Functional and phenotypic analysis of thymic CD34+CD1a- progenitor-derived dendritic cells: predominance of CD1a+ differentiation pathway

Whether thymic dendritic cells (DC) are phenotypically and functionally distinct from the monocyte lineage DC is an important question. Human thymic progenitors differentiate into T, NK, and DC. The latter induce clonal deletion of autoreactive thymocytes and therefore might be different from their monocyte-derived counterparts. The cytokines needed for the differentiation of DC from thymic progenitors were also questioned, particularly the need for GM-CSF. We show that various cytokine combinations with or without GM-CSF generated DC from CD34+CD1a- but not from CD34+CD1a+ thymocytes. CD34+ thymic cells generated far fewer DC than their counterparts from the cord blood. The requirement for IL-7 was strict whereas GM-CSF was dispensable but nonetheless improved the yield of DC. CD14+ monocytic intermediates were not detected in these cultures unless macrophage-CSF (M-CSF) was added. Cultures in M-CSF generated CD14-CD1a+ DC precursors but also CD14+CD1a- cells. When sorted and recultured in GM-CSF, CD14+ cells down-regulated CD14 and up-regulated CD1a. TNF-alpha accelerated the differentiation of progenitors into DC and augmented MHC class II transport to the membrane, resulting in improved capacity to induce MLR. The trafficking of MHC class II molecules was studied by metabolic labeling and immunoprecipitation. MHC class II molecules were transported to the membrane in association with invariant chain isoforms in CD14+ (monocyte)-derived and in CD1a+ thymic-derived DC but not in monocytes. Thus, thymic progenitors can differentiate into DC along a preferential CD1a+ pathway but have conserved a CD14+ maturation capacity under M-CSF. Finally, CD1a+-derived thymic DC and monocyte-derived DC share very close Ag-processing machinery.     

TNF skews monocyte differentiation from macrophages to dendritic cells

Monocytes represent a large pool of circulating precursors of APCs, both macrophages and dendritic cells (DCs). It is thus important to identify the mechanisms by which microenvironment regulates monocyte differentiation. We have previously shown that, upon contact with resting stromal cells such as fibroblasts, monocytes differentiate into macrophages in an IL-6/M-CSF-dependent fashion. Yet, in the inflamed tissue, monocytes need to yield DCs for the adaptive immunity to be induced. Inasmuch as TNF and IL-1 are present at the site of inflammation, we tested their capacity to modulate monocyte differentiation into either macrophages or DCs. TNF, but not IL-1, induce monocytes to become DCs despite the presence of fibroblasts. TNF-induced DCs contain Langerin-positive cells and are able to induce allogenic T cell proliferation. Then, TNF was found to decrease the expression and internalization of the M-CSF receptor, thus overriding the IL-6/M-CSF pathway. Thus, TNF facilitates the induction of adaptive immunity by promoting DC differentiation not only from CD34+ progenitors but also from CD14+ blood precursors.     

Macrophage colony-stimulating factor drives cord blood monocyte differentiation into IL-10(high)IL-12absent dendritic cells with tolerogenic potential

Immature dendritic cells (DCs) induce tolerance and mature DCs induce inflammatory immune responses. However, the likelihood of maturation of immature DCs in vivo limits its potential application for suppression of unwanted immune reactions in vivo. The aim of this study was to generate DCs with anti-inflammatory properties in both the immature and mature states. GM-CSF combined with IL-4 drives monocyte differentiation into DCs. As M-CSF is a critical cytokine in development of the monocytic lineage and its level is dramatically elevated in immunosuppressive conditions, we investigated whether M-CSF could replace GM-CSF and generate DCs with distinct functions from umbilical cord blood monocytes. Highly purified umbilical cord blood monocytes cultured with M-CSF and IL-4, in a GM-CSF-independent fashion, differentiated into IL-10(high)IL-12absent cells with a DC phenotype (termed M-DC). Single time stimulation with immature DCs (both M-DCs and DCs) derived from cord blood induced hyporesponsive and regulatory CD4+ T cells. In contrast to mature DCs, mature M-DCs induced decreased Th1 differentiation and proliferation of naive CD4+ T cells in both primary and secondary allogeneic MLR and showed tolerogenic potential. These results demonstrate an unrecognized role for M-CSF in alternative differentiation of monocytes into anti-inflammatory M-DCs and suggest that M-CSF-induced DCs may be of use for suppressing unwanted immune responses.     

Notch1 and IL-7 receptor interplay maintains proliferation of human thymic progenitors while suppressing non-T cell fates

Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34high c-kit+ flt3+ IL-7Ralpha+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitors.     

Lymphohematopoietic progenitors do not have a synchronized defect with age-related thymic involution

It has been speculated that aging lymphohematopoietic progenitor cells (LPC) including hematopoietic stem cells (HSC) and early T-cell progenitors (ETP) have intrinsic defects that trigger age-related thymic involution. However, using a different approach, we suggest that that is not the case. We provided a young thymic microenvironment to aged mice by transplanting a fetal thymus into the kidney capsule of aged animals, and demonstrated that old mouse-derived LPCs could re-establish normal thymic lymphopoiesis and all thymocyte subpopulations, including ETPs, double negative subsets, double positive, and CD4(+) and CD8(+) single positive T cells. LPCs derived from aged mice could turn over young RAG(-/-) thymic architecture by interactions, as well as elevate percentage of peripheral CD4(+)IL-2(+) T cells in response to costimulator in aged mice. Conversely, intrathymic injection of ETPs sorted from young animals into old mice did not restore normal thymic lymphopoiesis, implying that a shortage and/or defect of ETPs in aged thymus do not account for age-related thymic involution. Together, our findings suggest that the underlying cause of age-related thymic involution results primarily from changes in the thymic microenvironment, causing extrinsic, rather than intrinsic, defects in T-lymphocyte progenitors.     

IL-10 Acts As a Developmental Switch Guiding Monocyte Differentiation to Macrophages during a Murine Peritoneal Infection

The peritoneal wash of BALB/c or C57BL/6 mice contains two populations of macrophages that differ in their level of expression of MHC class II (MHC II). Although both populations efficiently phagocytose bacteria in vivo, only the MHC II(lo) population is effective at phagocytosing apoptotic cells in vivo and only the MHC II(hi) population is effective at presenting Ag to T cells in vitro. Soon after induction of a peritoneal infection both of these macrophage populations are lost from the peritoneal wash fraction. Blood monocytes then enter the inflamed peritoneum and develop into new peritoneal macrophages. Whether these monocytes develop into MHC II(lo) or into MHC II(hi) macrophages is crucially dependent on the cytokine IL-10, which is transiently elevated in the peritoneal wash during the early phase of infection. Monocytes from CD45.1 animals transferred early in infection when the IL-10 concentration is high into congenic CD45.2 recipients develop into the MHC II(lo) macrophage population. Monocytes transferred later, when the IL-10 concentration has fallen, develop into the MHC II(hi) population. In infected IL-10-deficient animals monocytes fail to develop into the MHC II(lo) population but can be induced to do so by exogenous application of IL-10. Finally, high numbers of wild-type monocytes injected into IL-10R1-deficient animals develop into MHC II(lo) macrophages and were able by a bystander effect to induce the differentiation of the endogenous monocytes to the same fate.     

Macrophage colony-stimulating factor pretreatment of bone marrow progenitor cells regulates osteoclast differentiation based upon the stage of myeloid development

Osteoclasts (OCs) are large, multinucleated bone resorbing cells originating from the bone marrow myeloid lineage, and share a common progenitor with macrophages and dendritic cells. Bone marrow cells (BMCs) are a common source for in vitro osteoclastogenesis assays but are a highly heterogeneous mixture of cells. Protocols for in vitro osteoclastogenesis vary considerably thus hindering interpretation and comparison of results between studies. Macrophage colony-stimulating factor (M-CSF) pretreatment is commonly used to expand OC progenitors (OCPs) in BMC cultures before in vitro differentiation. However, the failure of osteoclastogenesis of M-CSF primed bone marrow myeloid blasts has been reported. In this study, we used a simple method of differential adherence to plastic to enrich OCP from mouse BMCs. We found that M-CSF pretreatment of plastic-adherent BMCs (adBMCs) increased the number of CD11b-F4/80+ macrophages and decreased the number of CD11b+ monocytes resulting in decreased OC formation. M-CSF pretreatment of purified c-Kit+ progenitors weakly inhibited OC formation, whereas M-CSF pretreatment of purified c-Kit-CD11b+ progenitors promoted the formation of large OC. M-CSF pretreatment increased the proliferation of both purified c-Kit+ and c-Kit-CD11b+ cells and increased the percentage of CD11b-F4/80+ cells from c-Kit+ progenitors. In addition, M-CSF pretreatment increased the percentage of CD11b+ F4/80− cells from purified c-Kit-CD11b+ cells. M-CSF pretreatment increased the percentage of CD14 + CD16 + intermediate monocytes and subsequent OC formation from human 2adBMCs, and increased OC formation of purified CD14 + cells. Together, these results indicate that in vitro OCP expansion in the presence of M-CSF and bone marrow stromal cells is dependent upon the developmental stage of myeloid cells, in which M-CSF favors macrophage differentiation of multipotent progenitors, promotes monocyte maturation and supports differentiation of late-stage OCP cells.     

P-selectin enhances generation of CD14+CD16+ dendritic-like cells and inhibits macrophage maturation from human peripheral blood monocytes

Endothelial cells play a critical role in monocyte differentiation. Platelets also affect terminal maturation of monocytes in vitro. P-selectin is an important adhesion molecule expressed on both endothelial cells and activated platelets. We investigated its effects on human peripheral blood monocyte differentiation under the influence of different cytokines. Generation of dendritic-like cells (DLCs) from peripheral blood monocytes was promoted by immobilized P-selectin in the presence of M-CSF and IL-4 as judged by dendritic cell (DC) morphology; increased expression of CD1a, a DC marker; low phagocytic activity; and high alloreactivity to naive T cells. In contrast to typical DCs, DLCs expressed CD14 and FcgammaRIII (CD16). These features link the possible identity of DLCs to that of an uncommon CD14(+)CD16(+)CD64(-) monocyte subset found to be expanded in a variety of pathological conditions. Functionally, DLCs generated by P-selectin in combination with M-CSF plus IL-4 primed naive allogeneic CD4(+) T cells to produce significantly less IFN-gamma than cells generated by BSA in the presence of M-CSF and IL-4. P-selectin effects on enhancing CD14(+)CD16(+) DLC generation were completely abrogated by pretreatment of cells with the protein kinase C delta inhibitor rottlerin, but not by classical protein kinase C inhibitor G?6976. Immobilized P-selectin also inhibited macrophage differentiation in response to M-CSF alone as demonstrated by morphology, phenotype, and phagocytosis analysis. The effects of P-selectin on macrophage differentiation were neutralized by pretreatment of monocytes with Ab against P-selectin glycoprotein ligand 1. These results suggest a novel role for P-selectin in regulating monocyte fate determination.     

The development of macrophages from human CD34+ haematopoietic stem cells in serum-free cultures is optimized by IL-3 and SCF

The derivation of human macrophages from peripheral blood monocytes remains a convenient method for the study of macrophage biology. However, for macrophage differentiation, a significant proportion of development has occurred prior to the monocyte stage; monocyte subsets also have varying potential for differentiation. Differentiation of macrophages from a less mature precursor, such as CD34⁺ haematopoietic stem cells, can further inform with regard to the development of macrophage-lineage cells. CD34⁺ cells were cultured in serum-free medium containing Flt3L, SCF, IL-3, IL-6 and M-CSF. Using differing combinations of growth factors, the effect on cell proliferation and differentiation to adherent macrophage-like cells was determined. The proliferative response of CD34⁺ cells to M-CSF was determined during the initial phase of cell culture. Thirteen combinations of SCF, IL-3, IL-6 and M-CSF were then compared to determine the optimum combination for proliferation. Adherence was used to isolate mature macrophages, and the macrophage-like phenotype was confirmed by analyses of surface markers, histo-morphology and phagocytosis. This study refines the means by which large numbers of macrophages are obtained under serum-free conditions from haematopoietic precursors.     

Interleukin-34 induces monocytic-like differentiation in leukemia cell lines

Interleukin-34 (IL-34) is a cytokine consisting of a 39kD homodimer, shown to be a ligand for both the Macrophage Colony Stimulating Factor (M-CSF/CSF-1) receptor and the Receptor-like protein tyrosine phosphatase-zeta (RPTP-ƺ). IL-34 has been shown to promote monocyte viability and proliferation as well as the differentiation of bone marrow cells into macrophage progenitors. Published work on IL-34 involves its effects on normal hematopoietic and osteoclast progenitors. However, it is not known whether IL-34 has biologic effects in cancer, including leukemia. Here we report that the biological effects of IL-34 include induction of differential expression of Interleukins-1α and -1β as well as induction of differentiation of U937, HL-60 and THP-1 leukemia cell lines demonstrating monocyte-like characteristics. The ability of IL-34 to induce monocytic-like differentiation is supported by strong morphological and functional evidence. Cell surface markers of myeloid lineage, CD64 and CD86, remain constant while the levels of CD11b and CD71 decline with IL-34 treatment. IL-34 also induced increases in CD14 and CD68 expression, further supporting maturation toward monocytic character. IL-34-induced differentiated U937 and THP-1 cell lines exhibited biological functions such as endocytosis and respiratory burst activities. Collectively, we conclude that while IL-34 does not induce cell growth or proliferation, it is able to induce differentiation of leukemia cell lines from monoblastic precursor cells towards monocyte- and macrophage-like cells, mediated through the JAK/STAT and PI3K/Akt pathways. To our knowledge, this is the first report that IL-34 induces differentiation in human leukemic cells, let alone any cancer model.     

Macrophage colony stimulating factor (M-CSF) is essential for osteoclast formation in vitro

The op/op mouse, in which the M-CSF gene is mutated, has greatly reduced numbers of macrophages and osteoclasts. We assessed the ability of M-CSF to induce osteoclast and macrophage formation in op/op hemopoietic cells in vitro. Osteoclast production was undetectable in op/op cell cultures, but was restored by M-CSF at concentrations approximately an order of magnitude higher than those that induced macrophages. In normal hemopoietic tissue M-CSF similarly increased macrophage numbers, but inhibited osteoclast formation. Despite cure of the macrophage defect, neither interleukin 3 nor granulocyte-macrophage CSF were able to induce osteoclastic differentiation in op/op cells. The results suggest that M-CSF induces osteoclastic differentiation but that macrophages, which are also induced by M-CSF, suppress osteoclast differentiation. Macrophages induced by other cytokines seem unable to contribute to osteoclast-formation.     

T/NK bipotent progenitors in the thymus retain the potential to generate dendritic cells

We have previously shown that the earliest thymic progenitors retain the potential to generate T and NK cells and that they lose the bipotentiality to give rise to unipotent T and NK progenitors during the progression of intrathymic developmental stages. The present study examines the ability of these thymic progenitors for generation of dendritic cells (DC) with a new clonal assay that is capable of determining the developmental potential for DC in addition to T cells and NK cells. We found that the large majority of the T/NK bipotential progenitors in the earliest population of fetal thymus was able to generate DC. Although the DC potential is lost with the progression of the differentiation stage, some of the T/NK bipotential progenitors still retain their DC potential even at the CD44(+)CD25(+) stage.     

Cytokine production by M-CSF- and GM-CSF-induced mouse bone marrow-derived macrophages upon coculturing with late apoptotic cells

Recently, we found that resident peritoneal macrophages produce MIP-2, one of the major chemokines for neutrophils, upon coculturing with late apoptotic cells, and that intraperitoneal injection of late apoptotic cells into the peritoneal cavity causes neutrophil infiltration via MIP-2. It is not known, however, whether or not macrophages are heterogeneous in such MIP-2 production. In this study, we examined changes in the surface phenotype during the differentiation of bone marrow cells into macrophages due to M-CSF and GM-CSF, and then examined the production of cytokines, namely IL-12 p40, MIP-2, IL-10, and TGF-β, following phagocytosis of late apoptotic cells with these macrophages or LPS stimulation of these macrophages. GM-CSF and M-CSF induced macrophage populations with distinct but overlapping cell surface phenotype. Although these macrophages phagocytosed late apoptotic cells to a similar extent, they produced either IL-12 p40 or IL-10, whereas they produced MIP-2 to a similar extent after the coculture, raising the possibility that late apoptotic cells may induce neutrophil infiltration in any organs, such as the liver and lungs, where the macrophages are differentiated by either M-CSF or GM-CSF, respectively.     

Effect of different activation stimuli on the cytokine response of human macrophages to CD40L.

Here we show that CD40L (ligand for CD40) failed to induce the production of tumour necrosis factor alpha (TNF-alpha), interleukin (IL-)-1 beta, IL-10 and IL-12 in macrophages matured in vitro in the absence of growth factors or in the presence of macrophage colony-stimulating factor (M-CSF). In contrast, enzyme-linked immunoabsorbent assay (ELISA) testing and cytofluorimetric (FACS) analysis demonstrated significant production of TNF-alpha and IL-1 beta, but not of IL-10 and IL-12 in macrophages maturated in the presence of CD40L and re-stimulated with CD40L. The priming effect of CD40L on TNF-alpha and IL-1 beta production was related to induction of CD40 expression. Finally, CD40L priming did not modify the cytokine response of macrophages to lipopolysaccharide. In conclusion, our results suggest that CD40/CD40L interactions are important for the activation of macrophages as effector cells that mediate inflammation and tissue damage in T cell-mediated inflammatory processes.     

E2a/Pbx1 induces the rapid proliferation of stem cell factor-dependent murine pro-T cells that cause acute T-lymphoid or myeloid leukemias in mice

Oncoprotein E2a/Pbx1 is produced by the t(1;19) chromosomal translocation of human pre-B acute lymphoblastic leukemia. E2a/Pbx1 blocks differentiation of primary myeloid progenitors but, paradoxically, induces apoptosis in established pre-B-cell lines, and no transforming function of E2a/Pbx1 has been reported in cultured lymphoid progenitors. Here, we demonstrate that E2a/Pbx1 induces immortal proliferation of stem cell factor (SCF)-dependent pro-T thymocytes by a mechanism dependent upon both its transactivation and DNA-binding functions. E2a-Pbx1 cooperated with cytokines or activated signaling oncoproteins to induce cell division, as inactivation of conditional E2a/Pbx1 in either factor-dependent pro-T cells or pro-T cells made factor independent by expression of Bcr/Abl resulted in pro-T-cell quiescence, while reactivation of E2a/Pbx1 restored cell division. Infusion of E2a/Pbx1 pro-T cells in mice caused T lymphoblastic leukemia and, unexpectedly, acute myeloid leukemia. The acute lymphoblastic leukemia did not evidence further maturation, suggesting that E2a/Pbx1 establishes an early block in pro-T-cell development that cannot be overcome by marrow or thymic microenvironments. In an E2a/Pbx1 pro-T thymocyte clone that induced only pro-T acute lymphoblastic leukemia, coexpression of Bcr/Abl expanded its leukemic phenotype to include acute myeloid leukemia, suggesting that unique functions of cooperating signaling oncoproteins can influence the lymphoid versus myeloid character of E2a/Pbx1 leukemia and may cooperate with E2a/Pbx1 to dictate the pre-B-cell phenotype of human leukemia containing t(1;19).     

Chicken myeloid stem cells infected by retroviruses carrying the v-fps oncogene do not require exogenous growth factors to differentiate in vitro

To determine the function of c-fps in chicken macrophages and granulocytic cells we have infected chicken bone marrow cells with retroviruses containing the v-fps oncogene. Normal chicken macrophage progenitors, M-CFCs, give rise to macrophage colonies in semisolid cultures when macrophage colony stimulating factor (M-CSF) is added into the culture medium. Upon infection with v-fps bearing retroviruses, we observed that M-CFCs were induced to develop macrophage colonies in vitro without exogenous M-CSF. This activation results from a direct effect of v-fps on the M-CFCs. No leukemic transformation was observed in the infected colonies. By comparing the effects of several retroviruses, we showed that the induction of M-CFC development is specific to v-fps containing viruses and mediated by the v-fps protein. These observations support the hypothesis that the c-fps gene is involved in the control of proliferation and/or differentiation of myeloid cells.     

Differentiation of the IL-3-dependent NFS-60 cell line and adaption to growth in macrophage colony-stimulating factor

Macrophage CSF (M-CSF) induces responsive bone marrow precursors into rapid growth and differentiation to mature macrophages. Available cell lines that depend on M-CSF for growth are well differentiated and rather adherent. We investigated the effects of M-CSF on immature myeloid cell lines as models of the marrow precursors. The murine line NFS-60 requires IL-3 for growth and also responds to granulocyte-CSF and granulocyte-macrophage-CSF. Cultures of one NFS-60 subline, when switched from IL-3 to 10% L cell conditioned media, a source of M-CSF, or purified M-CSF, frequently acquired large, adherent cells. The adherent cells grew slowly in the presence of M-CSF, in contrast to the majority population of small, round, rapidly growing cells. The large cells had properties of differentiated macrophages that were absent in the nonadherent cells. Cells with macrophage phenotype were not observed in IL-3-supported cultures over many passages. A subline was derived from NFS-60 that grew rapidly and continuously in human or murine M-CSF as round, nonadherent cells. The line, called M-NFS-60, responded well to M-CSF and IL-3, weakly to granulocyte-CSF and not at all to murine granulocyte-macrophage-CSF, IL-4, or human IL-1. A mAb to human M-CSF specifically inhibited only M-NFS-60 proliferation induced by the human growth factor, whether produced by mammalian or bacterial cells. This study shows two effects of M-CSF on the IL-3-dependent NFS-60 line. Upon first exposure to M-CSF, cells may undergo global differentiation to slowly replicating macrophages in conditions we have not been able to define. The more common effect is rapid growth of immature myeloid cells like the bone marrow precursors, but with a block to differentiation. Thus, these cells may be useful as models of M-CSF-induced differentiation, and of permanently maintained macrophage precursors.