Novel Evidence That the Mannan-Binding Lectin (MBL) Pathway of Complement Activation Plays a Pivotal Role in Triggering Mobilization of Hematopoietic Stem/Progenitor Cells By Activation of Both the Complement and Coagulation Cascades

Background. The complement cascade (ComC), which is part of the innate immune system, exerts several pleiotropic effects, and, as we have demonstrated, it is required for mobilization of hematopoietic stem/progenitor cells (HSPCs) during infection or tissue/organ injury as well as in response to administration of pharmacological mobilizing agents, such as G-CSF or AMD3100 (Blood 2004, 103, 2071-2078). The ComC is activated by three pathways: the classical, mannan-binding lectin (MBL), and alternative pathways. Activation of the ComC and generation of cleavage fragments of the fifth component of the ComC (C5), such as C5a, _desArgC5a, and C5b, by classical C5 convertase initiates events that are required for egress of HSPCs from bone marrow (BM) into peripheral blood (PB) (Leukemia 2009, 23, 2052-2062). Recent results indicate that the coagulation cascade (CoaC) is activated in parallel with activation of the ComC during the mobilization process and plays a supportive role, because thrombin has "C5 convertase-like activity" (Leukemia 2014, 28, 2148-2154). While a requirement for ComC activation and the pivotal roles of the distal part of complement activation and the generation of C5 cleavage fragments have been previously demonstrated (Leukemia 2009, 23, 2052-2062), mice with mutations in components of the classical pathway (C1q^-/- mice), in which the distal pathway of C5 activation remained intact, do not show impairment of HSPC mobilization (Leukemia 2010, 24, 1667-1675). Aim of the study. Since no studies have yet been performed to address the role of the MBL pathway in triggering the mobilization of HSPCs, we became interested in its involvement in both ComC and CoaC activation after administration of G-CSF or AMD3100. The MBL pathway is homologous to the classical pathway but contains a soluble MBL receptor instead of C1q, and MBL functions as activator of the MBL-associated serine proteases, MASP-1 and MASP-2, which are activated downstream of both the ComC and the CoaC. Hypothesis. We hypothesized that the MBL-initiated ComC and CoaC activation pathways are involved in triggering mobilization of HSPCs and that MBL deficiency may result in poor mobilization efficiency.Materials and Methods. In our experiments, 2-month-old, MBL-deficient (MBL^-/-) and MASP-1-deficient (MASP-1^-/-) mice as well as their normal wild type (WT) littermates were mobilized with G-CSF or AMD3100. Following mobilization, we measured i) the total number of white blood cells (WBCs), ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors, and iii) the number of Sca-1⁺c-kit⁺lineage^- (SKL) cells in PB. In parallel, we evaluated activation of the ComC after administration of G-CSF and AMD3100 in experimental animals by employing C5a ELISA. To address the role of the CoaC in MBL-MASP-1- and MBL-MASP-2-induced mobilization, MBL^-/- mice were treated with inhibitors of the CoaC (refludan) in some of the experiments. Results. We found that the MBL-MASP ComC activation pathway is involved in pharmacological G-CSF- and AMD3100-induced mobilization of HSPCs. As predicted, MBL^-/- and MASP-1^-/- mice were found to be poor mobilizers. Furthermore, inhibition of the CoaC by refludan inhibited mobilization in wild type animals but did not generate the additional defects seen in MBL^-/- mice. Conclusions. We identified a previously unrecognized role for the MBL-MASP-1 pathway in triggering ComC and CoaC activation in the HSPC mobilization process. This finding explains the pivotal role of the MBL pathway in triggering activation of the proximal part of the ComC and explains why, even with a deficiency in activation of classical pathway components (C1q), mobilization of HSPCs proceeds normally as long as the MBL pathway is intact. Taking into consideration that ~10% of normal people are poor activators of the MBL pathway and that this percentage corresponds with the ~10% of the normal healthy population that are poor mobilizers, we are currently investigating whether MBL deficiency correlates with poor mobilization status in patients. MBL could be an important predictive parameter for identifying poor mobilizers.