthy donors, and that LTB4 inhibition by LTB4 receptor antagonists in murine model downregulate the G-CSF-induced HSCs mobilization. These indicate that LTB4 may be involved in the downstream pathway of G-CSF-induced HSCs mobilization. Through the our results, we hypothesize that G-CSF increase LTB4 level in plasma during HSCs mobilization, and LTB4 receptor activation by increased LTB4 level in plasma may contribute HSCs mobilization in vivo. Currently, we are investigating the cellular and molecular mechanism(s) of potential role of LTB4 during G-CSF induced peripheral blood progenitor cell mobilization.

No relevant conflicts of interest to declare.