Phenotypical analysis of murine macrophage differentiation

Hemopoietic cells perform many vital functions in the maintenance of homeostasis and integrity of the body. These functions include, among others, the transport of oxygen by erythrocytes, and the initiation of blood clotting by thrombocytes at sites of injury. Furthermore, hemopoietic cells are the primary effector cells in the defense against micro-organisms and tumor cells. This defense is mainly carried out by two different cell types: lymphocytes and phagocytes. The distinction between these cell types is, among other things, based upon the different mechanisms that lymphocytes and phagocytes use to recognize foreign material. Lymphocytes recognize invading substances by highly specific antigen receptor molecules which are different and specific for each antigen. Mononuclear and polymorphonuclear phagocytes, on the other hand, discern their foreign targets by receptor molecules with much broader specificity, such as lectin-like receptors recognizing carbohydrate moieties. From an evolutionary point of view, phagocytes are the most 'ancient' cells in host defense. All multicellular animals contain amoeboid phagocytic cells; in some invertebrates such cells are even the only blood cells present. In general, increasing evolutionary ancientry coincides with increasing phenotypical diversity. The heterogeneity among phagocytes, and especially among the mammalian mononuclear phagocytes, clearly demonstrates the validity of this general rule. In this thesis, an analysis is presented of the phenotypical differentiation of murine mononuclear phagocytes, i.e. macrophages and their precursors. The term 'differentiation' in this context includes both the process of maturation and of diversification. It should be realized, however, that the term 'differentiation' is also generally used as equivalent to maturation only. In the next section an introductory overview is given of our current knowledge of macrophage differentiation. The literature cited in this overview primarily deals with studies performed with mice as experimental animals, since the subsequent chapters describe results of experimental studies using murine cells

Bone marrow precursors of nonobese diabetic mice develop into defective macrophage-like dendritic cells in vitro

The NOD mouse spontaneously develops autoimmune diabetes. Dendritic cells (DC) play a crucial role in the autoimmune response. Previous studies have reported a defective DC generation in vitro from the NOD mouse bone marrow (BM), but a deviated development of myeloid precursors into non-DC in response to GM-CSF was not considered. In this study, we demonstrate several abnormalities during myeloid differentiation of NOD BM precursors using GM-CSF in vitro. 1) We found reduced proliferation and increased cell death in NOD cultures, which explain the previously reported low yield of DC progeny in NOD. Cell yield in NOR cultures was normal. 2) In a detailed analysis GM-CSF-stimulated cultures, we observed in both NOD and NOR mice an increased frequency of macrophages, identified as CD11c(+)/MHCII(-) cells with typical macrophage morphology, phenotype, and acid phosphatase activity. This points to a preferential maturation of BM precursors into macrophages in mice with the NOD background. 3) The few CD11c(+)/MHCII(high) cells that we obtained from NOD and NOR cultures, which resembled prototypic mature DC, appeared to be defective in stimulating allogeneic T cells. These DC had also strong acid phosphatase activity and elevated expression of monocyte/macrophage markers. In conclusion, in this study we describe a deviated development of myeloid BM precursors of NOD and NOR mice into macrophages and macrophage-like DC in vitro. Potentially, these anomalies contribute to the dysfunctional regulation of tolerance in NOD mice yet are insufficient to induce autoimmune diabetes because they occurred partly in NOR mice

Developmental stages of myeloid dendritic cells in mouse bone marrow

The lineage relationship of dendritic cells (DC) with other hematopoietic cell types has been studied extensively, resulting in the identification of different bone marrow (BM) progenitors that give rise to distinct DC types. However, the identity of the different maturation stages of DC precursors in the BM remains unclear. In this study we define the in vivo developmental steps of the myeloid DC lineage in mouse BM. To this end, BM cells were separated according to their expression of CD31 (ER-MP12), Ly-6C (ER-MP20) and ER-MP58 antigens, and stimulated to develop into myeloid DC, using granulocyte macrophage colony stimulating factor as a specific growth factor. DC developed from three BM subpopulations: ER-MP12(hi)/20(-) (early blast cells), ER-MP12(+)/20(+) (myeloid blasts) and ER-MP12(-)/20(hi) (monocytes). The kinetic and phenotypic features of DC developing in vitro indicate that the three populations represent successive maturation stages of myeloid DC precursors. Within the earliest ER-MP12(hi)/20(-) population, DC precursors exclusively occurred in the myeloid-restricted ER-MP58(hi) subset. By using switch cultures, we show that these BM precursor subpopulations, when stimulated to develop into macrophages using macrophage colony stimulating factor, retain the ability to develop into myeloid DC until advanced stages of maturation. Together, these findings support a common ER-MP12/20-defined differentiation pathway for both macrophages and myeloid DC throughout their BM development

Inhibition of proliferation and differentiation during early T cell development by anti-transferrin receptor antibody

Proliferating cells require iron and, therefore, express the transferrin receptor (CD71) that mediates cellular iron uptake. Cycling thymocytes, which have the CD4−8−3−, CD4−8+3−, or CD4+8+3− phenotypes, also express CD71. The importance of CD71-mediated iron uptake for proliferation and maturation of thymocytes was studied using fetal thymus organ cultures at day 14 of gestation and treating them for 7 days with a CD71 monoclonal antibody (mAb). The intracellular iron deficiency caused by this treatment, inhibits both proliferation and maturation of the thymocytes. Cell recovery was reduced by 60%, but cells still expanded tenfold during the culture. Remarkably, the final maturation of αβ T cells was completely blocked as no thymocytes with low or high CD3/αβTcR expression developed. Moreover, only few cells reached the CD4+8+3− stage of T cell development. CD4−8−3− thymocytes, however, as well as its CD44−25+ subset developed in normal numbers, suggesting that CD44−25+ CD4−8−3− cells, or their immediate progeny, were most vulnerable to CD71 mAb treatment. The development of γδ T cells, which also express CD71, was not affected in these cultures. This suggests that γδ T cells are either less iron-dependent or possess alternative iron-uptake mechanisms. Thus, our observation

The monoclonal antibody ER-BMDM1 recognizes a macrophage and dendritic cell differentiation antigen with aminopeptidase activity

Abstract Here we describe the reactivity of monoclonal antibody (mAb) ER-BMDM1, directed against a 160-kDa cell membrane-associated antigen (Ag) with aminopeptidase activity. The aminopeptidase recognized by ER-BMDM1 is present on various mouse macrophage (MΦ) and dendritic cell (DC) subpopulations as well as on microvillous epithelia. Analysis of ER-BMDM1 Ag expression in in vitro models of MΦ maturation revealed that the Ag is expressed at increasing levels upon maturation of MΦ. In vivo, high level expression of the ER-BMDM1 Ag occurs after thmonocytic stage of maturation, since bone marrow cells and peripheral blood monocytes are essentially ER-BMDM1 negative. Analysis of isolated-resident and elicited MΦ populations showed that ER-BMDM1 recognizes a specific subpopulation of mature MΦ: only some resident peritoneal and alveolar MΦ are ER-BMDM1 positive, whereas virtually all thioglycollate-elicited peritoneal exudate MΦ bind the mAb. In lymphoid organs, a subpopulation of MΦ is recognized as well as interdigitating cells (IDC) located in T cell areas. Phenotypic analysis of isolated DC- the in vitro equivalents of IDC - from spleen and lymph nodes confirmed that the majority of this important antigen-presenting cell population expresses the ER-BMDM1 aminopeptidase. The molecular characteristics of the ER-BMDM1 Ag suggest that it may represent the mouse homolog of human CD13

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 d

Facilitated engraftment of human hematopoietic cells in severe combined immunodeficient mice following a single injection of Cl²MDP liposomes

Transplantation of normal and malignant human hematopoietic cells into severe combined immunodeficient (SCID) mice allows for evaluation of long-term growth abilities of these cells and provides a preclinical model for therapeutic interventions. However, large numbers of cells are required for successful engraftment in preirradiated mice due to residual graft resistance, that may be mediated by cells from the mononuclear phagocytic system. Intravenous (i.v.) injection of liposomes containing dichloromethylene diphosphonate (Cl2MDP) may eliminate mouse macrophages in spleen and liver. In this study outgrowth of acute myeloid leukemia (AML) cells and umbilical cord blood (UCB) cells in SCID mice conditioned with a single i.v. injection of Cl2MDP liposomes in addition to sublethal total body irradiation (TBI) was compared to outgrowth of these cells in SCID mice that had received TBI alone. A two- to 10-fold increase in outgrowth of AML cells was observed in four cases of AML. Administration of 107 UCB cells reproducibly engrafted SCID mice that had been conditioned with Cl2MDP liposomes and TBI, whereas human cells were not detected in mice conditioned with TBI alone. As few as 2 x 104 purified CD34+ UCB cells engrafted in all mice treated with Cl2MDP liposomes. In SCID mice treated with macrophage depletion unexpected graft failures were not observed. Histological examination of the spleen showed that TBI and Cl2MDP liposomes i.v. resulted in a transient elimination of all macrophage subsets in the spleen, whereas TBI had a minor effect. Cl2MDP liposomes were easy to use and their application was not associated with appreciable side-effects. Cl2MDP liposome pretreatment in combination with TBI allows for reproducible outgrowth of high numbers of human hematopoietic cells in SCID mice

The interplay between critical transcription factors and microRNAs in the control of normal and malignant myelopoiesis

Myelopoiesis is a complex process driven by essential transcription factors, including C/EBPα, PU.1, RUNX1, KLF4 and IRF8. Together, these factors are critical for the control of myeloid progenitor cell expansion and lineage determination in the development of granulocytes and monocytes/macrophages. MicroRNAs (miRNAs) are expressed in a cell type and lineage specific manner. There is increasing evidence that miRNAs fine-tune the expression of hematopoietic lineage-specific transcription factors and drive the lineage decisions of hematopoietic progenitor cells. In this review, we discuss recently discovered self-activating and feed-back mechanisms in which transcription factors and miRNAs interact during myeloid cell development. Furthermore, we delineate how some of these mechanisms are affected in acute myeloid leukemia (AML) and how disrupted transcription factor-miRNA interplays contribute to leukemogenesis

Differential inhibition of macrophage proliferation by anti-transferrin receptor antibody ER-MP21: correlation to macrophage differentiation stage

Abstract Monoclonal antibodies (mAbs) directed against the transferrin receptor are known to inhibit proliferation of cells due to iron deprivation. Some cell types, however, escape from growth inhibition by a mechanism which is unclear at present. This mechanism is the subject of the present study. We investigated the differential growth inhibition caused by anti-transferrin receptor mAb ER-MP21 in connection with the differentiation of murine macrophages (Mφ). Therefore, we applied two models of Mφ differentiation, namely, culture of bone marrow cells in the presence of M-CSF and a panel of Mφ cell lines ordered in a linear differentiation sequence. In both models we observed that proliferation of Mφ precursors was strongly inhibited by ER-MP21. In contrast, proliferation of more mature stages of Mφ differentiation was hardly affected. Remarkably, iron uptake by Mφ precursor and mature Mφ cell lines was inhibited by ER-MP21 to the same extent. However, mature Mφ cell lines showed an iron uptake two-to threefold higher than that of Mφ precursor cell lines. These observations strongly suggest that mature Mφ escape from ER-MP21-mediated growth inhibition, because these cells take up more iron than is actually needed for proliferation. Furthermore, we found that enhanced iron uptake by mature Mφ is not necessarily accompanied by a higher cell surface expression of transferrin receptors, thus suggesting an increased recycling of transferrin receptors in mature Mφ

Dietary n-3 fatty acids increase spleen size and postendotoxin circulating TNF in mice; role of macrophages, macrophage precursors, and colony-stimulating factor-1

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