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Despite possessing marked structural similarities, the chemokines macrophage inflammatory protein-1α (MIP-1α; CCL3) and RANTES (CCL5) display differential activity in hematopoietic progenitor-cell-inhibitory assays, with MIP-1α being active and RANTES inactive in this context. We have sought to identify the key structural determinants of this property of MIP-1α. This has involved constructing MIP-1α/RANTES chimeras by swapping structural domains between the 2 proteins. Results indicate that, in contrast to other chemokine functions, neither the N nor the C termini are key determinants of inhibitory activity. The motif that appears to be most important for this activity lies between the second and fourth cysteines of MIP-1α and further domain swap analysis has narrowed this down to the 310helical turn preceding the first β-strand in MIP-1α. More detailed analysis has highlighted the role played by a specific dipeptide motif in the proliferation-inhibitory activity of chemokines. The involvement of the 310helical-turn motif in chemokine function is unprecedented and this study therefore identifies a novel, functionally essential motif within chemokines. In addition, this study further attests to the alternative mechanisms of action used by MIP-1α in inhibition of hematopoietic progenitor-cell proliferation and regulation of leukocyte migration.

The proliferative status of the stem cell compartment is thought to be controlled by both positive and negative regulators of proliferation. These regulators have obvious clinical potential in manipulating the integrity and proliferative status of the stem cell in vivo during patient treatment for neoplastic disease. We have tested the ability of the human recombinant homologue of murine macrophage inflammatory protein-la (rhMIP1α) to suppress the proliferation of primitive murine progenitors in vitro and in vivo. This recombinant protein (stem cell inhibitor, similar to the human homologue of MIPIα, LD78) is active in a dose-dependent manner in vitro on CFU-S measured at day 12 and to a slightly lesser extent on the more mature CFU-S that are measured at day 8. SCI/rhMIP1α is also active in vivo in separate models of bone marrow regeneration in which the high proliferative status of the CFU-S compartment is reduced to the quiescent state with a single inoculation of SCI/rhMIP1α. The inhibitory activity of the recombinant protein was then tested in a relevant therapeutic model in which the observed protection of part of the stem cell compartment is reflected by a significant improvement in the kinetics of neutrophil recovery. These results establish the feasibility of testing SCI /rhMIP1α in a range of chemotherapy regimes as a preliminary to clinical trials to attempt to protect the stem cell compartment during treatment for neoplastic disease.

The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL-11) produced a 50-fold amplification of clonogenic muhtipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4° BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11.