In this study, we identified important roles for the panleukocyte CD45 in key processes of immature hematopoietic cell function: its BM retention and release to the periphery, processes that are dramatically affected by stress conditions. By using the CD45KO mouse model, we investigated two major parameters involved in progenitor motility and location, including intrinsic properties and environmental regulation. First, we revealed autonomous defects in CD45KO cell motility into and out of the BM compartment, including reduced mobilization and homing of mature leukocytes and immature progenitor cell populations derived from different organs. We suggest that CD45 regulates two cellular processes that have key roles in the migration and retention of leukocytes in general, and progenitors in particular: proteolytic enzyme secretion and adhesion interactions. Lower secretion of MMP-9 by CD45KO BM MNCs after G-CSF stimulation implies that CD45 regulates MMP activation, and may thus further explain the reduced egress of CD45KO leukocytes. Corroborating our findings, mouse multiple myeloma CD45KO cells also secrete lower levels of MMP-9, correlating with their reduced invasive capacities compared with CD45+
BM-derived cells lacking CD45 have increased activation of β1 integrins and hyperinduction of adhesion properties, demonstrating that CD45 is a negative regulator of signaling cascades, inducing cell detachment and release. We found that Src kinase, the CD45 substrate, is a potential target by which CD45 regulates the migration of hematopoietic cells. Indeed, Src kinase inhibition enhanced CD45KO cell motility, demonstrating that Src activity is unbalanced in these cells. Several studies support the involvement of Src kinases in adhesion and motility properties. Src kinases were shown to regulate β1 and β2 integrins in different cells and cell lines (28
). Moreover, in mice deficient in members of the Src family, immature, hematopoietic Sca-1+
cells demonstrated increased homing (37
), and primitive, BM-derived SKL cells showed enhanced G-CSF–induced mobilization that was associated with elevated MMP-9 and accelerated breakdown of vascular cell adhesion molecule 1 (38
). In line with these studies, our results demonstrate the opposite effects when Src is hyperactive because of CD45 deficiency. Still, such fundamental defects in the motility of both CD45KO progenitors and maturing leukocytes strongly suggest that additional pathways are also imbalanced by the lack of CD45 function. This is especially apparent in the defective cell polarization in response to chemotactic signals of CD45KO c-Kit+
progenitors. Moreover, SDF-1 is considered as a survival factor for stem and progenitor cells (39
). Thus, the impaired ability of immature CD45KO c-Kit+
cells to normally respond to SDF-1 stimulation may further explain their inferior retention in the BM.
Previously, we suggested that interactions between hematopoietic stem and progenitor cells with their BM microenvironment are mutual (5
). We further reveal that CD45 also plays a role in progenitor mobilization by regulating components of the BM microenvironment. Reduced progenitor expansion and release in response to RANKL activation in CD45KO mice was associated with impaired modulation of the stem cell niche regulating components osteopontin and SCF. Osteopontin was shown to negatively regulate and limit the number of endosteal stem cells (40
). The impaired degradation of osteopontin in the endosteum of RANKL-treated CD45KO mice may explain the low numbers and reduced expansion of progenitors and stem cells in their BM. In addition, it was previously shown that shedding of membrane-bound SCF by MMP-9 shifts stem cells from a quiescent to a proliferative state, enabling their release from the BM (9
). The impaired resorption activity and the low secretion of MMP-9 by CD45KO osteoclasts may also contribute to the reduced progenitor expansion in the CD45KO BM, a prerequisite step for immature cell mobilization.
Osteoclasts derived from hematopoietic precursors in the BM of CD45KO mice show abnormal morphology and function both in vitro and in vivo, reflecting mild osteopetrosis. Of note, osteoclasts derived from precursors in the spleen exhibited the same defective phenotype (unpublished data), although the numbers of hematopoietic progenitors were higher in the CD45KO spleens. This demonstrates that the decreased osteoclast numbers are not caused by the lack of progenitor cells but rather an intrinsic defect in osteoclast differentiation. Our investigations further showed that CD45 regulates osteoclast formation via controlling Src kinase activity and DC-STAMP expression. In support of our findings, previous reports showed that osteoclasts derived from DC-STAMP−/−
mice were TRAP+
MNCs exhibiting a reduced bone-resorbing activity (17
). Interestingly, DC-STAMP−/−
osteoclasts demonstrated enhanced Src expression, suggesting a link between these two regulators (17
). Thus, our data propose a role for the CD45–Src axis in osteoclast fusion and maturation. In addition, low expression of MMPs in CD45KO osteoclasts showed that by regulating MMP-9 and MT1-MMP expression, CD45 is eventually involved in osteoclast motility and bone degradation activity (42
). These defects in osteoclasts may thus explain the poor mobilization observed in CD45KO mice. However, distinguishing between the environmental versus hematopoietic effects using chimera models revealed a parallel and perhaps additive impact of both compartments on progenitor retention and mobilization potentials.
The abnormal phenotype and activity of CD45KO osteoclasts are associated with lower numbers of trabecules in the femoral metaphysis, a region known to harbor stem cells (32
). Mouse models of severe osteopetrosis exhibit extramedular hematopoiesis, demonstrating lower levels of stem and progenitors cells in the BM caused by several bone structure defects, and higher levels of progenitor cells in the spleen (33
). CD45KO mice demonstrated a similar phenotype of the primitive SKL pool size and location driven by multiple defects of both the CD45KO primitive cells and their osteoclast progeny. The reduction in CD45KO primitive cells in the BM is complementary to previous findings showing that Lyn−/−
mice (members of the Src kinase family) display higher numbers of primitive SKL cells in the BM (45
), demonstrating the central role of the CD45–Src cascade in stem cell retention. Our findings indicate that stem and progenitor cells can modulate their CD45 expression and signaling via Src kinase, influencing their retention, survival, and motility. Moreover, CD45KO spleen progenitors, which are not directly influenced by osteoclasts, exhibited poor mobility and repopulation potentials, and an unusual distribution of these progenitors was observed between the spleen and the PB. However, previous studies showed that in normal settings, spleen progenitors reside in equilibrium with the blood, suggesting no barrier between these organs (46
), as opposed to the BM (6
). Hence, progenitor accumulation in the spleen may also be affected by the impaired intrinsic ability of CD45KO spleen progenitors to traffic to the circulation. Additional factors may also be involved, including increased survival and/or proliferation of these progenitor cells in extramedullary locations such as the spleen.
Our results suggest that hematopoietic stem cells and their leukocyte progeny have dual CD45-mediated self-regulation modes: their motility, proliferation, and adhesion are autonomously and dynamically regulated. In addition to stem cell regulation by the niche, functional CD45 is needed for osteoclast development and activity, which indirectly affect hematopoiesis and the progenitor pool size via interactions with the bone and BM stromal cells. This notion of a dynamic cross talk between all components of the system is also supported by a recent study, which shows that primitive signaling lymphocyte activation molecule stem and progenitor cells can directly regulate osteoblast development (47
). Collectively, our results reveal that hematopoietic stem and progenitor cells are involved in regulating their own levels and the dynamic BM microenvironment via their osteoclast progeny, which require modulated CD45 activity.