The Neurexin Iα/Neurexophilin1 Axis as a Myelosuppressive Factor in Human Cord Blood and Murine Bone Marrow Hematopoiesis.

Abstract 2545

Poster Board II-522

Neurexin Iα (NRXN1α) is a receptor protein upsteam of members of the Lin family and other developmental proteins. NRXN1α's role in neuronal development has been well characterized however its role in hematopoiesis remains undefined despite the close connection between neuronal and hematopoietic development. The stem cell database created by the Lemischka laboratory suggests that NRXN1α is present in a variety of hematopoietic populations including very primitive populations. We confirmed the presence of NRXN1α by probing Western blots created from murine bone marrow (Mu BM). Using the agonistic toxin alpha-latrotoxin, we assayed for functional activity of the receptor and found that it may play a role in progenitor cell survival under growth-factor starved conditions. These results prompted us to hypothesize that natural antagonists of the neurexophilin family may play a role in hematopoiesis, particularly because of the secretion of neurexophilins by macrophages during immune system fatigue. Using a variety of colony stimulating factors (CSF) both alone and in combination, we have found that continued contact with Neurexophilin1 (NXPH1) specifically inhibits the synergistic proliferation activity of granulocyte-macrophage colony stimulating factor (GM-SCF) and stem cell factor (SCF), on both human cord blood (Hu CB) and Mu BM hematopoietic progenitor cells (HPCs) in a dose dependent manner. In contrast, HPCs stimulated by GM-CSF and Flt3-ligand (FL), or interleukin 3 (IL-3) in combination with SCF and/or FL in hu CB CD34+ and Mu BM cells show no change in the presence of NXPH1. The selective activity of NXPH1 on GM-CSF and SCF stimulated synergistic proliferation stands in contrast to the effects of certain chemokines which have been shown to inhibit both SCF and FL mediated synergy in Mu BM, but not in Hu CB after both sustained and transient contact. To test whether the inhibitory effect of NXPH1 was truly due to sustained contact with NXPH1 or if some secondary factor is secreted in response to NXPH1 exposure, Hu CB cells were incubated overnight in the presence or absence of NXPH1 and the conditioned medium was used. The conditioned medium had no effect on the proliferation of Hu CB cells in the presence of GM-CSF and SCF. To elucidate the specificity of NXPH1, we exposed Mu BM cells to GM-CSF or IL-3 for either one hour or overnight before the protein lysate was collected. Western blots created from these protein lysates were probed for NRXN1α and we found that cells rapidly downregulate NRXN1a expression in a sustained manner after exposure to IL-3, but not to GM-CSF. We hypothesized that NXPH1 may also be active in vivo, so we then proceeded to assess the effects of intravenously injected C57bl/6 mice with NXPH1 in a dose-dependent and time-related fashion. Twenty-four hours after injection, BM was collected and progenitor activity was assayed using the in vitro colony assay where the HPCs were stimulated by a variety of CSFs, including IL-3. There was a 55% decrease in numbers of BM HPCs for all conditions. The decrease in absolute colony number was mirrored by a decrease in the cycling population of HPCs in mice treated with NXPH1 as measured by the high specificity activated tritiated thymidine kill assay. Additionally, the white blood cell and platelet counts were decreased in the peripheral blood of treated mice. However, the c-kit+ Sca1+ Lineage- (KSL), KSL CD34+ and KSL CD34- population levels were not affected. After forty-eight hours, the progenitor cell numbers from NXPH1 treated mice were minimally suppressed but the levels of the peripheral blood populations remained significantly reduced. These results have implications for a variety of elements in hematopoietic research: NXPH1 is able to inhibit the colony forming ability of hu CB cells, which other inhibitors of Mu BM such as chemokines have been heretofore unable to demonstrate; additionally, the specificity of the NXPH1-induced inhibition may shed light on the synergy of GM-CSF and SCF.