Both pharmacological mobilization of hematopoietic stem progenitor cells (HSPCs) and myeloablative conditioning for transplantation activate the complement cascade (CC) in bone marrow (BM), and in the previous funding period we focused on the role of the third complement component (C3) in mobilization and homing of HSPCs. In this competing renewal we will focus on the fifth complement component (C5), which is located downstream from C3. C5 cleavage leads to generation of the potent anaphylatoxins C5a and desArgC5a and generation of non-lytic and lytic C5b-C9, known as the membrane attack complex (MAC). Our recently published data support a crucial role for C5a/C5b-C9 in trafficking of HSPCs. In particular, C5-deficient mice are poor mobilizers in response to granulocyte colony stimulating factor (G-CSF) and exhibit delayed hematopoietic reconstitution after transplantation with wild type (WT) BM cells. Based on these findings, the central hypothesis of this competing renewal is that the activation of distal steps of the complement cascade and release of C5a and C5b-C9 (MAC) are important regulators of mobilization and homing of HSPCs. To address this hypothesis, we propose:
Specific Aim 1. The molecular basis of the mobilization defect in C5-deficient mice. Based on our observation that C5-deficient mice are poor G-CSF mobilizers, we will address whether C5 cleavage is i) required for circadian HSPC mobilization, ii) mobilization of HSPC induced by other agents and iii) whether C5 activation and S1P plasma level correlates with mobilization efficacy in patients. We will also focus on the role of erythrocyte-released S1P in mobilization by performing mobilization studies in CD55/CD59-deficient mice that are highly sensitive to C5b-C9 (MAC) lysis. Finally, we will focus on the role of C5b-C9-induced release of S1P in mobilization observed in patients with hemolytic syndromes.
Specific Aim 2. The molecular basis of the homing defect in C5-deficient mice. We found that the CC is activated in lethally irradiated mice and that C5-deficient mice engraft poorly with WT BM cells. To shed more light on the role of the distal part of the CC (C5a/C5b-C9) in homing/engraftment, we will study whether conditioning for transplantation by chemotherapy also activates the CC in BM and study the effect of C5a/C5b-C9 on expression of i) HSPC chemoattractants in BM, ii) adhesion/tethering molecules for HSPCs on BM endothelium, and ii) on secretion of factors facilitating homing.
Specific Aim 3. The C5a/C5b-C9-induced BM stroma-derived cathelicidin (LL-37) as a potent homing sensitizing factor. We found that LL-37, like C3a, is a very strong priming factor for SDF-1 that becomes upregulated in BM stroma after conditioning for transplantation in a C5a/C5b-C9-dependent manner. In an immunodeficient mouse model, we will test whether short exposure of HSPCs to LL-37 before transplantation increases homing of human BM- and umbilical cord blood (UCB)-derived HSPCs. We will also address whether LL-37 also primes the responsiveness of HSPCs to other homing factors and focus on the molecular mechanism of these phenomena.

Public Health Relevance

Section In this competing renewal, we will focus on the role of a major component of innate immunity, namely, activation of the complement cascade in trafficking of hematopoietic stem/progenitor cells. This application will allow us to better understand this process, as well as lead to the development of novel therapeutic strategies to treat patients with leukemias and inborn defects of hematopoiesis by transplantation of hematopoietic stem cells.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG1-VH-C (02))
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Bishop, Terry Rogers
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University of Louisville
Internal Medicine/Medicine
Schools of Medicine
United States
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Ratajczak, Mariusz Z; Suszynska, Malwina; Borkowska, Sylwia et al. (2014) The role of sphingosine-1 phosphate and ceramide-1 phosphate in trafficking of normal stem cells and cancer cells. Expert Opin Ther Targets 18:95-107
Grymula, Katarzyna; Tarnowski, Maciej; Piotrowska, Katarzyna et al. (2014) Evidence that the population of quiescent bone marrow-residing very small embryonic/epiblast-like stem cells (VSELs) expands in response to neurotoxic treatment. J Cell Mol Med 18:1797-806
Ratajczak, M Z; Zuba-Surma, E; Wojakowski, W et al. (2014) Very small embryonic-like stem cells (VSELs) represent a real challenge in stem cell biology: recent pros and cons in the midst of a lively debate. Leukemia 28:473-84
Klyachkin, Yuri M; Karapetyan, Anush V; Ratajczak, Mariusz Z et al. (2014) The role of bioactive lipids in stem cell mobilization and homing: novel therapeutics for myocardial ischemia. Biomed Res Int 2014:653543
Schneider, Gabriela; Bowser, Mark J; Shin, Dong-Myung et al. (2014) The paternally imprinted DLK1-GTL2 locus is differentially methylated in embryonal and alveolar rhabdomyosarcomas. Int J Oncol 44:295-300
Suszynska, Malwina; Zuba-Surma, Ewa K; Maj, Magdalena et al. (2014) The proper criteria for identification and sorting of very small embryonic-like stem cells, and some nomenclature issues. Stem Cells Dev 23:702-13
Schneider, Gabriela; Sellers, Zachariah Payne; Abdel-Latif, Ahmed et al. (2014) Bioactive lipids, LPC and LPA, are novel prometastatic factors and their tissue levels increase in response to radio/chemotherapy. Mol Cancer Res 12:1560-73
Wysoczynski, Marcin; Solanki, Mitesh; Borkowska, Sylwia et al. (2014) Complement component 3 is necessary to preserve myocardium and myocardial function in chronic myocardial infarction. Stem Cells 32:2502-15
Borkowska, S; Suszynska, M; Mierzejewska, K et al. (2014) Novel evidence that crosstalk between the complement, coagulation and fibrinolysis proteolytic cascades is involved in mobilization of hematopoietic stem/progenitor cells (HSPCs). Leukemia 28:2148-54
Suszynska, Malwina; Poniewierska-Baran, Agata; Gunjal, Pranesh et al. (2014) Expression of the erythropoietin receptor by germline-derived cells - further support for a potential developmental link between the germline and hematopoiesis. J Ovarian Res 7:66

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