Phorbol myristate acetate treatment of normal human myeloid blast cells promotes monopoiesis and inhibits granulopoiesis (original) (raw)
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Cancer research, 1982
A human diploid hematopoietic cell line, CM-S, has been established from the bone marrow of a patient with congenital hypoplastic (Diamond-Blackfan) anemia. The cells grow in sus pension in an almost undifferentiated form but express some enzyme markers of monocytic and granulocytic cells. Upon treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)2 (10~7 M concentrations or higher), the cells become rapidly adherent to glass or plastic surfaces and show, by transmission electron microscopy, an increase in the rough endoplasmic reticulum, an apparent increase in the number and size of swollen mitochondria, and numerous granules, rarely seen in the absence of TPA. Fc and C3 receptors increase, and the cells become capable of phagocytosis and immunoerythrophagocytosis.
Modulation of the maturation of human leukemic promyelocytes (HL-60) to granulocytes or macrophages
Leukemia Research, 1982
Abstract~The relationship between the effects of two types of inducers on the maturation of a line of human promyelocytic cells (HL-60) was studied. The dual potentiality of these promyelocytes was demonstrated by the ability of isolated colonies to differentiate into granulocytes, following induction by dimethylsulphoxide (DMSO) or express properties specific to macrophages following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). The differentiation process involved an irreversible step which occurred 48 h after exposure to DMSO, and a few h after exposure to TPA. This implies that the presence of the inducer in the culture medium is no more required for con'tpletion of the differentiation programme. Removal of the inducer prior to this step resulted in reversal of all the inducer-mediated effects. During the period of non-commitment the specific pathway of maturation was still undetermined; cells in which differentiation was initiated by exposure to DMSO were able to develop into macrophages after substitution of DMSO by TPA. Moreover, pre-exposure to DMSO and other inducers of granulocyte differentiation resulted in higher sensitivity to TPA, as indicated by the cell response to low TPA concentrations and more rapid expression of macrophage specific properties. These results suggest that the early stages in HL-60 differentiation are probably common to the granulocyte and the macrophage pathways.
PLoS ONE, 2010
Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD 3 ) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD 3 and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells. Citation: Daigneault M, Preston JA, Marriott HM, Whyte MK, Dockrell DH (2010) The Identification of Markers of Macrophage Differentiation in PMA-Stimulated THP-1 Cells and Monocyte-Derived Macrophages. PLoS ONE 5(1): e8668.
Journal of Immunological Methods, 2005
Culture medium conditioned by the L929 murine fibroblast cell line contains macrophage colony-stimulating factor (M-CSF), providing an alternative to recombinant M-CSF for in vitro generation of murine macrophages. No such alternative has been described for in vitro studies requiring human macrophages. We tested the differentiation of human blood monocytes into mature macrophages by culturing in media conditioned by the human KPB-M15 cell line, which produces M-CSF and interleukin 6 (IL-6). The phenotypes of macrophages cultured in KPB-M15 conditioned media and recombinant M-CSF were compared by examining viability, expression of cell surface markers, phagocytic/pinocytic activity, and cytokine/chemokine secretion in response to bacterial lipopolysaccharide (LPS). In conditioned medium, monocytes differentiated into a homogeneous population of large cells that exhibited higher expression of CD14 and the macrophage mannose receptor (CD206) than did M-CSF-cultured cells. Cells matured in KPB-M15 conditioned medium exhibited macrophage morphology, were phagocytic, and were activated in response to LPS. These data demonstrate that KPB-M15 conditioned medium can be used to differentiate human blood monocytes into macrophages in vitro.
Research in Immunology, 1992
Macrophages (MAC) are important effector cells of the immune system. They arise from circulating blood monocytes (MO), which undergo further maturation upon leaving the vasculature and migrating into the various tissues and body cavities. A similar differentiation process can be followed in vitro when monocytes are cultured in the presence of serum. In this study, different factors and serum proteins, either alone or in combination, were tested for their ability to promote the survival and/or maturation of blood MO in the absence of serum. Elutriation-purified MO cultured for 8 days on hydrophobic teflon foils in the presence of 5 % human serum differentiated into large, well-spread MAC, whereas in the absence of serum, MO rapidly died. The serum-induced maturation of MAC was accompanied by a strong expression of CD1 6, CD14 and MAX antigens. Secretion of TNF-alpha and neopterin increased about IO-fold as compared with freshly isolated MO. The replacement of serum by either M-CSF (100 nglml) or immunoglobulin (0.5-5 mglmll had a marked effect on MO survival (about 50 % of serum-cultured MO), but cells were smaller, less spread out and had low expression of CD1 6, CD14 and MAX antigens. Their functional competence in terms of TNF-alpha and neopterin release was reduced to IO-20 % as compared with MAC cultured in the presence of serum. Both albumin (0.5-5 mglml) and 1,25-dihydroxyvitamin D, (1,25(OH),D,) (10e8 M) as substitutes for serum were less effective in terms of MO survival (20-30 % of serum-cultured MO), but were comparable to serum with respect to morphology and phenotype; however, they induced MAC with lower secretory activity. A combination of 1,25(OHI,D, or albumin (0.5 mglml) with immunoglobulin (0.5 mg/ml) resulted in MAC showing serum-cultured characteristics in terms of phenotype, but lower secretory capacity and survival rate. However, the combination of the three factors together could substitute for serum in all parameters tested. The same result was obtained by cultivation of MO with high concentrations of albumin (5 mglml) and immunoglobulin (5 mglml). Other factors tested had no effect on the MO into MAC differentiation process (transferrin, vitamin A, fibronectin, vitamin D,). In summary, we describe defined serum-free culture conditions for the generation of distinct types of MAC, which differ in terms of phenotype, morphology and function.
Parameters affecting the in vitro maturation of human monocytes to macrophages
The International Journal of Cell Cloning, 2000
In vitro maturation of human monocytes to macrophages was characterized by morphological criteria, cell size and lysosomal enzymes activity. Purified populations of monocytes were maintained in culture at either adherent or nonadherent conditions and their maturation to macrophages was observed in both cases. The addition of external factors such as hydrocortisone and vitamin D3 inhibited monocyte maturation. In the absence of external factors, nonadherent monocytes were inhibited in their maturation for up to 10 days when plated at crowded cell concentrations. In addition, the presence of human serum in the culture media had a higher inhibitory activity than similar concentrations of fetal calf serum. Supernates from crowded macrophages were also inhibitory for monocyte maturation. We suggest the possibility that cell crowding, as well as soluble factors found in the Serum and probably secreted by macrophages, participate in the regulation of monocyte development by inhibiting their maturation. Once released from this inhibitory signal or environment, the monocytes mature to macrophages.
British Journal of Haematology, 2002
Mature blood neutrophils have a short lifespan in vitro and are not easily transfectable. We obtained terminally mature neutrophils after differentiation of immature transfectable PLB-985 myeloid cells by treatment with dimethylformamide (0AE5%), Nutridoma SP (1%) and fetal calf serum (0AE5%). Maturation was shown by functional degranulation, in response to bacterial N-formyl peptide (fMLP), of specific granules and secretory vesicle contents; the latter emerge during the last step of normal neutrophil differentiation into bone marrow. These differentiated cells also produced quantities of superoxide anion similar to those produced by blood neutrophils, in response to physiological stimuli (fMLP); in addition, the fMLPinduced respiratory burst was primed by the proinflammatory cytokine granulocyte-macrophage colony-stimulating factor. Thus, in our experimental conditions, PLB-985 cells transformed into fully differentiated neutrophils capable of fine regulation by inflammatory agents. This cell model will help in the understanding of the molecular mechanisms underlying neutrophil functions.