As mentioned above, the biological activity of CXCL12 may decrease in BM due to the induction of a proteolytic microenvironment after conditioning for transplantation, as seen, for example, after lethal irradiation 29, 30
. However, as shown in Figure ,
several factors are released from the damaged tissues that ameliorate this effect. These molecules are produced during the process of complement cascade (CC) activation, secreted from stromal and myeloid cells, or released from damaged cells, and cooperatively enhance or sensitize the responsiveness of HSPCs to a decreasing CXCL12 gradient. This process of sensitizing the responsiveness of cells to a shallow CXCL12 gradient in the presence of external modulatory molecules is known as the priming effect and can easily be evaluated in vitro
in the Transwell migration assay, where two chambers (an upper chamber containing the tested cells and a lower chamber containing chemoattractant) are separated by a porous membrane that allows transmigration of cells that respond to the chemotactic gradient (Figure
). Cells that respond to this gradient migrate and subsequently accumulate in the lower chamber.
Figure 2 A priming effect increases the responsiveness of HSPCs to shallow CXCL12 gradients. The overall scheme of chemotactic assays performed in the Transwell system to evaluate the HSPC priming phenomenon (Panel A). In the presence of a priming agent (e.g., (more ...) Figure
illustrates the findings that chemotaxis of HSPCs in response to a shallow CXCL12 gradient is significantly enhanced in the presence of several of these priming factors. This situation occurs also in vivo
when the concentrations of these factors increases in a BM microenvironment damaged by myeloablative treatment and collectively enhance the responsiveness of transplanted HSPCs circulating in PB to an CXCL12 gradient 31-38
- C3 cleavage fragments as priming factors.
It has been demonstrated that the CC, an evolutionarily ancient danger-sensing mechanism, becomes activated during conditioning for transplantation by radio- and chemotherapy 29, 31, 32
. The third component of the CC (C3) is an abundant protein in PB plasma (1 mg/ml) that becomes cleaved during CC activation by both classical and alternative pathways 39
. C3 cleavage leads to release of liquid-phase cleavage fragments, the C3a and des-Arg
C3a anaphylatoxins 40
. C3a has a short half-life in plasma and is processed by serum carboxypeptidase N to des-Arg
C3a, which is a long-half-life cleavage product.
However, C3 cleavage fragments alone do not chemoattract HSPCs, our previous work on C3-/-
mice revealed that animals lacking C3 display a significant delay in hematopoietic recovery from either sub-lethal irradiation or transplantation of wild type (WT) HSPCs 40
. Specifically, we observed that transplantation of histocompatible wild type (WT) Sca-1+
cells into C3-/-
mice resulted in a delay in hematological reconstitution in all hematopoietic lineages 40
. The fact that HSPCs from C3-/-
mice engrafted normally into irradiated WT mice suggests that there was a defect in the hematopoietic environment of C3-/-
mice and not an autonomous defect in the C3-/-
mouse-derived HSPCs 40
mice cannot activate or cleave C3, the C3 cleavage products C3a and des-Arg
C3a were examined for a role in modulating the responsiveness of HSPCs to an CXCL12 homing gradient 35, 40
. We observed that both short-half-life C3a and long-half-life des-Arg
C3a significantly enhanced migration of HSPCs at a low or threshold level of CXCL12 in a Transwell migration assay 31
. The molecular explanation for this phenomenon has been identified as a C3a- and des-Arg
C3a-mediated increase in CXCR4 incorporation into membrane lipid rafts 35, 40
Lipid rafts are membrane domains rich in sphingolipids and cholesterol, which form a lateral assembly in a saturated glycerophospholipid environment of cell surface membranes. The raft domains are known to serve as moving platforms on the cell and are also good sites for crosstalk between various cell surface molecules (e.g., CXCR4) and proteins that form intra-cellular signaling pathways. For example, it has recently been reported that small guanine nucleotide triphosphatases (GTPases), such as Rac-1 and Rac-2, which are crucial for engraftment of hematopoietic cells after transplantation, are associated with lipid rafts on migrating HSPCs 41-45
. Therefore, since the CXCR4 receptor is a lipid raft-associated protein, its signaling ability is enhanced if it is incorporated into membrane lipid rafts, where it can better interact with several signaling molecules, including the small GTPase Rac-1. This co-localization of CXCR4 and Rac-1 in lipid rafts facilitates GTP binding and activation of Rac-1 35
. Thus, the generation of C3 cleavage fragments in the BM microenvironment may somehow act as a protective mechanism that increases the responsiveness of HSPCs to an CXCL12 gradient when it is degraded by a proteolytic microenvironment after myeloablative conditioning for transplantation 29, 30
. In C3-deficient mice this phenomenon is attenuated, explaining why these animals show delayed engraftment after hematopoietic transplantation.
In this context, activation of the CC, which leads to increases in C3a or des-Arg
C3a levels in BM after myeloablative conditioning 29
, can be envisioned as one of the mechanisms that promotes homing of HSPCs (Figures -
). In fact, this relatively simple strategy for enhancing responsiveness of the HSPCs to be transplanted, which is based on short ex vivo
exposure of HSPCs before transplantation to C3a, has been proposed for use in the clinic to enhance engraftment of HSPCs, and an appropriate FDA-approved clinical trial has been initiated for patients undergoing umbilical cord blood (UCB) transplantations.
- Cationic antimicrobial peptides (CAMPs) as priming factors.
CAMPs are host-defense peptides and are an evolutionarily conserved component of the innate immune response 46-48
. Interestingly, the C3a and des-Arg
C3a anaphylatoxins mentioned above share several properties with classical peptides from the CAMP family 47
. CAMPs have been demonstrated to perforate prokaryotic cell membranes and thus kill bacteria, enveloped viruses, and fungi, but affect only the organization and not the viability of eukaryotic cell membranes. The selective effects of these natural antibiotics (i.e., prokaryotic killing and eukaryotic membrane perturbation) are known to be dependent on the characteristics of cell membranes 46-48
. While prokaryotic cell membranes are susceptible to strong electrostatic and hydrophobic interactions with these “natural antibiotics”, which may lead to membrane perforation, the cell membranes of eukaryotic cells are more resistant to the toxic effects of these peptides, because of their high cholesterol content and weak hydrophobic interactions with these peptides 46-48
One of the interesting properties of CAMPs that we identified is their ability to enhance or prime the responsiveness of cells to an CXCL12 gradient (Figure
). Therefore, cathelicidin (LL-37) and β2-defensin, which, like C3a, belong to the CAMP family, positively prime the responsiveness of HSPCs to an CXCL12 homing gradient (Figures -
Like C3 cleavage fragments, CAMPs also enhance incorporation of CXCR4 into lipid rafts so that CXCR4 is activated more efficiently in the presence of low doses of CXCL12, which facilitates its signaling. Interestingly, LL-37 and β-2 defensin, first described as being secreted by myeloid cells, are also secreted by BM stromal cells and, like CXCL12, their expression is regulated by HIF-1α 48
. However, while LL-37 and β-2 defensin are also peptides, they seem to be more resistant to proteolytic enzymes and are thus more stable in a proteolytic microenvironment than CXCL12.
- Other priming molecules.
In addition to the CAMPs, hylauronic acid 34
, fibrinogen 35
, and cell membrane-derived microvesicles 38
have also been reported to increase the responsiveness of HSPCs to an CXCL12 gradient. The exact mechanism of this priming effect, however, has still not been elucidated, but is most likely mediated by the interaction of these factors with integrin receptors on HSPCs. Activation of these receptors may promote lipid raft formation, again leading to incorporation of CXCR4 into lipid rafts 35
. This putative phenomenon has been confirmed for fibrinogen 35, 40
and the small spherical membrane fragments shed and secreted from the activated cells called microvesicles 35, 38
Thus, incorporation of CXCR4 into membrane lipids rafts, which ensures its most optimal signaling, is a mechanism shared by several factors that positively modulate the CXCL12-CXCR4 axis, including CC cleavage fragments, CAMPs, and molecules related to coagulation/inflammation (e.g., fibrinogen and hyaluronic acid).
That priming molecules (e.g., C3 cleavage fragment, CAMPs, and microvesicles) are generated during harvest of mobilized HSPCs from PB during the process of leucophoresis, may explain why, in certain situations, mobilized PB HSPCs engraft faster than HSPCs harvested by aspiration under steady-state conditions BM 49
. This mechanism, which is involved in trafficking of CXCR4+
HSPCs, may also play an important role in migration of other types of CXCR4+
cells, such as lymphocytes or macrophages in tissues affected by inflammation.