Beta Glucan Enhances Hematopoietic Stem Cell Mobilization.

Background: Peripheral blood stem cell infusion is the preferred method for establishing hematopoiesis in transplantation. Use of G-CSF is now the most commonly used mobilizing agent. Despite advances in stem cell techniques and agents, studies have shown that up to 20–25% of patients exhibit poor mobilization and are not able to proceed with autotransplantation. Strategies to improve mobilization include using chemotherapy alone or in conjunction with growth factor or novel agents such as AMD3100. β-glucan PGG is a soluble yeast beta glucan with a molecular mass of 150kD comprised of a β-D-(1–3)-linked glucopyranosyl backbone with a β-D-(1–6)-linked β(1–3) side chains. In previous studies, β-glucan PGG has been shown to induce hematopoietic stem and progenitor cell (HSPC) mobilization to the periphery. In this study, we examined β-glucan PGG’s ability to mobilize HSPC alone and in conjuction with G-CSF and explored its mechanism of action. Methods: Prior to our study, dose kinetic studies were done and showed peak stem cell mobilization at 24 hours and maximum results using the 9.2 mg/kg dose with β-glucan PGG alone. In this study, four groups of wildtype (WT) C57BL/6 mice (6 mice/group) were used; control (saline injection × 4 days), G-CSF only (125ug/d × 4 days), PGG only (4.8 or 9.2mg/kg × one dose), and G-CSF/PGG combination. In the combination group, G-CSF injections were given daily for four days and one PGG injection on day three. Four hours after the last G-CSF injection, the mice were sacrificed and final white blood cell count were collected. Blood was assayed for in vitro mobilization in methycellulose culture. Peritoneal macrophage were stimulated with PGG and supernatant was harvested at times indicated and concentration of MMP-9 was determined using ELISA (R&D Systems). Results: All treated groups showed increased mobilization of all major cell lines (CFU-GM, CFU-M, and CFU-G). β-glucan PGG alone was able to mobilize peripheral stem cells at both doses (4.8mg/kg–9.3 CFU/200000 PBL and 9.2mg/kg–14 CFU/200000 PBL). The combination group (G-CSF/PGG-4.8mg/kg) showed an almost two-fold increase in CFU compared to G-CSF alone (G-CSF-30.42 CFU/200000 PBL, G-CSF/PGG(4.8)-57 CFU/200000 PBL, p=0.008). Initial in vitro chemotaxis assays revealed β-glucan PGG induces HPSC mobilization independent of SDF-1 (stromal derived factor) gradient. Our previous studies have demonstrated that β-glucan can enhance bone marrow engraftment via a CR3 dependent mechanism. However, our current study indicated that β-glucan mobilized stem cells via a CR3 independent mechanism and did not induce appreciable levels of cytokine secretion. To further explore its mechanism of action, we stimulated peritoneal macrophages with β-glucan PGG. Strikingly, β-glucan PGG stimulated macrophages to produce significant amounts of matrix metalloproteinase-9 (MMP-9). Similarly, β-glucan PGG also stimulated bone marrow-derived macrophages to secrete MMP-9. Conclusion: β-glucan PGG is an agent that enhances stem cell mobilization alone and has a synergistic effect when used in conjunction with G-CSF. The mechanism of mobilization by β-glucan PGG involves MMP-9, which results from release of pro-MMP-9 from marrow macrophages. The efficacy of β-glucan PGG and lack of proinflammatory activity make it an attractive agent to supplement mobilization with G-CSF.