It has been reported that proteolytic enzymes are upregulated in BM after conditioning for transplantation and, by removal of three N-terminal amino acids from stromal derived factor-1 (SDF-1), abrogate its chemotactic activity, even if the SDF-1 peptide remains detectable by ELISA (Cancer Res. 2010;70:3402). We also reported that cationic antimicrobial peptides (CAMPs), such as complement cascade C3 protein cleavage fragment C3a, b2-defensin, and cathelicidin (LL-37) (Leukemia 2012, 26, 736), potently enhance the migration and homing responses of HSPCs to a low SDF-1 gradient, which is critical for retention of SDF-1 chemotactic activity in the highly proteolytic microenvironment of BM induced by conditioning for transplantation. This priming effect depends on incorporation of the CXCR4 receptor into membrane lipid rafts, a phenomenon that allows for more efficient interaction of the CXCR4 receptor with downstream signaling proteins (Blood 2005;101:3784). However, we were aware that inhibition of lipid raft formation by b-methylcyclodextrin inhibits only ∼50% of the CAMP priming effect, which strongly indicates the involvement of other mechanisms. Interestingly, it was reported recently that LL-37, by involving pannexin channels, enhances the release of ATP from cells (J Biol. Chem. 2008;263:30471) that had been described to be an autocrine chemottractant secreted at the leading edge of migrating macrophages and neutrophils (Science 2006;314:1792). Exogenous ATP has also been reported in Tranwell assays to chemoattract HSPCs (Blood 2004;104:1662).
We hypothesized that HSPCs, like monocytes and neutrophils, release at their leading edge ATP, and respond to this autocrine chemoattractant. Thus, this autocrine ATP migration-enhancing phenomenon could provide not only new insight into mechanisms regulating the migration of HSPCs but also better explain the priming effect of CAMPs to a low or decreasing SDF-1 gradient.
First, we tested the responsiveness of murine and human BM-, mobilized peripheral blood (mPB)-, and human umbilical cord blood (UCB)-derived HSPCs to different extracellular nucleotides including ATP by performing i) Transwell migration assays, ii) MAPKp42/44 and AKT phosphorylation studies, and iii) RQ-PCR expression of different purinergic receptors on normal HSPCs. The priming effect of CAMPs on autocrine secretion of ATP by HSPCS was studied in functional chemotaxis assays by employing the broad-spectrum ATP receptor antagonist suramin and apyrase, an enzyme that degrades extracellular ATP.
We noticed that for all the nucleotides tested (ATP, UTP, GTP, TTP, and CTP), ATP has the strongest chemotactic activity against murine and human BM-derived HSPCs, which correlated with the phosphorylation of MAPKp42/44 and AKT. In contrast to BM-derived HSPCs, the chemotactic effect of ATP and its induction of signaling were significantly attenuated for mPB- and UCB-derived HSPCs, which suggests a desensitization of purinergic surface receptors by free extracellular nucleotides circulating in blood plasma. Most importantly, we found that exposure of HSPCs to suramin or apyrase significantly diminished (by ∼50%) the priming effect of CAMPs in enhancing the responsiveness of these cells to a low SDF-1 gradient. Finally, we found that both ATP and CAMPs are secreted from irradiated BM stromal cells, which provides a heretofore underappreciated homing signal for HSPCs.
We show that ATP secreted both as a paracrine factor by BM stromal cells during conditioning for transplantation and by migrating HSPCs at their leading edge enhances migration and homing of HSPCs to BM niches. Most importantly, our data demonstrates, for first time, the involvement of an autocrine ATP–purinergic receptor migration regulatory loop, which better explains the pro-migratory priming effect seen after exposure of HSPCs to CAMPs. Since CAMPs, which enhance migration of HSPCs to a low SDF-1 gradient are upregulated like ATP in irradiated BM, all these factors together orchestrate the responsiveness of HSPCs to a decreasing SDF-1 gradient in the proteolytic microenvironment of BM conditioned for transplantation. These mechanisms should be further explored to improve the homing of HSPCs after transplantation, and we are currently testing this possibility in appropriate animal models.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.