Heme oxygenase-1 derived carbon monoxide permits maturation of myeloid cells

Critical functions of the immune system are maintained by the ability of myeloid progenitors to differentiate and mature into macrophages. We hypothesized that the cytoprotective gas molecule carbon monoxide (CO), generated endogenously by heme oxygenases (HO), promotes differentiation of progenitors into functional macrophages. Deletion of HO-1, specifically in the myeloid lineage (Lyz-Cre:Hmox1^flfl), attenuated the ability of myeloid progenitors to differentiate toward macrophages and decreased the expression of macrophage markers, CD14 and macrophage colony-stimulating factor receptor (MCSFR). We showed that HO-1 and CO induced CD14 expression and efficiently increased expansion and differentiation of myeloid cells into macrophages. Further, CO sensitized myeloid cells to treatment with MCSF at low doses by increasing MCSFR expression, mediated partially through a PI3K-Akt-dependent mechanism. Exposure of mice to CO in a model of marginal bone marrow transplantation significantly improved donor myeloid cell engraftment efficiency, expansion and differentiation, which corresponded to increased serum levels of GM-CSF, IL-1α and MCP-1. Collectively, we conclude that HO-1 and CO in part are critical for myeloid cell differentiation. CO may prove to be a novel therapeutic agent to improve functional recovery of bone marrow cells in patients undergoing irradiation, chemotherapy and/or bone marrow transplantation.     

Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells

In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin(-)CD34(+)CD43(+)CD45(+) multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5-19 d of culture with cytokines, depending on the cell type.     

Functional maturation of murine B lymphocyte precursors. II. Analysis of cells required from the bone marrow microenvironment

The development of mature B cells in cultures of early B cell precursors depends on the presence of a confluent adherent bone marrow (aBM) cell layer. Adherent and sIgM+ cell-depleted bone marrow (BM) from untreated or 5-fluorouracil-pretreated donors or day 12 fetal liver cells were used as precursor cell populations. When adherent cells from thymus or highly enriched BM-derived macrophages were co-cultured with precursor cells, mature B cells were not developed. Similarly, aBM cell layers generated in the presence of hydrocortisone and horse serum were unable to support aBM cell-dependent precursor differentiation, even though cortisone was removed before the addition of precursor cells. In contrast, this type of microenvironment promoted the differentiation of precursor of myeloid cell lineages. Repeated treatment of established aBM cell populations with a monoclonal anti-macrophage antibody (31.3, known to recognize a surface marker on a subset of BM macrophages) and complement abolished the capacity of otherwise functional aBM cells to sustain the development of B cell precursors. Macrophage-depleted aBM cells regained their function after supplementation with highly enriched BM-derived macrophages grown in vitro. Limiting dilution analysis of aBM cells in microcultures containing saturating numbers of early B cell progenitors also suggests the participation of more than one cell type in the BM cell population. In conclusion, differentiation of early B cell progenitors requires macrophages in addition to at least one additional cell type contained in the aBM cell population.     

Increased generation of dendritic cells from myeloid progenitors in autoimmune-prone nonobese diabetic mice

Aberrant dendritic cell (DC) development and function may contribute to autoimmune disease susceptibility. To address this hypothesis at the level of myeloid lineage-derived DC we compared the development of DC from bone marrow progenitors in vitro and DC populations in vivo in autoimmune diabetes-prone nonobese diabetic (NOD) mice, recombinant congenic nonobese diabetes-resistant (NOR) mice, and unrelated BALB/c and C57BL/6 (BL/6) mice. In GM-CSF/IL-4-supplemented bone marrow cultures, DC developed in significantly greater numbers from NOD than from NOR, BALB/c, and BL/6 mice. Likewise, DC developed in greater numbers from sorted (lineage(-)IL-7Ralpha(-)SCA-1(-)c-kit(+)) NOD myeloid progenitors in either GM-CSF/IL-4 or GM-CSF/stem cell factor (SCF)/TNF-alpha. [(3)H]TdR incorporation indicated that the increased generation of NOD DC was due to higher levels of myeloid progenitor proliferation. Generation of DC with the early-acting hematopoietic growth factor, flt3 ligand, revealed that while the increased DC-generative capacity of myeloid-committed progenitors was restricted to NOD cells, early lineage-uncommitted progenitors from both NOD and NOR had increased DC-generative capacity relative to BALB/c and BL/6. Consistent with these findings, NOD and NOR mice had increased numbers of DC in blood and thymus and NOD had an increased proportion of the putative myeloid DC (CD11c(+)CD11b(+)) subset within spleen. These findings demonstrate that diabetes-prone NOD mice exhibit a myeloid lineage-specific increase in DC generative capacity relative to diabetes-resistant recombinant congenic NOR mice. We propose that an imbalance favoring development of DC from myeloid-committed progenitors predisposes to autoimmune disease in NOD mice.     

Leukotriene B4 mediates neutrophil migration induced by heme

High concentrations of free heme found during hemolytic events or cell damage leads to inflammation, characterized by neutrophil recruitment and production of reactive oxygen species, through mechanisms not yet elucidated. In this study, we provide evidence that heme-induced neutrophilic inflammation depends on endogenous activity of the macrophage-derived lipid mediator leukotriene B(4) (LTB(4)). In vivo, heme-induced neutrophil recruitment into the peritoneal cavity of mice was attenuated by pretreatment with 5-lipoxygenase (5-LO) inhibitors and leukotriene B(4) receptor 1 (BLT1) receptor antagonists as well as in 5-LO knockout (5-LO(-/-)) mice. Heme administration in vivo increased peritoneal levels of LTB(4) prior to and during neutrophil recruitment. Evidence that LTB(4) was synthesized by resident macrophages, but not mast cells, included the following: 1) immuno-localization of heme-induced LTB(4) was compartmentalized exclusively within lipid bodies of resident macrophages; 2) an increase in the macrophage population enhanced heme-induced neutrophil migration; 3) depletion of resident mast cells did not affect heme-induced LTB(4) production or neutrophil influx; 4) increased levels of LTB(4) were found in heme-stimulated peritoneal cavities displaying increased macrophage numbers; and 5) in vitro, heme was able to activate directly macrophages to synthesize LTB(4). Our findings uncover a crucial role of LTB(4) in neutrophil migration induced by heme and suggest that beneficial therapeutic outcomes could be achieved by targeting the 5-LO pathway in the treatment of inflammation associated with hemolytic processes.     

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1-3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.     

1 alpha, 25-Dihydroxyvitamin D3 augments clonal growth of macrophages from rat bone marrow progenitor cells and modulates their function

1 alpha, 25-Dihydroxyvitamin D3 (1,25(OH)2D3) was shown to enhance (approximately 2 fold) the colony-stimulating factor-dependent clonal growth of macrophage colonies and clusters from rat bone marrow progenitor cells. The proliferative capacity of macrophage progenitors in liquid cultures was likewise augmented (2-3 fold). Mononuclear phagocytes (macrophages, for simplicity) developing in the presence of 1,25(OH)2D3 showed a reduced capacity of migration. 1,25(OH)2D3 administered at bone marrow culture initiation led to augmentation of the phagocytic capability of macrophages in four-day cultures and to its suppression in macrophages in seven-day cultures. The observed patterns of modulation of differentiation and function by 1,25(OH)2D3 differ from the patterns we found for mouse bone marrow cells. The results suggest that the differential response to hormones observed in different species may include responses to 1,25(OH)2D3.     

Contribution of Monocyte/Macrophage Differentiation to the Stromal Layer in Human Long-Term Bone Marrow Cultures

The stroma of bone marrow is a poorly understood tissue which is believed to have important roles in haemopoietic stem cell maintenance and differentiation. We have undertaken immunohistochemical studies of bone marrow stroma in human long-term bone marrow cultures (hLTBMC) and biopsy specimens in order to characterise the cell and matrix components present. We have found two morphological variants of macrophages to be present consistently in hLTBMC stroma, adherent to a substratum of myofibroblastic cells. Large round macrophages appear to actively phagocytic and are formed in hLTBMC regardless of successful establishment of a myofibroblastic cell layer. Elongated macrophages with dendritic processes appear to be non-phagocytic and form only in the presence of a well-established layer of myofibroblasts. Although the functions of these macrophages are not yet known, they have counterparts within intact human bone marrow and their presence in hLTBMC shows some association with the haemopoietic capacity of the cultures.     

The cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), can deliver Bcl-XL as an extracellular fusion protein to protect cells from apoptosis and retain differentiation induction

Bcl-XL, a member of the Bcl-2 protein family, is able to suppress cell death induced by diverse stimuli in many cell types, including hematopoietic cells. Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that promotes the proliferation and maturation of neutrophils, eosinophils, and macrophages from bone marrow progenitors. We fused GM-CSF to Bcl-XL and examined the capacity of this chimera to bind human cells through the GM-CSF receptor and prevent apoptosis. We found that the chimeric protein increased the proliferation of human monocytes in culture from 24 h until at least 72 h. In the presence of different apoptotic agents, GM-CSF-Bcl-XL protected cells from induced cell death and promoted proliferation, whereas GM-CSF alone was completely inhibited. In the presence of cytarabine, GM-CSF-Bcl-XL was able also to promote the differentiation of the CD34+ myeloid precursor whereas Lfn-Bcl-XL, lacking the GM-CSF domain-stimulated cell proliferation and not differentiation. We conclude that recombinant GM-CSF-Bcl-XL binds the GM-CSF receptor on human monocyte/macrophage cells and bone marrow progenitors inducing differentiation and allowing Bcl-XL entry into cells where it blocks cell death and allows amplified cell proliferation. This fully human fusion protein has potential to prevent monocytopenia and represents a new strategy for engineering anti-apoptotic therapeutics.     

MLL-ENL cooperates with SCF to transform primary avian multipotent cells

The MLL gene is targeted by chromosomal translocations, which give rise to heterologous MLL fusion proteins and are associated with distinct types of acute lymphoid and myeloid leukaemia. To determine how MLL fusion proteins alter the proliferation and/or differentiation of primary haematopoietic progenitors, we introduced the MLL-AF9 and MLL-ENL fusion proteins into primary chicken bone marrow cells. Both fusion proteins caused the sustained outgrowth of immature haematopoietic cells, which was strictly dependent on stem cell factor (SCF). The renewing cells have a long in vitro lifespan exceeding the Hayflick limit of avian cells. Analysis of clonal cultures identified the renewing cells as immature, multipotent progenitors, expressing erythroid, myeloid, lymphoid and stem cell surface markers. Employing a two-step commitment/differentiation protocol involving the controlled withdrawal of SCF, the MLL-ENL-transformed progenitors could be induced to terminal erythroid or myeloid differentiation. Finally, in cooperation with the weakly leukaemogenic receptor tyrosine kinase v-Sea, the MLL-ENL fusion protein gave rise to multilineage leukaemia in chicks, suggesting that other activated, receptor tyrosine kinases can substitute for ligand-activated c-Kit in vivo.     

Interactions of tumor necrosis factor with granulocyte-macrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34+ progenitors in human bone marrow.

Colonies of CD1a+ HLA-DR+/DQ+ CD4+ cells with the functional and some of the structural attributes of Langerhans cells are observed in human bone marrow cultures in semi-solid media and are assumed to be the progeny of an early progenitor, the dendritic/Langerhans cell CFU (CFU-DL). The cytokine-regulated growth of these cells has been studied using a chemically defined serum-free system to culture both unfractionated and highly enriched bone marrow progenitor cell populations. Although unfractionated cell growth was optimal in serum replete cultures with PHA-stimulated leukocyte-conditioned medium (PHA-LCM) suboptimal proliferation of CFU-DL was observed in serum even in the absence of PHA-LCM. No colonies were observed under serum-free conditions when granulocyte-macrophage CSF (GM-CSF), IL-3, granulocyte CSF (G-CSF), and macrophage CSF (M-CSF) were present at levels optimal for granulocyte colony-forming unit (CFU-G) and macrophage colony-forming unit (CFU-M) growth. Addition of IL-1 alpha to these cytokines stimulated a small number of CFU-DL. However, in the presence of GM-CSF and IL-3, TNF-alpha or TNF-beta (5 U/ml) were both highly effective in promoting growth up to 82% of optimal and CFU-G growth was also enhanced at these concentrations. TNF was only active during the first 3 days of culture and higher concentrations of TNF-alpha but not TNF-beta were inhibitory for both CFU-DL and CFU-G. CD34+ cell-enriched populations were also enriched for both myeloid progenitors (CFU-G + CFU-M) and CFU-DL to 36- and 48-fold, respectively, and single cell cultures of CD34+ cells yielded single colonies containing both CD1a+ dendritic cells and CD1a- macrophages. Thus dendritic/Langerhans progenitors in the bone marrow expresses CD34, have a capacity for both macrophage and dendritic cell differentiation, and depend on hemopoietic growth factors and TNF for their further development in vitro.     

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.     

Dendritic accessory cells derived from rat bone marrow precursors under chemically defined conditions in vitro belong to the myeloid lineage

Serum-free conditions have been developed to differentiate dendritic cells from a non-adherent fraction of rat bone marrow precursors by action of the multipotential and macrophage colony-stimulating factors further supplemented with linoleic acid, vitamin E, and vitamin D3. Accessory activity was demonstrated by the high potency of the dendritic cells to stimulate autologous T cell proliferation, whereas such cells were negative for Fc receptor-dependent phagocytosis, a characteristic macrophage feature. While the dendritic cells were weakly positive for alpha-naphtylbutyrate esterase, they strongly expressed RT.1 class II antigens. Apparently, these cells represent a more differentiated phenotype since they expressed the nuclear A/C lamins. By addition of serum to the cultures, the dendritic cells developed into macrophages, which were also lamin A/C-positive as well as strongly positive for alpha-naphtylbutyrate esterase. Thus, these dendritic cells belong to the myeloid lineage, and it appears as if serum factor(s) control differentiation at a mature level. Suitable conditions could also be established for large-scale cultures of dendritic cells, which would be useful for applications requiring higher numbers of cells.     

Factors affecting the generation of mast cells from multipotential colony-forming cells

The cells responsible for the long-term in vitro generation of murine mast cells have been examined. Sequential analysis of all colony types obtained from cultures of spleen or bone marrow cells showed that only colonies derived from multipotential cells (mixed-erythroid colonies) or mast cell progenitors, contained cells responsible for mast cell generation in liquid cultures. Primary colony growth and subsequent maintenance of mast cells in liquid cultures was dependent upon pokeweed mitogen-stimulated spleen cell-conditioned medium (SCM). Mixed-erythroid colonies from 14-day cultures of spleen cells had the greatest capacity for mast cell generation. Analysis by clone splitting and transfer to high (20%) and low (2.5%) concentrations of SCM showed that the concentration of SCM used in either the primary colony culture or subsequent liquid culture phase altered both the proliferative capacity of the mast cells generated and the frequency of mast cell progenitors within individual mixed-erythroid colonies. Thus, mixed-erythroid colonies stimulated with 2.5% SCM contained the highest proportion of mast cell progenitors (34% of colonies) and when stimulated with 20% SCM, approximately fourfold higher numbers of mast cells were produced at weekly intervals from liquid cultures maintained in 2.5% SCM compared to parallel liquid cultures containing 20% SCM. These studies confirm the hemopoietic origin of mast cells and demonstrate that a factor(s) in SCM is able to modulate their proliferative potential.     

Haematopoietic stem cells in spleen have distinct differentiative potential for antigen presenting cells

Dendritic cells (DC) are known to develop from macrophage dendritic progenitors (MDP) in bone marrow (BM), which give rise to conventional (c)DC and monocytes, both dominant antigen presenting cell (APC) subsets in spleen. This laboratory has however defined a distinct dendritic‐like cell subset in spleen (L‐DC), which can also be derived in long‐term cultures of spleen. In line with the restricted in vitro development of only L‐DC in these stromal cultures, we questioned whether self‐renewing HSC or progenitors exist in spleen with restricted differentiative capacity for only L‐DC. Neonatal spleen and BM were compared for their ability to reconstitute mice and to give rise to L‐DC, as well as other splenic APC. Neonatal spleen cells were transplanted into allotype‐distinct lethally irradiated hosts along with host‐type competitor BM cells, and assayed over 8 to 51 weeks for haematopoietic reconstitution of L‐DC and cDC subsets, along with other lymphoid and myeloid cells. In this study, neonatal spleen showed multilineage haematopoietic reconstitution in mouse chimeras, rather than specific or restricted ability to differentiate into L‐DC. However, the representation of individual APC subsets was found to be unequal in chimeras partially reconstituted with donor cells, such that more donor‐derived progeny were seen for L‐DC than for myeloid and cDC subsets. The ability of HSC in spleen to develop into L‐DC was indicated by a strong bias in the subset size of these cells over other splenic APC subsets. This type of evidence supports a model whereby spleen represents an important site for haematopoiesis of this distinct DC subset. The conditions under which haematopoiesis of L‐DC occurs in spleen, or the progenitors involved, will require further investigation.     

Hemopoiesis in long-term stroma-dependent cultures from lymphoid tissue: Production of cells with myeloid/dendritic characteristics

Summary A long-term stroma-dependent culture system (LTC) has been developed which continuously produces hemopoietic cells providing an in vitro system for the study of cell differentiation. These nonadherent cell populations contain a large subpopulation of dendritic cells (DC). LTC producing DC were easily generated from spleen, but could also be established from bone marrow (BM) and lymph node with less success. It was difficult to establish DC-producing LTC from thymus. The properties of splenic and thymic stroma have been compared. Spleen stroma developed more complicated networks of fibroblasts, endothelial cells, macrophages, and DC. Thymic stromal monolayers were dominated by epithelial cells and fibroblasts, with a lower proportion of macrophages and endothelial cells. They had a relatively sparse structure of cell networks compared with spleen stroma. Cells with dendritiform morphology first appeared in cultures by 2–3 wk. The majority of cells produced were large cells which expressed DC-specific cell surface markers, major histocompatibility complex (MHC) Class II molecules, and the CD80/CD86(B7) costimulator. A high proportion of cells also expressed myeloid cell markers. No T or B lymphoid cells or granulocytes were present in the cultures. LTC continued to produce nonadherent cells resembling myeloid/DC for long periods, even after passage of stromal cells and stem cells at about 3–4 mo. after culture establishment. The LTC system offers potential to study the in vitro differentiation of myeloid/DC.     

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis

The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.