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

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

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

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