An Unexpected Role for the Complement C5b-C9 Membrane Attack Complex (MAC) In Trafficking of Hematopoietic Stem/Progenitor Cells - a Novel Unexpected Link Between Innate Immunity and Hematopoiesis

Abstract 555

We previously reported that complement cascade (CC) is activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs; Blood 2003;101:3784; Blood 2004;103:2071; and Blood 2005;105:40) and that C5 cleavage fragments direct egress of HSPCs from BM into peripheral blood (PB) (Leukemia 2009;23:2052 and Leukemia 2010;24:976). Accordingly, C5 cleavage fragments (C5a and _desArgC5a) stimulate myeloid cells in BM to secrete proteolytic enzymes and chemoattract granulocytes into peripheral blood (PB). Therefore, granulocytes form a first wave of cells that permeabilize the BM-PB endothelial barrier and prime it for subsequent egress of HSPCs. We have also observed that activation of the distal part of the complement cascade (CC), which leads to formation of C5b-C9 (also known as the membrane attack complex [MAC]), is crucial for egress/mobilization of HSPCs. It is known that proteins that form MAC can be inserted into cell membranes, resulting in cell lysis, or may remain in biological fluids as soluble MAC (sMAC) and in this “non-lytic” form may interact with target cells. We have already reported that sMAC releases bioactive lipid - sphingosine-1 phosphate (S1P) from erythrocytes, which is a major chemoattractant in mobilized peripheral blood (mPB) for HSPCs (Leukemia 2010;24:976). Since the level of sMAC increases in PB during mobilization as well as following conditioning for transplantation, we became interested in whether this protein complex affects the biology of normal HSPCs. First, we observed that, while sMAC does not affect proliferation and viability of clonogenic progenitors, it activates phosphorylation of MAPKp42/44 and AKT in both human CD34⁺ and murine SKL cells. Furthermore, sMAC primes and enhances chemotactic responsiveness of HSPCs to S1P and SDF-1 gradients and increases adhesiveness of these cells to BM stroma and endothelium. This effect is probably lipid raft mediated, because exposure of cells to methylo-b-cyclodextrin before chemotaxis abrogates this phenomenon. We also found that HSPCs, as well as PB mononuclear cells exposed to sMAC, secrete increased levels of PGE2 and metalloproteinases, which indicates that an increase in sMAC level in PB after conditioning for transplantation may enhance the homing properties of HSPCs. Thus, our results in toto provide novel evidence that sMAC is an underappreciated and potent regulator of HSPC trafficking and plays an important role, both direct and indirect (via released from cells S1P), in mobilization and homing of HSPCs after transplantation. In support of this notion, we found that mice displaying defects in CC activation and sMAC generation display a defect in homing of HSPCs. Thus, our data provide yet more evidence that innate immunity and the complement cascade regulate trafficking of HSPCs by (1) releasing active C3 and C5 cleavage fragments that increase the level of bioactive lipids chemoattractants in PB and BM and by (2) modulating the migratory properties of HSPCs with sMAC. We propose modulation of CC as a novel strategy for controlling both mobilization and homing of HSPCs.