Process Of Macrophage Differentiation Research Articles

Abstract LB201: ENIGMAC platform enables at-scale CRISPRi screenings for macrophage target discovery

Abstract RNA sequencing has improved our understanding of cellular heterogeneity by providing in-depth transcriptome information at the single cell level. However, this “dataset infodemic” did not translate into an abundance of new validated targets due to the difficulties in performing gene-to-function studies. The main goal of the ENIGMAC™ discovery platform is to support gene-to-function studies and to deliver new validated macrophage targets. ENIGMAC™ combines custom, disease relevant assays and unique genome editing methods supported by multi-omics bioinformatics analyses yielding selected gene sets for interrogation. We use a human Induced Pluripotent Stem Cell (iPSC) line that yields macrophages phenotypically and functionally similar to MDM. By employing this iPSC system, we can produce millions of macrophages per week, which allows multiple high throughput assays to support target validation and drug discovery. Furthermore, we developed several proprietary technologies allowing fast and reliable gene editing of macrophages, including gene Knock In (KI), Knock Out (KO) and Knock Down (KD) with high efficiency both at iPSC and macrophage level while maintaining expression/silencing during macrophage differentiation. Here we report preliminary results of the CRISPRi screen, aiming to identify novel regulators of INFg-mediated PD-L1 upregulation on macrophages, a surrogate readout for a suppressive phenotype. To this aim, we developed a proprietary human iPSC line that constitutively expresses dCAS9-KRAB protein, allowing for a highly gene specific guide RNA (sgRNA)-mediated method of silencing of gene transcription. Notably, our proprietary anti-silencing system allows to maintains dCAS9-KRAB expression throughout the entire macrophage differentiation process. During the screen, 100 million iPSC-derived microphages were transduced with a 6500 gRNAs library targeting 1300 genes selected from disease-relevant macrophage datasets. Macrophages were subsequently stimulated with INFg and sorted for loss of PD-L1 expression using flow cytometry. Sequencing of the PD-L1 low macrophage population successfully identified PD-L1 regulators such as IFNGR1, IFNGR2, JAK1, STAT1 and IRF1 among other hits. The novel hits are being now individually validated, and selected ones will be progressed to drug discovery. Importantly, this macrophage CRISPRi screen provides technical proof of concept for the ENIGMAC™ platform to perform screens at scale and to identify novel hits. In conclusion, the ENIGMAC™ platform represents a unique tool for gene to function studies using human macrophages. Furthermore, it is disease agnostic and can be integrated with a variety of disease-specific conditions and phenotypic readouts. Citation Format: Thomas W. Monteiro Crozier, Martha Lopez-Yrigoyen, Helena Engman, Moritz Haneklaus, Samuel Witham, Krzystof B. Wicher, Steven Myatt, Luca Cassetta, Carola Ries. ENIGMAC platform enables at-scale CRISPRi screenings for macrophage target discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB201.

Optimizing the Method for Differentiation of Macrophages from Human Induced Pluripotent Stem Cells.

Macrophage is a very promising cell type for cancer immunotherapy, yet it is difficult to obtain enough functional macrophages for clinical cell therapy. Herein, we descibe a reliable method to produce functional macrophages through the differentiation of human induced pluripotent stem cells (hiPSCs). By optimizing the size control of embryoid bodies (EBs), we accelerated the differentiation process of macrophages and increased the production of macrophages without attenuating macrophage functions. Our final yield of macrophages was close to 50-fold of starting iPSCs. The macrophages showed phagocytic capacity in vitro and a xenograft tumor model. M0 macrophages could be further polarized into M1 and M2 subtypes, and M1 cells exhibited typical proinflammatory characteristics. Moreover, we found that hematopoietic differentiation originated from the outside of EB and matured inward gradually. Taken together, our protocol provides an effective method for the generation of macrophages comparable to blood-derived macrophages, which provides potential value for cell therapy and gene editing studies.

The effect of ketorolac and triamcinolone acetonide on adipogenic and hepatogenic differentiation through miRNAs 16/15/195: Possible clinical application in regenerative medicine

ObjectiveThe role of miR-16/15b/195 and complementary compounds in the commitment of mesenchymal stem cells (MSCs) to different lineages is unknown. The aim of this work was to investigate the effect of ketorolac and triamcinolone acetonide on adipogenic and hepatogenic processes, respectively. Also, miR-16/15 and miR-195 expression levels were evaluated under different induction conditions. MethodsMSCs were isolated, expanded and directed using adipogenesis medium or medium supplemented with ketorolac, and also subjected to hepatogenic differentiation using a cocktail of hepatocyte growth factor (HGF) and Oncostatin M (OSM) either with or without triamcinolone acetonide. Periodic acid-Schiff (PAS) staining, Oil red O staining, albumin and adiponectin protein secretion were evaluated. MiR-16 family expression level was assessed using qRT-PCR in four induced groups as compared to non-induced cells. ResultsThe expression levels of miR-16 and miR-15 increased significantly from adipose derived stem cells to adipocytes (p<0.01). Positive stimulatory effects of ketorolac and triamcinolone on adipogenic and hepatogenic induction, respectively, were observed. Ketorolac stimulated upregulation of miR-16/15b in the adipogenic induced stem cells. Interestingly, triamcinolone caused upregulation of miR-15b and miR-195 in hepatic commitment. ConclusionsTwo highly effective stimulators were identified. Ketorolac is similar to indomethacin and affects adipogenic differentiation. Triamcinolone is involved in hepatogenic commitment of stem cells like differentiation process of macrophages, adipocytes and osteocytes. The alteration of miR-16 family members’ expression indicates that this family may play a possible role in directing stem cell fate.

PMA-Induced THP-1 Macrophage Differentiation is Not Impaired by Citrate-Coated Platinum Nanoparticles.

The innate immune system consists of several complex cellular and molecular mechanisms. During inflammatory responses, blood-circulating monocytes are driven to the sites of inflammation, where they differentiate into tissue macrophages. The research of novel nanomaterials applied to biomedical sciences is often limited by their toxicity or dangerous interactions with the immune cell functions. Platinum nanoparticles (PtNPs) have shown efficient antioxidant properties within several cells, but information on their potential harmful role in the monocyte-to-macrophage differentiation process is still unknown. Here, we studied the morphology and the release of cytokines in PMA-differentiated THP-1 pre-treated with 5 nm PtNPs. Although NP endocytosis was evident, we did not find differences in the cellular structure or in the release of inflammatory cytokines and chemokines compared to cells differentiated in PtNP-free medium. However, the administration of PtNPs to previously differentiated THP-1 induced massive phagocytosis of the PtNPs and a slight metabolism decrease at higher doses. Further investigation using undifferentiated and differentiated neutrophil-like HL60 confirmed the harmlessness of PtNPs with non-adherent innate immune cells. Our results demonstrate that citrate-coated PtNPs are not toxic with these immune cell lines, and do not affect the PMA-stimulated THP-1 macrophage differentiation process in vitro.

The study of the influence of micro-environmental signals on macrophage differentiation using a quantitative Petri net based model

Abstract The complexity of many biological processes, which, thanks to the development of many fields of science, becomes for us more and more obvious, makes these processes extremely interesting for further analysis. In this paper a quantitative model of the process of macrophage differentiation, which is essential for many phenomena occurring in the human body, is proposed and analyzed. The model is expressed in the language of Petri net theory on the basis of one of the three hypotheses concerning macrophage differentiation existing in the literature. The performed analysis allowed to find an importance of individual factors in the studied phenomenon.

Nitro-oleic acid regulates growth factor-induced differentiation of bone marrow-derived macrophages

Many diseases accompanied by chronic inflammation are connected with dysregulated activation of macrophage subpopulations. Recently, we reported that nitro-fatty acids (NO2-FAs), products of metabolic and inflammatory reactions of nitric oxide and nitrite, modulate macrophage and other immune cell functions. Bone marrow cell suspensions were isolated from mice and supplemented with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with NO2-OA for different times. RAW 264.7 macrophages were used for short-term (1–5min) experiments. We discovered that NO2-OA reduces cell numbers, cell colony formation, and proliferation of macrophages differentiated with colony-stimulating factors (CSFs), all in the absence of toxicity. In a case of GM-CSF-induced bone marrow-derived macrophages (BMMs), NO2-OA acts via downregulation of signal transducer and activator of transcription 5 and extracellular signal-regulated kinase (ERK) activation. In the case of M-CSF-induced BMMs, NO2-OA decreases activation of M-CSFR and activation of related PI3K and ERK. Additionally, NO2-OA also attenuates activation of BMMs. In aggregate, we demonstrate that NO2-OA regulates the process of macrophage differentiation and that NO2-FAs represent a promising therapeutic tool in the treatment of inflammatory pathologies linked with increased accumulation of macrophages in inflamed tissues.

Soluble human leukocyte antigen G5 polarizes differentiation of macrophages toward a decidual macrophage-like phenotype

What are the actions of soluble human leukocyte antigen G5 (sHLAG5) on macrophage differentiation? sHLAG5 polarizes the differentiation of macrophages toward a decidual macrophage-like phenotype, which could regulate fetomaternal tolerance and placental development. sHLAG5 is a full-length soluble isoform of human leukocyte antigen implicated in immune tolerance during pregnancy. Low or undetectable circulating level of sHLAG5 in first trimester of pregnancy is associated with pregnancy complications such as pre-eclampsia and spontaneous abortion. Decidual macrophages are located in close proximity to invasive trophoblasts, and are involved in regulating fetomaternal tolerance and placental development. Human peripheral blood monocytes were differentiated into macrophages by treatment with granulocyte macrophage colony-stimulating factor in the presence or absence of recombinant sHLAG5 during the differentiation process. The phenotypes and the biological activities of the resulting macrophages were compared. Recombinant sHLAG5 was produced in Escherichia coli BL21 and the protein identity was verified by tandem mass spectrometry. The expression of macrophage markers were analyzed by flow cytometry and quantitative PCR. Phagocytosis was determined by flow cytometry. Indoleamine 2,3-dioxygenase 1 expression and activity were measured by western blot analysis and kynurenine assay, respectively. Cell proliferation and cell cycling were determined by fluorometric cell proliferation assay and flow cytometry, respectively. Cytokine secretion was determined by cytokine array and ELISA kits. Intracellular cytokine expression was measured by flow cytometry. Cell invasion and migration were determined by trans-well invasion and migration assay, respectively. sHLAG5 drove the differentiation of macrophages with 'immuno-modulatory' characteristics, including reduced expression of M1 macrophage marker CD86 and increased expression of M2 macrophage marker CD163. sHLAG5-polarized macrophages showed enhanced phagocytic activity. They also had higher expression and activity of indoleamine 2,3-dioxygenase 1, a phenotypic marker of decidual macrophages, which inhibited proliferation of autologous T-cells via induction of G0/G1 cell cycle arrest. In addition, sHLAG5-polarized macrophages had an increased secretion of interleukin-6 and C-X-C motif ligand 1, which inhibited interferon-γ production in T-cells and induction of trophoblast invasion, respectively. Most information on the phenotypes and biological activities of human decidual macrophages are based on past literatures. A direct comparison between sHLAG5-polarized macrophages and primary decidual macrophages is required to verify the present observations. This is the first study on the role of sHLAG5 in macrophage differentiation. Further study on the mechanism that regulates the differentiation process of macrophages would enhance our understanding on the physiology of early pregnancy. This work was supported in part by the Hong Kong Research Grant Council Grant HKU774212 and the University of Hong Kong Grant 201309176126. The authors have no competing interests to declare. Nil.

MiRNA modulation of M1/M2 macrophage phenotype (CAM5P.231)

Abstract After spinal cord injury (SCI), “M1” (pro-inflammatory) and “M2” (anti-inflammatory) macrophages populate the site of injury. M1 macrophages are neurotoxic and hamper neuroregeneration while M2 macrophages have a pro-regenerative, anti-inflammatory phenotype. Therefore, therapeutic strategies that suppress M1 and enhance M2 macrophages after SCI are actively sought. Micro RNAs (miRNA) are small, non-coding RNAs, approximately 19-24 nucleotides in length, which regulate gene expression at the transcriptional level. miRNA are known to regulate inflammation in autoimmune disease and inflammatory disorders. We therefore hypothesized that miRNA play a key role in differentiation process of macrophages into the pro-inflammatory M1 phenotype. The relative expression of several miRNA was analyzed in vitro in bone marrow-derived macrophages undergoing differentiation into M1 versus M2 phenotypes. We found that M1 macrophages expressed significantly higher levels of miR-155 than M2 macrophages. In addition, miR-155 expression was increased in the injured spinal cord mirroring the increase in M1 macrophages at the site of injury. miR-155 deficiency or silencing resulted in blunted iNOS, IL-1β and TNFα up-regulation in M1 macrophages, among others. In contrast, Arginase-1 expression remained stable in M2 macrophages. These results indicate that miR-155 plays an important role in the differentiation of macrophages into an M1 phenotype.

Platelet-derived Growth Factor-C (PDGF-C) Induces Anti-apoptotic Effects on Macrophages through Akt and Bad Phosphorylation

PDGF-C, which is abundant in the malignant breast tumor microenvironment, plays an important role in cell growth and survival. Because tumor-associated macrophages (TAMs) contribute to cancer malignancy, macrophage survival mechanisms are an attractive area of research into controlling tumor progression. In this study, we investigated PDGF-C-mediated signaling pathways involved in anti-apoptotic effects in macrophages. We found that the human malignant breast cancer cell line MDA-MB-231 produced high quantities of PDGF-C, whereas benign MCF-7 cells did not. Recombinant PDGF-C induced PDGF receptor α chain phosphorylation, followed by Akt and Bad phosphorylation in THP-1-derived macrophages. MDA-MB-231 culture supernatants also activated macrophage PDGF-Rα. PDGF-C prevented staurosporine-induced macrophage apoptosis by inhibiting the activation of caspase-3, -7, -8, and -9 and cleavage of poly(ADP-ribose) polymerase. Finally, TAMs isolated from the PDGF-C knockdown murine breast cancer cell line 4T1 and PDGF-C knockdown MDA-MB-231-derived tumor mass showed higher rates of apoptosis than the respective WT controls. Collectively, our results suggest that tumor cell-derived PDGF-C enhances TAM survival, promoting tumor malignancy.

The podosome marker protein Tks5 regulates macrophage invasive behavior.

Tks5 is a Src substrate and adaptor protein previously recognized for its regulation of cancer cell invasion through modulation of specialized adhesion structures called podosomes/invadopodia. Here we show for the first time that Tks5 localizes to the podosomes of primary macrophages, and that Tks5 protein levels increase concurrently with podosome deposition during the differentiation of monocytes into macrophages. Similar results are reported for model THP-1 cells, which differentiate into macrophages and form proteolytically active podosomes in response to a PKC signaling agonist (PMA) and with sensitivity to a PKC inhibitor (bisindolylmaleimide). Genetic manipulation of Tks5 expression (silencing and overexpression) in stable THP-1 cell lines does not independently alter this macrophage differentiation process. Nor do these cells lose the ability to focalize F-actin and its accessory proteins into podosome-like structures following PMA treatment. However, Tks5 directly controls podosome-associated gelatin degradation and invasion through collective changes in adhesion, chemotaxis, and the expression/proteolytic activity of MMP9. The Src family kinase-dependent phosphorylation of Tks5 is also implicated in the regulation of THP-1 macrophage invasive behavior. These results therefore define a previously unappreciated function of Tks5 signaling specific to the functional attributes of the macrophage podosome in adhesion, motility, and extracellular matrix-remodeling.

Colony-stimulating factor-1–induced oscillations in phosphatidylinositol-3 kinase/AKT are required for caspase activation in monocytes undergoing differentiation into macrophages

AbstractThe differentiation of human peripheral blood monocytes into resident macrophages is driven by colony-stimulating factor-1 (CSF-1), which upon interaction with CSF-1 receptor (CSF-1R) induces within minutes the phosphorylation of its cytoplasmic tyrosine residues and the activation of multiple signaling complexes. Caspase-8 and -3 are activated at day 2 to 3 and contribute to macrophage differentiation, for example, through cleavage of nucleophosmin. Here, we show that the phosphatidylinositol-3 kinase and the downstream serine/threonine kinase AKT connect CSF-1R activation to caspase-8 cleavage. Most importantly, we demonstrate that successive waves of AKT activation with increasing amplitude and duration are required to provoke the formation of the caspase-8–activating molecular platform. CSF-1 and its receptor are both required for oscillations in AKT activation to occur, and expression of a constitutively active AKT mutant prevents the macrophage differentiation process. The extracellular receptor kinase 1/2 pathway is activated with a coordinated oscillatory kinetics in a CSF-1R–dependent manner but plays an accessory role in caspase activation and nucleophosmin cleavage. Altogether, CSF-1 stimulation activates a molecular clock that involves phosphatidylinositol-3 kinase and AKT to promote caspase activation. This oscillatory signaling pathway, which is coordinated with extracellular receptor kinase 1/2 oscillatory activation, involves CSF-1 and CSF-1R and controls the terminal differentiation of macrophages.

Differentiation of monocytic U937 cells under static magnetic field exposure

We present here a morphological, cytochemical and biochemical study of the macrophagic differentiation of human pro-monocytic U937 cells exposed to moderate intensity (6 mT) static magnetic fields (MF). It was found that the following substances induced differentiation in U937 cells to a progressively lower degree: 50 ng/mL 12-0-tetradecanoyl-13-phorbol acetate (TPA), low concentration of glutamine (0,05 mM/L), 10% dimethyl sulfoxide (DMSO) and 100 mM/L Zn++. Differentiated U937 cells shift from a round shape to a macrophage-like morphology, from suspension to adhesion growth and acquire phagocytotic activity, the cytoskeleton adapting accordingly. Exposure to static MF at 6 mT of intensity decreases the degree of differentiation for all differentiating molecules with a consequent fall in cell adhesion and increased polarization of pseudopodia and cytoplasmic protrusions. Differentiation alone, or in combination with exposure to static MFs, affects the distribution and quantity of cell surface sugar residues, the surface expression of markers of macrophage differentiation, and phagocytotic capability. Our results indicate that moderate-intensity static MFs exert a considerable effect on the process of macrophage differentiation of pro-monocytic U937 cells and suggest the need for further studies to investigate the in vivo possible harmful consequences of this.

Global effects of inorganic arsenic on gene expression profile in human macrophages

Inorganic arsenic, a major environmental contaminant, exerts immunosuppressive effects towards human cells. We previously demonstrated that relevant environmental concentrations of inorganic arsenic altered morphology and functions of human primary macrophages, suggesting interference with macrophage differentiation program. The goal of this study was to determine global effect of low concentrations of arsenic trioxide (As2O3) on gene expression profile in human primary macrophages, in order to identify molecular targets of inorganic arsenic, especially those relevant of macrophage differentiation process. Using a pan-genomic microarray, we demonstrate that exposure of human blood monocyte-derived macrophages to 1μM As2O3 for 72h, a non-cytototoxic concentration, results in up-regulation of 32 genes and repression of 91 genes. Among these genes, 26 are specifically related to differentiation program of human macrophages. Particularly, we validated that As2O3 strongly alters expression of MMP9, MMP12, CCL22, SPON2 and CXCL2 genes, which contribute to major macrophagic functions. Most of these metalloid effects were reversed when As2O3-treated macrophages were next cultured in arsenic-free medium. We also show that As2O3 similarly regulates expression of this macrophagic gene subset in human alveolar macrophages, the phenotype of which closely resembles that of blood monocyte-derived macrophage. In conclusion, our study demonstrates that environmentally relevant concentrations of As2O3 impair expression of macrophage-specific genes, which fully supports interference of metalloid with differentiation program of human macrophages.

Expression of GM1, a marker of lipid rafts, defines two subsets of human monocytes with differential endocytic capacity and lipopolysaccharide responsiveness

Monocytes constitute 5-10% of total human peripheral blood leucocytes and remain in circulation for several days before replenishing the tissue macrophage populations. Monocytes display heterogeneity in size, granularity and nuclear morphology, and in the expression of cell membrane molecules, such as CD14, CD16, CD32, CD64, major histocompatibility complex class II, CCR2, CCR5, among others. This has led to the suggestion that individual monocyte/macrophage populations have specialized functions within their microenvironments. This study provides evidence for the occurrence of two peripheral blood monocyte subpopulations on the basis of their differential expression of GM1, a sphingolipid found mostly in lipid rafts, a CD14(+) GM1(low) population and a CD14(+) GM1(high) population comprising about 97.5% and 2.5% of total CD14(+) cells, respectively. GM1 expression correlates with functional differences in terms of endocytic activity, susceptibility to mycobacterial infection, and response to lipopolysaccharide (LPS) (modulation of Toll-like receptor-4 expression). CD14(+) GM1(low) cells proved to be less endocytic and more responsive to LPS, whereas CD14(+) GM1(high) cells are more endocytic and less responsive to LPS. In addition, during monocyte to macrophage differentiation in vitro, the percentage of CD14(+) GM1(high) cells increases from about 2.5% at day 1 to more than 50% at day 7 of culture. These results suggest that GM1(low) and GM1(high) monocytes in peripheral blood, represent either different stages of maturation or different subsets with specialized activities. The expression of CD16 on GM1(high) favours the first possibility and, on the other hand that up-regulation of GM1 expression and probably lipid rafts function is involved in the monocyte to macrophage differentiation process.

Regulatory properties and cellular redistribution of zinc during macrophage differentiation of human leukemia cells

Many proteins require the binding of trace metals such as Ca, Fe, Cu, or Zn, which may modulate their structure, function, or activity. To determine if there were any overall changes in metalloprotein distribution or metal concentration during the process of macrophage differentiation we induced human myeloid HL-60 leukemia cells with phorbol 12-myristate 13-acetate (PMA) and quantitatively mapped their metal content using hard X-ray fluorescence micro-analysis. We found a transient increase in the zinc content of HL-60 cell nuclei during the early stages of differentiation induction. This finding was confirmed by spectrofluorometry in HL-60 cells and extended to U-937 leukemia cells. A role for protein kinase C-β (PKC-β) in this process was established by examining zinc content in an HL-60 variant, HL-525, which is PKC-β deficient, and in HL-525 cells in which PKC-β was restored by stable overexpression. Chemical chelation of both Cu and Zn served to inhibit macrophage differentiation in HL-60 cells, indicating a requirement for these metals during this process. Finally, we demonstrate that growth of HL-60 cells in a low-zinc environment removes their susceptibility to PMA-induced differentiation, and that this capacity can be partially restored by the addition of exogenous zinc.

Inhibition of human immunodeficiency virus type 1 infection in macrophages by an alpha-v integrin blocking antibody

Macrophages are key cells for HIV infection and HIV spreading inside the organism. Macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (M-CSF). In the monocyte to macrophage differentiation process with M-CSF, αv-integrins are upregulated concomitantly with the capacity of HIV to generate a productive virus infection. In the present study we show that an anti-αv antibody, 17E6, inhibited HIV-1 infection of primary macrophages. The effect of 17E6 on HIV-1 BaL replication in acutely infected macrophages was dose-dependent, with a 50% effective concentration (EC 50) of 17 ± 2 μg/ml in the absence of cytotoxicity. Similarly, a monoclonal antibody targeting the αvβ6 integrin (14D9.F8) also inhibited HIV-1 BaL infection in this cell type. 17E6 reduced the detection of HIV-1 BaL proviral DNA in acutely infected macrophages, but was completely ineffective against HIV-1 BaL production in chronically infected macrophages, suggesting that 17E6 inhibited HIV infection at an early stage of the virus cycle. Finally, a small molecular weight antagonist of the αvβ6 integrin, EMD 409849, reduced HIV replication at subtoxic concentrations. Therefore, our results suggest that αv-containing integrins could play a role in HIV replication in macrophages and suggest that small-molecular-weight compounds might interfere with HIV replication in macrophages through the interaction with αv integrins.

A Novel Switch Culture System for Proliferation and Differentiation of Monocytic Cells: ERK Activation Is Not Sufficient for Macrophage Differentiation by M-CSF.

M-CSF, a ligand for Fms, is an essential cytokine for proliferation and differentiation of monocytes/macrophages. However, molecular events responsible for M-CSF/Fms-mediated differentiation and their differences to proliferation-inducing signals are poorly understood. To clarify the difference, it is required to establish cell culture systems, in which distinct responses (proliferation versus differentiation) can be assessed independently in same cells. Here, we report the establishment of a novel culture system useful for analyzing the differences. TF-1-fms leukemia cells proliferate in response to M-CSF, but tend to differentiate into macrophages in the presence of a phorbol ester TPA. Interestingly, in the presence of TPA, M-CSF was likely to hardly yield the initiation of proliferation signal but markedly accelerate the TPA-triggered macrophage differentiation process. The combination of both TPA and M-CSF caused more drastic morphologic change than the treatment with TPA alone. The cells cultured with TPA and M-CSF showed increased granularity and higher phagocytic activity than those cultured with TPA alone. However, there was no significant difference in the numbers of viable cells and in the percentage of PI-positive dead cells between the two treatments, suggesting that the differentiation-accelerating effect did not reflect the survival-enhancing activity of M-CSF but the factor actively transmitted differentiation signals to TF-1-fms in the presence of TPA. The differentiation by TPA/M-CSF apparently correlated with elevated phosphorylation of ERK, a possible regulator of differentiation in a number of cell systems. However, and of importance, a specific MEK inhibitor, PD98059, inhibited the ERK phosphorylation but not macrophage differentiation. The result raised the possibility that elevated ERK activation was not necessarily sufficient for macrophage differentiation of TF-1-fms cells mediated by M-CSF. The experiments with TF-1-fms cells expressing HIV-1 Nef protein further supported the hypothesis: TF-1-fms cells over-expressing Nef showed impaired macrophage differentiation response but increased ERK phosphorylation (Suzu S et al., Blood 2005). Therefore, our switch culture system provides a useful tool for identifying molecules regulating the M-CSF/Fms-mediated macrophage differentiation.

Differentiation of human monocytic cell lines confers susceptibility to Bacillus anthracis lethal toxin

Anthrax lethal toxin (LT) is comprised of protective antigen and lethal factor. Lethal factor enters mammalian cells in a protective antigen-dependent process and cleaves mitogen-activated protein kinase kinases. Although LT has no observable effect on many cell types, it causes necrosis in macrophages derived from certain mouse strains and apoptosis in activated mouse macrophages. In this study, we observed that LT treatment of three different human monocytic cell lines U-937, HL-60 and THP-1 did not induce cell death. Cells did become susceptible to the toxin, however, after differentiation into a macrophage-like state. Treatment with LT resulted in decreased phosphorylation of p38, ERK1/2 and JNK in both undifferentiated and differentiated HL-60 cells, suggesting that the change in susceptibility does not result from differences in toxin delivery or substrate cleavage. Death of differentiated HL-60 cells was accompanied by chromosome condensation and DNA fragmentation, but was not inhibited by the pan-caspase inhibitor Z-VAD-FMK. In addition, we observed that the macrophage differentiation process could be inhibited by LT. Our results indicate that LT-mediated death of mouse and human macrophages may occur through distinct processes and that the differentiation state of human cells can determine susceptibility or resistance to LT.

P2X7 receptor induces gene expression

The concentration of extracellular nucleotides is known to rise significantly under inflammatory conditions in vivo. In normal tissues, extracellular nucleotides are maintained at low concentrations and tightly regulated. However, activated lymphocytes, macrophages, microglia, and platelets, as well as cells undergoing necrosis or apoptosis, release high concentrations of different nucleotide diphosphates and triphosphates into the extracellular space. It has also been shown that P2 nucleotide receptors ligands modulate IL‐1β‐induced transcription factor activation and differentially regulate inflammatory gene expression in primary human astrocytes. In previous work using cDNA microarray technology and RT–PCR, we found that chronic exposure of human monocytic U937 cells to UTP modulated the expression of a large number of genes related to the monocyte to macrophage differentiation process, including the tumor necrosis factor (TNF‐α). Here we present the effect of chronic exposure to extracellular nucleotides and nucleotide analogs on the expression of genes in 1321 N1 astrocytoma cells expressing recombinant P2X7 receptors. Our findings will help us better understand the role of nucleotide receptors in the inflammatory response in the nervous system, information that is bound to have pharmacological applications and clinical significance.

Transcription factors Sp1 and AP-2 mediate induction of acid sphingomyelinase during monocytic differentiation

Cells from the human monocytic leukemia cell line THP-1 differentiate towards a macrophage-like phenotype when stimulated with phorbol 12-myristate-13-acetate (PMA), 1,25-dihydroxy-vitamin D3, and various other agents. We demonstrate here that the expression of the lysosomal enzyme acid sphingomyelinase (ASM; E.C. 3.1.4.12) is induced during this process and is strongly elevated in differentiated THP-1 cells, as well as in differentiated human mononuclear phagocytes. Using Northern blotting, RNase protection assay, and nuclear run-on analyses, we show that the up-regulation of ASM expression is regulated mainly at the level of transcription and that new protein synthesis is required for enhanced ASM activity. This cell-type specific induction by PMA treatment was further investigated with respect to transcriptional control. A series of 5′ deletion derivatives of the upstream regulatory region were used in transient transfection assays to identify promoter elements required for basal and inducible gene expression. A PMA responsive element was localized to a region between –319 and –219 bp upstream of the initiation codon and co-transfections with transcription factor expression plasmids for AP-2 and Sp1 resulted in augmented ASM promoter activity, which was abolished when the binding sites for these two factors were deleted. Using electrophoretic mobility shift assays and supershift assays we demonstrate that this region is specifically bound by Sp1 and AP-2. These factors are present in nuclear extracts prepared from both induced and uninduced THP-1 cells. However, the intensity of the complex formed appeared to increase when nuclear extracts from PMA-treated cells were used. From these studies, we conclude that a concerted action of the transcription factors AP-2 and Sp1 is essential for the up-regulation of ASM expression during the process of macrophage differentiation.—Langmann, T., C. Buechler, S. Ries, A. Schaeffler, C. Aslanidis, M. Schuierer, M. Weiler, K. Sandhoff, P. J. de Jong, and G. Schmitz. Transcription factors Sp1 and AP-2 mediate induction of acid sphingomyelinase during monocytic differentiation. J. Lipid Res. 1999. 40: 870–880.