The process of HSPC mobilization is still not fully understood and a significant number of patients, particularly those with previous histories of chemotherapy, are poor G-CSF mobilizers. Several host-related factors have been described to regulate mobilization, including: i) release of proteases in the BM microenvironment by activated myeloid cells, which perturb interactions of SDF-CXCR4,23,25
and KL-c-KitR axes49–51
; ii) release of neurotransmitters in the BM microenvironment by neural fibers that innervate BM tissue52
; iii) down-regulation of SDF-1 expression in osteoblastic stem cell niches53,54
; iv) activation of osteoclasts55
; and v) up-regulation of serpin family protease inhibitors.56
Recent evidence from our laboratory demonstrates that mobilization of HSPCs is regulated by several components of innate immunity including CC and granulocytes.3,41
Accordingly, we reported that, e.g., G-CSF and cyclophosphamide strongly activate CC in BM by a classical pathway due to exposure during mobilization of neo-epitope on the surface of damaged BMNCs.36,37,57,58
This neo-epitope binds naturally occurring (N)Abs and subsequently neo-epitope-NAb complexes circulating in PB via C1q to trigger the classical Ig-dependent pathway of CC activation. In contrast, zymosan as a polysaccharide may directly activate CC by employing factor B and D involving the Ig-independent alternative CC activation pathway. The crucial role of CC in mobilization of HSPCs was demonstrated in experiments in mice with various defects of CC activation (e.g., Ig-deficient SCID and Rag−/−
, and C5−/−
animals).Granulocytes as a source of proteolytic enzymes in the BM that perturb HSPC retention signals (SDF-CXCR4, VLA-4-VCAM-1, and KL-c-KitR axes) are another important component of innate immunity required for HSPC mobilization. To support this, it was reported that mobilization of HSPCs is severely reduced in neutropenic mice.13,14,39
The pivotal role of CC activation and granulocyte activation/egress was recently reported by us to be perturbed in C5-deficient mice. Accordingly, we described activation of CC in response to a mobilizing agent (G-CSF or zymosan) leads to C5 clevage and that C5 clevage fragments C5a and desArg
C5a via BM-granulocytes orchestrate egress of HSPCs into PB. We noticed that C5a stimulates BM-granulocytes to secrete proteases, which contribute with other factors to establish a proteolytic environment on BM.40,41
C5a activated in BM sinusoids chemoattracts granulocytes that egress from BM as a first wave of cells to “pave the way” for HSPCs that follow. Finally, granulocytes mobilized into PB release several cationic peptides that increase responsiveness of HSPCs to SDF-1.
AMD3100 (plerixafor) is a bicyclam with two 1,4,8,11-tetraazacyklotetradecyne (cyclam) rings linked by 1,4-phenylenbis(methylene) moiety that is currently employed as an efficient mobilizing agent alone or in combination with other drugs (e.g., G-CSF). Based on its different molecular action as compared to G-CSF or zymosan, we asked whether optimal AMD3100-induced mobilization also depends on CC activation. To address this question, we employed mice that possess a defect in the activation of the proximal (SCID, Rag−/−, and C2.Cfb−/− mice) as well as distal (C5−/− mice) steps of CC. We also directly evaluated activation of CC at the proximal C3 and distal C5 levels by employing an ELISA assay to determine the presence of cleavage fragments in plasma of AMD3100-mobilized mice. We learned that mice with a defect in the activation of the proximal steps of CC mobilize normally; however, mobilization was still impaired in C5−/− mice. Furthermore, our ELISA studies revealed that after AMD3100 administration, C5, but not C3, is cleaved/activated in the plasma of these animals.
Based on these observations, we hypothesized that during AMD3100 mobilization, C5 could be cleaved in a cascade independent, non-canonical manner. We also paid close attention to Gr-1+
phagocytic cells (granulocytes and monocytes), which are able cleave/activate C5 as reported.44–47
In fact, we noticed that granulocytes and monocytes are the first cells that egress from BM during AMD3100 mobilization.
To address this issue, we studied the influence of AMD3100 stimulation on Gr-1+
cells sorted from BM that are enriched in granulocytes and monocytes and found that AMD3100 activates phosphorylation of MAPKp42/44 in these cells. This could be explained by AMD3100 being a partial agonist of CXCR4.48
Of importance, we identified for the first time that AMD3100 stimulates expression/secretion of several proteases (e.g., MMP-9, cathepsin, elstaze) in a Gr-1+
population. More importantly, in a direct set of experiments, we observed that C5 is cleaved/activated in plasma by Gr-1+
cells and that stimulation of these cells by AMD3100 enhances this effect. Altogether, these observations support the idea that mobilized/activated granulocytes activate the distal steps of CC in PB by inducing cleavage of C5. Thus, our data again confirm that C5 clevage fragments C5a and desArg
C5a orchestrate optimal mobilization of HSPCs after AMD3100 administration.
Based on these observations, we envision a flowing scenario in mobilization and CC activation induced by AMD3100 (). First, at the BM microenvironment level, AMD3100 blocks the interaction of CXCR4+ granulocytes and CXCR4+ HSPCs with SDF-1 expressed in the BM microenvironment. At the same time, AMD3100 stimulates secretion in BM of MMPs and other proteolytic enzymes from activated granulocytes that help to perturb another axis crucial in HSPCs retention, i.e., VLA-4-VCAM-1. Next, granulocytes are the first cells to egress from BM and they “pave the way” for HSPCs to cross the BM-endothelial barrier. Finally, granulocytes activated/mobilized by AMD3100 into PB activate/cleave C5 and release C5a and desArgC5a that subsequently orchestrate/facilitate the final steps of HSPC egress.
AMD3100-directed HSPC mobilization depends on activation of CC by AMD3100 mobilized/activated granulocytes
In conclusion, our data: i) better explain molecular mechanisms directing AMD3100-induced mobilization; ii) support a central role of innate immunity; and iii) provide evidence that C5 is activated/cleaved in plasma in a Gr-1+ cell-dependent manner. Granulocytes and C5 cleavage fragments orchestrate this process and, in light of our current data, mobilization of HSPCs could be envisioned as a part of the innate immunity response. However, further studies are needed to address if, in addition to C5a and desArgC5a cleavage fragments, other elements downstream from C5 of CC, e.g., C5b-C9 and/or membrane attack complex (MAC), are involved in the mobilization process.