Identification and Characterization of Circulating Variants of CXCL12 from Human Plasma: Effects on Chemotaxis and Mobilization of Hematopoietic Stem and Progenitor Cells

Abstract

Mobilization of hematopoietic stem and progenitor cells (HPCs) is induced by treatment with granulocyte-colony stimulating factor, chemotherapy, or irradiation. We observed that these treatments are accompanied by a release of chemotactic activity into the blood. This plasma activity is derived from the bone marrow, liver, and spleen and acts on HPCs via the chemokine receptor CXCR4. A human blood peptide library was used to characterize CXCR4-activating compounds. We identified CXCL12[22–88] and N-terminally truncated variants CXCL12[24–88], CXCL12[25–88], CXCL12[27–88], and CXCL12[29–88]. Only CXCL12[22–88] could effectively bind to CXCR4 and induce intracellular calcium flux and chemotactic migration of HPCs. CXCL12[25–88] and CXCL12[27–88] revealed neither agonistic nor antagonistic activities in vitro, whereas CXCL12[29–88] inhibited CXCL12[22–88]-induced chemotactic migration. Since binding to glycosaminoglycans (GAG) modulates the function of CXCL12, binding to heparin was analyzed. Surface plasmon resonance kinetic analysis showed that N-terminal truncation of Arg₂₂-Pro₂₃ increased the dissociation constant K_D by one log10 stage ([22–88]: K_D: 5.4±2.6 μM; [24–88]: K_D: 54±22.4 μM). Further truncation of the N-terminus decreased the K_D ([25–88] K_D: 30±4.8 μM; [27–88] K_D: 23±1.6 μM; [29–88] K_D: 19±5.4 μM), indicating increasing competition for heparin binding. Systemic in vivo application of CXCL12[22–88] as well as CXCL12[27–88] or CXCL12[29–88] induced a significant mobilization of HPCs in mice. Our findings indicate that plasma-derived CXCL12 variants may contribute to the regulation of HPC mobilization by modulating the binding of CXCL12[22–88] to GAGs rather than blocking the CXCR4 receptor and, therefore, may have a contributing role in HPC mobilization.