To examine the potentials of agonists and antagonists of CXCR4, we have monitored directed migration, podia formation, adhesion behavior, and proliferation of human HPC under the influence of SDF-1α, a peptide agonist of CXCR4 (CTCE-0214), a peptide antagonist (CTCE-9908), and a nonpeptide antagonist (AMD3100). Despite the rationale that all of the SDF-1α analogs act on the same CXCR4 receptor, we have demonstrated that these compounds might have differential effects on different functional properties of SDF- 1α. It might be speculated that the SDF-1α analogs induce different conformational changes of the CXCR4 receptor or that other coreceptors are involved. These results suggest that the signal cascade induced by SDF-1α is not a monocausal succession (SDF-1α binding to CXCR4 activating G-proteins further activating downstream mediators) but rather a complex network [6, 20]. Analysis of the downstream targets in signal cascades such as calcium flux or MAPKp42/42 activation is concurrently under way and might help to clarify some of the effects of CXCR4 agonists and antagonists. SDF-1α is a powerful chemoattactant for primitive human hematopoietic progenitor cells [20, 41, 42]. Chemotaxis of CD34+ cells can be enhanced by SDF-1α in a dose-dependent manner in concentrations ranging from 0.01 μg/mL to 0.5 μg/mL [9, 41]. Zhong et al. have previously reported that the peptide agonist CTCE-0214 can also enhance migration of CD34+ cells in a transwell migration assay . These authors used concentrations of CTCE-0214 up to 100 μg/mL and they described a six-fold increase in migration of CD34+ cells from mobilized peripheral blood as compared to unstimulated cells. In analogy, we observed a moderate increase in cell migration using 100 μg/mL CTCE- 0214. However, using the same concentration range as for SDF-1α (0.01 μg/mL–0.5 μg/mL), we demonstrated that neither CTCE-0214 nor CTCE-9908 or AMD3100 exerted significant effects on migration of CD34+ cells from umbilical cord blood. Thus, in comparison to SDF-1α and on a μg to μg basis, none of the agonists and antagonists were a potent chemoattractant.
We have previously demonstrated that the primitive fraction of slow dividing cells has a higher proportion of elongated cells with a prominent uropod . Furthermore, HPCs adhere to supportive feeder layer cells with their uropod at the trailing edge . We and others have previously demonstrated that SDF-1α affects podia formation in various AML cell lines [9, 10]. In this study, we have shown that polarization and formation of an prominent uropod can be increased in CD34+ cells in a dose-dependent manner by SDF-1α. This effect can probably be attributed to cytoskeleton rearrangements of actin-containing protrusions . Surprisingly the peptide agonist and the peptide antagonist also had a significant impact on podia formation although they did not induce directed migration. Thus, directed migration in chemotaxis does not directly correlate with podia formation.
Survival of CD34+ cells in culture could bemaintained to a limited extent by cytokines such as thrombopoietin (TPO), stem cell factor (SCF), or flt-3 ligand alone or in combination. Some studies have indicated that SDF-1α also might have a moderate effect on survival of CD34+ cells and this effect was significantly enhanced in combination with other cytokines [11–13, 15]. In analogy, it has been shown that CTCE-0214 alone did not increase the viability of CD34+ cells, whereas a synergistic activity of CTCE-0214 in conjunction with other growth factors has been described .
In this study, we have demonstrated that neither the native molecule SDF-1α nor the agonist nor the antagonists alone had a significant impact on proliferation or survival of CD34+ cells. We cannot exclude the possibility that SDF-1α agonists or antagonists might induce proliferation when applied in conjunction with other chemokines as described before. It has been reported that AMD3100 was able tomobilize a CD34+ population with higher proliferative potential than upon mobilization with G-CSF . These observations are most likely due to different subfractions of CD34+ cells mobilized by AMD3100 as we did not observe a significant effect of this compound on proliferation.
Human BM-MSC represents a surrogate in vitro model for studying the specific molecular mechanisms of adhesion of HPC towards the cellular niche . The adhesion assay described here was used to test different compounds in parallel and it might be suitable for testing new chemical compounds that play a role on heterotypic cell-cell adhesion. We have demonstrated in this study that adhesion of CD34+ cells to BM-MSC was significantly reduced upon treatment with SDF-1α, CTCE-0214, or AMD3100. In contrast, the antagonist CTCE-9908 did not affect cell adhesion. Other authors demonstrated that cell adhesion is increased by SDF-1α [43, 45, 46]. However, in these studies adhesion was usually analyzed upon interaction with extracellular matrix components such as fibronectin, and nonadherent cells were separated by a washing step at one time point. In contrast, in our standardized adhesion assay without shear stress, gravitational force is affecting cell-cell interaction over a time course of one hour. An explanation for the increased migratory activity upon treatment with SDF-1α is that cell adhesion is temporarily loosened and these cells would then detach and add to the no-adherent fraction. Furthermore, it has been reported that CXCR4 activation is also important for mediating specificmigration of bone marrow stromal cells although this receptor seems to be only present at very low levels at the surface of MSC [47–49]. Thus, in contrast to previous studies that analyzed adhesion of HSC to fibronectin, we have analyzed adhesion to MSC with and without the addition of SDF-1α, its agonists, and antagonists. Our results are compatible with the observation that both agonist and antagonists of the SDF-1α/CXCR4 axis have been shown to effectively mobilize primitive HPC to the peripheral blood. Elevated plasma levels of SDF-1α induce mobilization of HPC to the peripheral blood . In the murine system, injection of CTCE-0214 led to an increase of primitive HPC in the peripheral blood . AMD3100 has been reported to mobilize HSC from the bone marrow to peripheral blood efficiently . The increase of HPC is rapid after a single injection and the corresponding clinical trials have been conducted successfully in patients who have failed to respond to granulocyte colony stimulating factors alone.
In our previous work, we provided evidence that more primitive fractions of HPC adhere significantly more than their more differentiated counterparts (CD34+ versus CD34−, CD34+CD38− versus CD34+CD38+, slow dividing fraction versus fast dividing fraction of CD34+CD38− cells) . Furthermore, there is evidence that AMD3100 and GCSF might mobilize a more primitive and hence multipotent HPC population than G-CSF alone [26, 27]. Thus, it could be speculated that CXCR4 agonists and antagonists affect the specific interaction of primitive HPCs and their cellular microenvironment. The proportion of CXCR4+/CD34+ cells was reduced upon stimulation with SDF-1α and AMD3100. Buul et al. have previously demonstrated that SDF-1α induced redistribution and internalization of a CXCR4-fusion protein in KG1a cells . This phenomenon has also been shown for other leukemic cell lines . Dar et al. reported that CXCR4-dependent internalization of and resecretion of SDF-1α by endothelial and stromal cells played an essential role to establish an SDF-1α gradient that assisted directed migration of CD34+ cells . Multiple residues within the CXCR4 C-terminal tail appeared to mediate this receptor internalization . In this context, our data have provided evidence that CXCR4 is also internalized in CD34+ cells upon stimulation with SDF-1α. Hatse et al. reported long-term interaction of AMD3100 with CXCR4 that blocked binding of the same antibody (clone 12G5) at the cell membrane . Furthermore, they described that AMD3100 inhibits SDF- 1α-induced internalization of CXCR4 in U87-CD4 cells. This is in line with our observation that CXCR4 surface detection by the 12G5 antibody is abolished by AMD3100, whereas additional washing steps increased the detection of CXCR4. Thus, further analysis of internalized receptor revealed no effect of AMD3100 treatment on intracellular CXCR4 detection. On the other hand, our results provide evidence that CTCE-0214 does not affect receptor internalization, whereas the peptide antagonist CTCE-9908 enhanced the proportion of CXCR4+ cells, and this observation is compatible with reduced receptor internalization. The G-protein coupled receptor CXCR4 is not an adhesion protein itself. However, recently crosstalk between the CXCR4/SDF-1α axis and other adhesion proteins such as VLA-4, VLA-5, and CD164 has been shown , and hence internalization of CXCR4 upon stimulation with agonists and antagonists might be associated with redistribution of these adhesion proteins, and thereby reducing intercellular adhesion.
Peptide and nonpeptide analogs have several advantages compared to the native molecules. The ease of synthesis, lower manufacturing costs, improved bioavailability, and lower immunogenicity of peptides or analogs may make them more accessible for clinical applications. Recent results have also suggested that the SDF-1α/CXCR4 axis is also used by cancer cells for metastatic dissemination in many types of solid tumours [52, 53]. In addition to mobilization of HPC, CXCR4 agonists and antagonists might have the potential for treatment ofmetastatic diseases. If confirmed, precise knowledge of homing and adhesion and their specific manipulation might have significant therapeutic potentials and implications.
We would like to thank Ms. KatrinMiesala for excellent technical assistance in cell culture and Christopher Schwab for the artwork of Figure 1. This work was supported by the GermanMinistry of Education and Research (BMBF) within the National Genome Research Network NGFN-2 (EP-S19T01) and within the Supporting Program “Cell-Based Regenerative Medicine” (START-MSC), the German Research Foundation DFG (HO 914/2-3), and the Joachim Siebeneicher- Stiftung, Germany. A. Faber and C. Roderburg contributed equally to this paper. The author of correspondence is professor A. D. Ho.