CD45 Phosphatase Is Involved in Motility and Development of Hematopoietic Stem and Maturing Cells by the Regulation of Cell Adhesion and Cytokine Signaling.

The phosphatase CD45 is a key regulator of antigen receptor signaling in lymphocytes. Still, CD45 is highly expressed in all hematopoietic lineages at most stages of development, suggesting that this phosphatase also regulates other cells and processes. During development, as well as in clinical transplantation, hematopoietic stem cells (HSCs) migrate through the circulation to the bone marrow (BM) and repopulate it. Migration and development of HSCs are multi-step processes, which are tightly regulated by interplays between cytokines, chemokines, adhesion molecules and proteolytic enzymes; however, not all the related key players have been fully identified.

In this study we explored the involvement of CD45 in hematopoietic cell motility and development, its role in cytokine signaling and adhesion interactions. The roles of CD45 were tested by either blocking the function of CD45 expressed on human and murine HSCs by neutralizing antibodies, or by utilizing CD45 knockout (KO) mice.

Our results show that blocking CD45 completely prevented homing of human CD34+ enriched progenitors to the murine BM, consequently abrogating repopulation in transplanted NOD/SCID mice. In addition, CD45 neutralization impaired the capacity of human progenitors to migrate in-vitro towards a gradient of SDF-1, suggesting a cross-talk between SDF-1 and CD45 signaling. Furthermore, blocking CD45 on human G2 cells (pre-B ALL line) activated signaling pathways, including an increase in phosphorylation of MAP kinase and the tyrosine kinase Pyk2, which are involved in cell adhesion and migration. This activation enhanced cell adhesion to stromal and endothelial cell lines in-vitro. Importantly, blockage of CD45 in human progenitors resulted in cell aggregation, which inhibited cell proliferation and impaired the capacity to form colonies in-vitro. In an additional set of experiments we tested the role of CD45 in cell mobilization. In-vivo studies in normal mice demonstrated that neutralization of CD45 function inhibited the release of mature leukocytes and progenitor cells from the BM to the circulation both under steady state conditions and in stress-induced recruitment by stimulation with G-CSF or LPS. More importantly, BM derived mononuclear cells from CD45KO mice displayed a significant reduction in in-vivo homing and in-vitro migration compared to their wild type counterparts. Furthermore, G-CSF induced mobilization was impaired in CD45KO mice, and accompanied by a reduction in MMP-9 secretion from blood-derived leukocytes. Unexpectedly, the ability of CD45KO progenitors to form colonies in-vitro was impaired in the absence of in-vivo BM environment, documenting a crucial role for phosphatases such as CD45 in stem cell differentiation.

Taken together, our findings demonstrate that functional CD45 is essential for human and murine hematopoietic cell migration and development (both homing and mobilization) by the regulation of adhesion and cytokine-induced signaling machineries. We suggest that in these cells CD45 may act as a negative regulator of major signaling pathways controlling adhesion properties and maintaining the balance between anchorage and release. We reveal a novel and dual role for CD45 in regulation of hematopoietic cell trafficking in general and progenitor cell motility and development in particular.