AMD3100 Enhances Engraftment Figure 5. Post transplant administration of AMD3100 mobilizes recipient residual HSCs, increasing marrow niche availability. Lethally irradiated C57BL/6 CD45.2 Thy1.2 mice (10.5 Gy) were injected via tail vein with sorted KTLS hematopoietic stem cells (2500 cells/mouse) obtained from C57BL/Ka Thy1.1 donor mice. At 48 hours after transplantation, recipient mice were injected subq with a single dose of AMD3100 at 5 mg/kg or PBS control. Twenty-four hours after this injection, the mice were sacrificed, and the 2 femurs and the spleen were harvested. Recipient-origin stem cells (CD45.2+ KLS cells) and donor derived stem cells (CD45.1+ KLS cells) in the spleen and in the marrow were determined. Marrow CFUs-GM and Bursting forming units of erythrocytes (BFUs-E) were measured using MethocultH GF 3434 (16105 cells/dish, triplicate dishes per individual mouse). Percent donor stem cell homing efficiency to the spleen was calculated by dividing the absolute number of CD45.1+ KTLS stem cells in the recipient’s spleen by the number of injected KTLS stem cells. The graphs are representative of 2 separate sets of experiments with 5–10 mice in each group of each experiment. A: total number of recipient CD45.2+ KLS stem cells in 2 femurs. B: marrow CFU-GM and BFU-E assay (numbers of donor CFU-GM and BFU-E/36105 marrow cells). C: total number of recipient stem cells in the spleen. D: % donor stem cell homing efficiency in the spleen. E: Measurement of hematopoietic stem/ progenitor cell mobilization after prolonged AMD3100 administration. Transplanted mice that were given AMD3100 at 5 mg/kg body weight or PBS subcutaneously every other day beginning at day +2 for 60 days and na¨ıve normal control mice were injected with AMD3100 subcutaneously at 5 mg/kg body weight or PBS. Blood samples were collected immediately prior to the injection and 4 hours after injection, and colony-forming units and white blood cell counts determined. Colony-forming units were represented as colony numbers/30 ml blood (n = 5 in each group. Open bar: before AMD3100 injection. Filled bar: 4 hours after AMD3100 injection). *: p,0.05. NS: not statistically significant. doi:10.1371/journal.pone.0011316.g005 to 66613% in the mice that received bone marrow from PBS- injected primary transplant mice (Fig. 7; p.0.05). These data demonstrated that post transplant administration of AMD3100 preserved the long-term repopulating HSC pool. Discussion In our current study, we utilized a specific and reversible CXCR4 antagonist to explore the biological roles of SDF-1/ CXCR4 interaction in hematopoiesis and HSC engraftment following HCT. Unlike other chemokines, SDF-1 binds to one single chemokine receptor, CXCR4, which itself is not recognized by any other ligands. CXCR4 knockout mice die in utero and are defective in vascular development, hematopoiesis and cardiogen- esis[36]. AMD3100 selectively antagonizes CXCR4 receptor and does not bind to other chemokine receptors. The extreme specificity of AMD3100 makes it a very useful and powerful tool to be used to investigate the role of SDF-1/CXCR4 in HSC engraftment and reconstitution. AMD3100 has a very short half- life, and is rapidly eliminated with an estimated distribution half- life of 0.3 hours and a terminal half-life of 5.3 hours[37]. In our current study, AMD3100 was given every other day. Our results suggest that even a short period of transient CXCR4 blockade could have dramatic impact on animal survival and on HSC engraftment following HCT. One of our most striking findings of the study was that AMD3100 given after transplant significantly improves survival in lethally irradiated mice transplanted with limited number of hematopoietic stem cells. Interestingly, the recovery of peripheral blood count appears to lag behind the survival benefit (Fig. 1 and 2). It is not uncommon to see a temporal discrepancy between survival benefit and peripheral blood cell count in transplantation animal models. For example, Chen et al transplanted T cell- depleted bone marrow cells into lethally irradiated allogeneic mice and observed survival benefit at 2 weeks when the peripheral blood counts were still depressed[38]. Most recently, we also observed survival benefit in transplanted mice treated with growth hormone at a time when the peripheral blood count just started to recover[39]. There are 2 possible explanations for this time gap between survival benefit and peripheral blood cell count recovery. First, it is possible that the threshold for peripheral blood count to achieve survival benefit may be low and a slight increase in blood counts may have dramatic impacts on animal survival. Secondly, the peripheral blood count may underestimate the degree and the speed of hematological recovery. In the early recovery phase following transplantation, bone marrow, spleen and other organs are the predominant sites for hematopoiesis. The differences in blood nucleated cell counts between AMD3100-treated mice and PBS- injected mice became signifi- cant at ,5 weeks after transplantation (Fig. 2). The slow peripheral blood hematological recovery and the delayed effects of AMD3100 on hematological reconstitution are likely due to very limited number of KTLS stem cells transplanted in these mice. The mice received approximately 250 KTLS stem cell per mouse in our study. Using a limiting dilution assay, Uchida et al found that engraftment kinetics depended on the number of transplanted HSCs and that the most rapid engraftment occurred when the mice received $5000 KTLS cells per mouse[40]. Compared to B cell recovery, AMD3100 had less effect on T cell reconstitution (Fig. 2). This discrepancy in the effects of AMD3100 on T and B cell reconstitution results from the different mechanisms of B-cell and T cell regenerations following transplantation[41]. B cell recovery after transplantation recapit- ulates normal B-cell ontogeny and derives directly from bone marrow lymphoid progenitors. In contrast, T cell reconstitution occurs through both thymic and extrathymic pathways. The contribution of bone marrow lymphoid progenitor cells in T cell reconstitution is limited and highly variable. Our study suggests that augmented hematological recovery and reduced inflammatory cytokine storm both contribute to the survival benefit observed in AMD3100-treated mice. However, the extent and the relative contribution of each of these 2 mechanisms to animal survival remain to be elucidated. Although our data suggested that hematopoietic stem cells are essential in rescuing these mice from lethal irradiation (Fig. 3D), it is possible that AMD3100 enhances hematological recovery by altering the levels of cytokines/chemokines in marrow microenvironment. Recent studies found that many cytokines and chemokines in the niche microenvironment are involved in hematopoiesis following transplantation[5,6]. Further dissection of the benefit of these 2 mechanisms in survival will require further studies including the use of knockout mice such as MyD88 mice (toll-like receptor knockout mice that fail to elicit a cytokine storm)[42]. Our results demonstrate that the CXCR4 chemokine receptor is involved in transplant-related cytokine storm and subsequent multi-organ toxicity. Blocking CXCR4 with AMD3100 improves animal survival. In addition to HSCs, CXCR4 is also expressed on T cells, B cells, macrophages, dendritic cells, eosinophils, fibroblasts and other inflammatory cells. SDF-1/CXCR4 interac- tion recruits inflammatory cells to the injury site, exacerbates inflammation and thus plays an important role in the pathogenesis of autoimmune diseases[31,32], idiopathic lung fibrosis[43], bleomycin-induced lung injury[44], and possibly in acute graft versus host disease[45]. Consistent with the important role of CXCR4 in these disease processes, blocking CXCR4 with AMD3100 attenuates autoimmune responses[31,32] and improves lung functions[46]. The observation of the decrease in the inflammatory cytokines and improved animal survival by PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e11316