Interleukin-3Rα+ Myeloid Dendritic Cells and Mast Cells Develop Simultaneously from Different Bone Marrow Precursors in Cultures with Interleukin-3

The distinct developmental routes of dendritic cells and mast cells from murine bone marrow cultures with interleukin-3 are unclear. We found that short-term bone marrow cultures with interleukin-3 after 8–10 d consist of about 10%–30% dendritic cells and 70%–90% mast cell precursors, and only after 4–6 wk do homogeneous populations of mast cells emerge. Phenotypical and functional analysis of interleukin-3/dendritic cells revealed a high similarity with myeloid dendritic cells generated with granulocyte-macrophage colony stimulating factor in the expression of myeloid dendritic cell markers (CD11c+ B220– CD8α– CD11b+), major histocompatibility complex II and costimulatory molecules, endocytosis, maturation potential, interleukin-12 production, and T cell priming. Interleukin-3/dendritic cells expressed higher levels of interleukin-3 receptor, however. To dissect the interleukin-3/dendritic cell and mast cell development, we sorted fresh bone marrow cells into six subsets by the antibodies ER-MP12 (CD31) and ER-MP20 (Ly-6C). Both interelukin-3/dendritic cells and granulocyte-macrophage colony stimulating factor/dendritic cells develop from the same bone marrow populations, including the ER-MP12neg, ER-MP20high bone marrow monocytes. In contrast, mast cells only developed from ER-MP12int+high, ER-MP20neg bone marrow cell subsets, indicating that different precursors exist for interleukin-3/dendritic cells and mast cells. Established mast cell cultures could not be converted to dendritic cells or stimulated to express major histocompatibility complex II molecules in vitro or home to lymph node T cell areas in vivo. In summary, we show that dendritic cells generated from bone marrow precursors with interleukin-3 are clearly myeloid and develop via a different pathway compared to bone marrow mast cells

ER-MP12 antigen, a new cell surface marker on mouse bone marrow cells with thymus-repopulating ability: I. Intrathymic repopulating ability of ER-MP12-positive bone marrow cells

We searched for new cell surface markers that allow a positive identification of thymusrepopulating cells in the bone marrow (BM) of the mouse. Recently we raised two rat monocional antibodies (ER-MP12 and ER-MP20) that recognize cell surface antigens expressed by mouse haematopoietic progenitor cells, among which are progenitor cells of the macrophage lineage. Here we show that the ER-MP12 antigen, but not the ER-MP20 antigen, is also expressed by BM cells with thymus-repopulating ability. Using ER-MP12 and ER-MP20 in two-colour immunofluorescence analysis six subpopulatlons of BM cells can be identified. The thymusrepopulating ability of each BM subpopulation was assessed after fluorescence-activated cell sorting and subsequent intrathymic injection into sublethally irradiated Thy-1 congenic recipient mice. Thymus-repopulating activity appeared to be exclusively confined to two subsets of BM cells expressing either high or intermediate levels of the ER-MP12 antigen, but lacking ER-MP20 antigen expression. These BM subsets comprised 1-2% and 30% of total nucleated BM cells respectively. The frequency of thymus-repopulating cells was maximal in the minor BM subpopulation with the highest level of ER-MP12 antigen expression. We conclude that ER-MP12 detects a hitherto unknown cell surface marker expressed by BM cells with thymus-repopulating ability

Markers of mouse macrophage development detected by monoclonal antibodies

In this review, we present and discuss a selected panel of antibody-defined markers expressed during different stages of mouse macrophage development. We distinguish four categories of markers, which are characteristic of: (1) macrophage precursors and immature macrophages (ER-MP12, ER-MP20, ER-MP54, ER-MP58); (2) mature macrophages in general (F4/80, BM8, Mac-1, Mac-2, ER-BMDM1); (3) macrophage subsets (ER-HR3, ER-MP23, ER-TR9, Forssman antigen, MOMA-1, MOMA-2, Monts-4, SER-4), and (4) IFN-γ-stimulated macrophages (H-2Ia, LFA-1, ICAM-1, 158.2, MBR-2, TM-2, TM-4, and TM-5).  It should be noted that many of the markers in this last category are inducible by other stimuli as well. The rigid classification of markers into four separate groups should be regarded as a digitalization of a continuum, thus inevitably implicating a simplification of the complex phenotypic changes that occur during mononuclear phagocyte development. Nevertheless, the current selection of antibodies against markers for different developmental stages of macrophages constitutes an important tool for characterization of mouse macrophages which participate in various biological processes

Distinct mouse bone marrow macrophage precursors identified by differential expression of ER‐MP12 and ER‐MP20 antigens

The characterization of early branch points in the differentiation of leukocytes requires identification of precursor cells in the bone marrow. Recently, we produced two monoclonal antibodies, ER‐MP12 and ER‐MP20, which in two‐color flow‐cytometric analysis divide the murine bone marrow into six defined subsets. Here we show, using fluorescence‐activated cell sorting followed by macrophage colony‐stimulating factor‐stimulated culture in soft agar, that precursors of the mononuclear phagocyte system reside only within the ER‐MP12hi20−, ER‐MP12+20+ and ER‐MP12−20hi bone marrow subsets. Together, these subsets comprise 15% of nucleated bone marrow cells. Furthermore, we provide evidence that the macrophage precursors present in these subsets represent successive stages in a maturation sequence where the most immature ER‐MP12hi20− cells develop via the ER‐MP12+20+ stage into ER‐MP12−20hi monocytes

ER-MP12 antigen, a new cell surface marker on mouse bone marrow cells with thymus-repopulating ability: II. Thymus-homing ability and phenotypic characterization of ER-MP12-positive bone marrow cells

In the accompanying paper we showed that six distinct subsets of bone marrow (BM) cells can be identified using the mAb ER-MP12 and ER-MP20 in two-colour immunofluorescence analysis. Upon intrathymic transfer into sublethally irradiated mice thymus-repopulating ability was restricted to ER-MP20- BM cells expressing either high or intermediate levels of the ER-MP12 antigen (1-2% and -30% of BM nucleated cells respectively). The highest frequency of thymus-repopulating cells was found in the minor subset of ER-MP12++20- BM cells. In the present study we demonstrate that upon intravenous transfer, thymus-homing and-repopulating BM cells are exclusively confined to the ER-MP12++20- and ER-MP12+20- subpopulations, the highest frequency being detected among ER-MP12++20- BM cells. Analysis of the peripheral blood leucocytes of reconstituted mice showed that not only prothymocytes but also progenitorcells of the B cell lineage as well as the myelold lineage were present within both subsets. Three-colour flow cytometric analysis revealed that ER-MP12++20- BM cells in particular were phenotyplcally heterogeneous with respect to the expression of the cell surface markers Thy-1, Sca-1, CD44, B220 and c-kit. Taken together our data demonstrate that ER-MP12 positively identifies BM cells with the ability to home to and repopulate the thymus. The phenotypic heterogeneity displayed by the ER-MP12++20- BM subset, containing the highest frequency of thymus-homing and-repopulating cells, provides a basis for further separation of prothymocyte activity from other haematopoietic activities in the BM of the mouse

High-level expression of the ER-MP58 antigen on mouse bone marrow hematopoietic progenitor cells marks commitment to the myeloid lineage

Studies on the early events in the differentiation of the nonspecific immune system require the identification and isolation of myeloid-committed progenitor cells. Using the monoclonal antibodies (mAb) ER-MP12 and ER-MP20, generated against immortalized macrophage precursors, we have shown previously that the earliest macrophage colony-stimulating factor (M-CSF)-responsive cells in the bone marrow have the ER-MP12(hi)20- phenotype. In addition, we found that the ER-MP12(hi)20- subset (comprising about 2% of total nucleated marrow) contains progenitor cells of all hematopoietic lineages. Aiming at the identification and purification of the myeloid progenitor cells within the ER-MP12(hi)20- subset, we used ER-MP58, a marker expressed at high level by all M-CSF-responsive bone marrow progenitors. With this marker the ER-MP12(hi)20- cell population could be divided into three subfractions: one with absent or low level ER-MP58 expression, one with intermediate, and one with high ER-MP58 expression. These subfractions were isolated by fluorescence-activated cell sorting and tested in vitro and in vivo for their differentiation capacities. In addition, the expression of ER-MP58 on stem cell subsets was examined in the cobblestone area-forming cell (CAFC) assay. Our data indicate that in the ERMP12(hi)20- subpopulation myeloid-committed progenitors are characterized by high-level expression of the ER-MP58 antigen, whereas cells with other or broader differentiation capacities have an ER-MP58 negative/low or intermediate phenotype. These myeloid-committed progenitors have no significant repopulating ability in vivo, in contrast to the ER-MP58 intermediate cells. Primitive CAFC-28/35, corresponding to cells providing long-term hematopoietic engraftment in vivo, also did not express the ER-MP58 Ag at a high level. Thus, cells committed to the myeloid lineage can be separated from progenitor cells with other differentiation capacities by means of multiparameter cell sorting using ER-MP58 in combination with ER-MP12 and ER-MP20

Dietary n-3 Fatty Acids Increase Spleen Size and Postendotoxin Circulating TNF in Mice; Role of Macrophages, Macrophage Precursors, and Colony-Stimulating Factor-1

In experimental studies in mice, dietary supplementation with n-3 fatty acids (FA) alleviates inflammation and increases resistance to infection. Nevertheless, TNF production capacity was found to be increased in n-3 FA-fed mice. We previously found increased relative spleen weights in n-3 FA-fed mice. In this study, the nature of this increased spleen size was further investigated. Spleen cellularity was increased significantly in mice fed n-3 FA (fish oil 15% w/w), compared with controls fed corn oil (15%) or normal lab chow (p < 0.05). Experiments with T cell-deficient nude mice and experiments using macrophage depletion through liposomal dichloromethylene-biphosphonate revealed that the increase in spleen cellularity is T cell independent and largely due to macrophage accumulation in the spleen. Accumulation of marginal zone and red pulp macrophages was histologically and immunohistochemically confirmed. n-3 FA induced peripheral blood monocytosis and an aspecific increase in bone marrow cellularity. Postendotoxin circulating TNF concentrations were increased significantly in n-3 FA-fed mice compared with controls. Splenectomy did not abolish this increase in circulating TNF. However, after macrophage depletion through liposomal dichloromethylene-biphosphonate, circulating TNF was not detectable after endotoxin challenge. Circulating concentrations of CSF-1 did not differ between the various experimental groups. It is suggested that the cellular changes observed relate to increased constitutive production of TNF