Induction Of Monocytic Differentiation Research Articles

Hepatitis C Virus-Induced Monocyte Differentiation Into Polarized M2 Macrophages Promotes Stellate Cell Activation via TGF-β

Background & AimsMonocyte and macrophage (MΦ) activation contributes to the pathogenesis of chronic hepatitis C virus (HCV) infection. Disease pathogenesis is regulated by both liver-resident MΦs and monocytes recruited as precursors of MΦs into the damaged liver. Monocytes differentiate into M1 (classic/proinflammatory) or M2 (alternative/anti-inflammatory) polarized MΦs in response to tissue microenvironment. We hypothesized that HCV-infected hepatoma cells (infected with Japanese fulminant hepatitis-1 [Huh7.5/JFH-1]) induce monocyte differentiation into polarized MΦs.MethodsHealthy human monocytes were co-cultured with Huh7.5/JFH-1 cells or cell-free virus for 7 days and analyzed for MΦ markers and cytokine levels. A similar analysis was performed on circulating monocytes and liver MΦs from HCV-infected patients and controls.ResultsHuh7.5/JFH-1 cells induced monocytes to differentiate into MΦs with increased expression of CD14 and CD68. HCV-MΦs showed M2 surface markers (CD206, CD163, and Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN)) and produced both proinflammatory and anti-inflammatory cytokines. HCV-induced early interleukin 1β production promoted transforming growth factor (TGF)β production and MΦ polarization to an M2 phenotype. TGF-β secreted by M2-MΦ led to hepatic stellate cell activation indicated by increased expression of collagen, tissue inhibitor of metalloproteinase 1, and α-smooth muscle actin. In vivo, we observed a significant increase in M2 marker (CD206) expression on circulating monocytes and in the liver of chronic HCV-infected patients. Furthermore, we observed the presence of a unique collagen-expressing CD14+CD206+ monocyte population in HCV patients that correlated with liver fibrosis.ConclusionsWe show an important role for HCV in induction of monocyte differentiation into MΦs with a mixed M1/M2 cytokine profile and M2 surface phenotype that promote stellate cell activation via TGF-β. We also identified circulating monocytes expressing M2 marker and collagen in chronic HCV infection that can be explored as a biomarker.

MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia

AbstractAlthough microRNAs (miRNAs) are increasingly linked to various physiologic processes, including hematopoiesis, their function in the myeloid development is poorly understood. We detected up-regulation of miR-29a and miR-142-3p during myeloid differentiation in leukemia cell lines and CD34+ hematopoietic stem/progenitor cells. By gain-of-function and loss-of-function experiments, we demonstrated that both miRNAs promote the phorbol 12-myristate 13-acetate–induced monocytic and all-trans-retinoic acid-induced granulocytic differentiation of HL-60, THP-1, or NB4 cells. Both the miRNAs directly inhibited cyclin T2 gene, preventing the release of hypophosphorylated retinoblastoma and resulting in induction of monocytic differentiation. In addition, a target of miR-29a, cyclin-dependent kinase 6 gene, and a target of miR-142-3p, TGF-β–activated kinase 1/MAP3K7 binding protein 2 gene, are involved in the regulation of both monocytic and granulocytic differentiation. A significant decrease of miR-29a and 142-3p levels and an obvious increase in their target protein levels were also observed in blasts from acute myeloid leukemia. By lentivirus-mediated gene transfer, we demonstrated that enforced expression of either miR-29a or miR-142-3p in hematopoietic stem/progenitor cells from healthy controls and acute myeloid leukemia patients down-regulated expression of their targets and promoted myeloid differentiation. These findings confirm that miR-29a and miR-142-3p are key regulators of normal myeloid differentiation and their reduced expression is involved in acute myeloid leukemia development.

Overexpression of Oncogenic KRAS G12V in Human Stem and Progenitor Cells Enhances Proliferation and Initiates Monocytic Differentiation Via Intrinsic and Extrinsic Pathways.

Abstract 3975Poster Board III-911In human hematopoietic malignancies, Ras mutations are frequently present in monocytic and T-cell leukemias. In this study we investigated KRAS G12V-induced phenotypes in human stem and progenitor cells and identified signal transduction pathways that are involved. Using a retroviral expression system, KRAS G12V was introduced to human CD34+ cord blood (CB) cells and proliferation, differentiation and stem cell/progenitor frequencies were evaluated. Overexpression of constitutively active KRAS G12V induced a strong increase in cell expansion over 5-fold in MS5 bone marrow stromal cocultures as well as in cytokine-driven liquid cultures, which coincided with increased early cobblestone formation and induction of monocytic differentiation. Erythroid progenitors were greatly reduced by introduction of KRAS G12V and Q-PCR analysis revealed that expression of PU.1 was increased in conjunction with reduced GATA1 expression in KRAS G12V cells. Progenitor frequencies were increased 6-fold in KRAS-transduced cells within 1 week after plating on MS5. By week three progenitors were exhausted and KRAS-transduced cells were terminally differentiated into monocytes/macrophages. These results were in line with the strong reduction in LTC-IC frequencies at week 5, indicating that also the stem cell pool was exhausted. Intriguingly, when KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, we observed that the non-transduced cells also displayed a strong growth advantage, coinciding with enhanced early cobblestone formation. Furthermore, the addition of conditioned medium from KRAS G12V-transduced cells grown on MS5 to non-transduced CB cells induced a strong growth advantage and formation of early CAFCs. These observations indicate that, besides intrinsic pathways, secreted factor(s) play an important role in the phenotypes induced by KRAS G12V in human CB CD34+ cells. Current studies include mass-spectroscopy analysis of the secretome of KRAS G12V-transduced CB CD34+ cells to identify the factor(s) that are involved.In order to elucidate signal transduction pathways that mediate KRAS G12V-induced phenotypes, Western-blot analysis was performed. These experiments revealed an increase in phospho-ERK1/2, phospho-p38 and phospho- STAT5 (Y694) levels in KRAS-transduced cells, whereas phospho-JNK was not induced and phospho-C/EBPa (S21) levels were slightly reduced. Induction of STAT5 Y649 phosphorylation by KRAS G12V was confirmed by intracellular phosphoFACS analysis, whereby both in HSCs as well as in more committed MPPs KRAS-induced phosphorylation of STAT5 was observed. KRAS-transduced cells did not show GM-CSF hypersensitivity in any measured cell population upon activation. Inhibition of the ERK/MAPK pathway using the MEK inhibitor U0126 resulted in strongly reduced expansion in MS5 cocultures, whereby both intrinsically induced proliferation as well as proliferation induced via secreted factor(s) were impaired. KRAS G12V-induced monocytic differentiation was not significantly affected by MEK inhibition. While inhibition of the JNK pathway hardly affected proliferation and differentiation of KRAS G12V cells, inhibition of the p38 pathway using SB203580 inhibitor impaired both proliferation and differentiation. When KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, inhibition of p38 predominantly affected the transduced cells but not the non-transduced cells, suggesting that the p38 pathway particularly mediates intrinsic phenotypes imposed by KRAS G12V.In conclusion, we show that overexpression of oncogenic KRAS G12V in human CD34+ cells enhances proliferation and initiates monocytic differentiation via intrinsic and extrinsic pathways. Disclosures:No relevant conflicts of interest to declare.

The Vitamin D3/Hox-A10 Pathway Supports MafB Function during the Monocyte Differentiation of Human CD34+ Hemopoietic Progenitors

Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hemopoiesis, the conclusions of such studies are quite controversial given that they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion, whereas in others they implicate this transcription factor in the induction of monocyte-macrophage differentiation. To clarify this issue, we analyzed the biological effects and the transcriptome changes determined in human primary CD34(+) hemopoietic progenitors by retroviral transduction of a full-length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of the MafB gene, recently identified as the master regulator of such a maturation pathway. By using a combined approach based on computational analysis, EMSA experiments, and luciferase assays, we were able to demonstrate the presence of a Hox-A10-binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Stimulation of the same cells with the vitamin D(3) monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving vitamin D(3) receptor, Hox-A10, and MafB transcription factors. Altogether, these data allow one to conclude that the vitamin D(3)/Hox-A10 pathway supports MafB function during the induction of monocyte differentiation.

A new simple multi-well plate-based assay for monocyte differentiation using human monocytic leukemia THP-1 cells

A new simple multi-well plate-based assay to determine monocyte differentiation-inducing activity was developed. It is based on the increase of macrophage adherence after the induction of monocyte differentiation. The assay was conducted using a human monocytic leukemia cell line, THP-1, treated with either 12- O-tetradecanoylphorbol 13-acetate or cytokines, such as tumor necrosis factor-α and interferon-γ. This method is sensitive and easy to perform, especially in case of analyzing a large number of samples.

Monocyte CD64 or CD89 targeting by surfactant protein D/anti‐Fc receptor mediates bacterial uptake

We recently showed that a chimeric protein, consisting of a recombinant fragment of human surfactant protein D (rfSP-D) coupled to a Fab' fragment directed against the human Fcalpha receptor (CD89), effectively targets pathogens recognized by SP-D to human neutrophils. The present study evaluates the effectiveness of chimeric rfSP-D/anti-Fc receptor proteins targeting Escherichia coli to CD89 or to the Fcgamma receptor I (CD64) on monocytes. Both chimeric rfSP-D/anti-Fc receptor proteins increased internalization of E. coli by the human promonocytic cell line U937, but only after induction of monocytic differentiation, despite the fact that the expression levels of CD64 and CD89 on undifferentiated cells were at least as high as on differentiated cells. The two chimeric rfSP-D/anti-Fc receptor proteins did not enhance each other's effect on E. coli uptake. Targeting to differentiated U937 cells was inhibited by blocking the interaction either between the rfSP-D part of the chimeric molecule and E. coli, or between the anti-Fc receptor Fab' fragment and the Fc receptor on the U937 cell. In conclusion, both CD64 and CD89 on U937 cells prove to be suitable for targeting by rfSP-D/anti-Fc receptor proteins. However, in addition to mere Fc receptor expression, effective targeting requires monocytic differentiation.

Combined treatment of leukemia cells with vitamin K2 and 1α,25-dihydroxy vitamin D3 enhances monocytic differentiation along with becoming resistant to apoptosis by induction of cytoplasmic p21CIP1

Vitamin K2 (VK2) effectively induces apoptosis in leukemia cell lines, including HL-60 and U937. However, combined treatment of cells with VK2 plus 1alpha,25-dihydroxy vitamin D3 (VD3) resulted in suppression of VK2-inducing apoptosis and pronounced induction of monocytic differentiation as compared with that by VD3 alone. After achieving monocytic differentiation by pre-exposure to VK2 and VD3, the cells became resistant to various apoptotic stimuli including VK2- and H2O2-treatment and serum deprivation. Accumulation of cytoplasm p21CIP1 along with disappearance of nuclear p21CIP1 was detected in cells in response to 96-h treatment with VK2 plus VD3. A stable transfectant, U937-deltaNLS-p21CIP1, which lacked the nuclear localization signal of p21CIP1 and showed overexpression of cytoplasm p21CIP1 without monocytic differentiation, was resistant to apoptosis. These data suggest that a change of intracellular distribution of p21CIP1 from nucleus to cytoplasm along with differentiation appears to be anti-apoptotic. Clinical benefits of using VK2 for treatment of patients with leukemia and myelodysplastic syndrome (MDS) have been reported. Our data suggest that VK2 plus VD3 may be an effective combination for differentiation-based therapy for leukemia and also MDS whose cytopenias are mediated though apoptosis.

In Vitro Biomarker Discovery for Atherosclerosis by Proteomics

The purpose of this study was to identify in vitro and then prioritize a tractable set of protein biomarker candidates of atherosclerosis that may eventually be developed to measure the extent, progression, regression, and stability of atherosclerotic lesions. A study was conducted using an in vitro"foam cell" model based on the stimulation of differentiated THP1 cells with oxidized low-density lipoprotein (oxidized LDL) as compared with low-density lipoprotein (LDL). Analysis of the proteins contained in the cell supernatant using proteome scanning technology identified 59 proteins as being increased, 57 with no statistically measurable difference, and 17 decreasing in abundance following treatment with oxidized LDL, as compared with LDL. From the up-regulated list, proteins were prioritized based on their analytical confidence as well as their relevance to atherosclerosis pathways. Within the group of increased abundance, seven families of proteins were of particular interest: fatty acid-binding proteins, chitinase-like enzymes, cyclophilins, cathepsins, proteoglycans, urokinase-type plasminogen activator receptor, and a macrophage scavenger receptor.

Microarray Analysis of the Short Isoform of Ikaros Overexpressed in Patients with Hematologic Malignancies.

We analyzed expression of the transcription factor Ikaros in human hematologic malignancies and found overexpression of the short isoform of Ikaros, Ik-6, in patients with blast crisis of chronic myeloid leukemia and pre-B cell acute lymphoblastic leukemia. We have made several stable transfectants which overexpress Ik-6 and analyzed the consequence of Ik-6 overexpression.First, overexpression of Ik-6 in FDH-1, which was established from a patient with early pre-B acute lymphoblastic leukemia, and Ramos conferred resistance to dexamethasone- and anti-IgM-induced apoptosis, respectively. Upon treatment with dexamethasone, FDH-1 cells demonstrated massive apoptosis measured by staining with Annexin V. In contrast to mock transfectants, Ik-6 transfectants showed less marked apoptosis (mock 86.4% vs Ik-6 34.4–73.6%, p=0.01). We also used Ramos as a model of anti-IgM-induced apoptosis, and flow cytometric analysis was performed with Annexin-V staining. In contrast to mock transfectants, Ik-6 transfectants showed less pronounced apoptosis (mock 40.4% vs Ik-6 23.1–27.4%, p=0.03). Next, overexpression of Ik-6 in Nalm6 lead to impaired cell growth and cell cycle progression. Cell growth and cell cycle were analyzed and compared with mock transfectants and Ik-6 transfectants using MTT assay and DNA content analysis with PI staining, respectively. MTT assay demonstrated that, after three days of growth, Ik-6 transfectants grew more slowly than mock transfectants (Ik-6/mock ratio 0.62–0.67; p=0.01). DNA content analysis showed that, after three days of growth, more Ik-6 transfectants underwent G0–G1 arrest than mock transfectants (Ik-6/mock ratio 1.15–1.21; p=0.01), and less Ik-6 transfectants entered into S phase than mock transfectants (Ik-6/mock ratio 0.84–0.90; p=0.05).Finally, overexpression of Ik-6 promoted all-trans retinoic acid (ATRA)-induced granulocytic differentiation, and, on the other hand, inhibited vitamin D3 (VD3)-induced monocytic differentiation of HL-60. Upon induction of granulocytic differentiation by ATRA, mock transfectants showed 74.5% positivity of myeloperoxidase (MPO) staining. In contrast, Ik-6 transfectants showed 51.5–56.8% of positivity of MPO staining (p=0.04). NBT reduction assay demonstrated 43.8% positivity with mock transfectants and 62.3–67.3% positivity with Ik-6 transfectants (p=0.001). Upon induction of monocytic differentiation by VD3, mock transfectants showed 51.0% positivity of nonspecific esterase (NSE) staining. In contrast, Ik-6 transfectants showed 26.8–36.0% of positivity of NSE staining (p=0.03).To understand the underlying mechanisms, we report the results of microarray analysis of Ik-6 transfectants.

Jun N-terminal kinase pathway enhances signaling of monocytic differentiation of human leukemia cells induced by 1,25-dihydroxyvitamin D3.

Recent studies revealed that the MEK/ERK module of the mitogen-activated protein kinase (MAPK) signaling cascades is up-regulated in the early stages of 1alpha,25-dihydroxyvitamin D(3) (1,25D(3))-induced monocytic differentiation of human leukemia cells HL60. In the present study, we investigated whether another MAPK module, the JNK pathway, also participates in this form of differentiation. We found that the dependence on the concentration of the inducer, the vitamin-hormone 1,25D(3), in two types of human leukemia cells, HL60 and U937, and the kinetics of monocytic differentiation in HL60 cells, parallel the degree of the activation of the JNK pathway. A blockade of JNK signaling by a stable expression of dominant negative (dn) JNK1 mutant in U937 cells resulted in reduced c-jun phosphorylation, and the differentiation of these cells was markedly decreased. Similarly, inhibition of JNK1 and JNK2 activities by the selective inhibitor SP600125 led to both dose-dependent reduction of c-jun and ATF-2 phosphorylation, and of the differentiation of HL60 cells. In addition, we found that JNK activity is essential for the AP-1 DNA binding induced by 1,25D(3) in HL60 and U937 cells. The results indicate that in cultured human leukemia cells, the JNK pathway participates in the induction of monocytic differentiation by 1,25D(3), probably by activating the AP-1 transcription factor.

Lineage-dependent NF-kappaB activation contributes to the resistance of human macrophages to apoptosis.

Granulocytes and mononuclear phagocytes develop from the same myeloid progenitor cells in the bone marrow via distinct differentiation pathways. Yet, it is known that mature macrophages are more resistant than granulocytes to spontaneous apoptosis in cultures without hematopoietic growth factors. This fact suggests that the development of resistance to apoptosis during myeloid differentiation is differentially regulated by a lineage-dependent mechanism. Using primary cultures of human bone marrow cells, we now report that induction of monocytic differentiation into mature macrophages with M-CSF was correlated with a steady and gradual increase in the levels of X-chromosome-linked inhibitor of apotosis (XIAP) and Bcl-2, while induction of granulocytic differentiation with G-CSF had no significant effects on the expression of these proteins. Consistent with this, NF-kappaB activation is linked to monocytic, but not granulocytic differentiation, while ERK or STAT3 activation is not lineage-dependent. Blockade of NF-kappaB activation in mature macrophages resulted in a marked decrease in the levels of XIAP and Bcl-2, which was accompanied with cell death through an apoptotic mechanism. Thus lineage-dependent activation of NF-kappaB is responsible at least in part for the resistance of mature macrophages to 'spontaneous' apoptosis in vitro.

Importance of MEK-1/-2 signaling in monocytic and granulocytic differentiation of myeloid cell lines

Activation of the MEK/ERK/MAP kinase signaling pathway promotes the proliferation and survival of hematopoietic cells. The kinases MEK-1, MEK-2, ERK-1/MAPK and ERK-2/MAPK are activated by phosphorylation at specific sites, and these events can be monitored using phospho-specific antibodies. In this report we examined the importance of the MEK/ERK/MAP kinase pathway in the monocytic and granulocytic differentiation of myeloid cell lines. Induction of monocytic differentiation in HL-60 cells by treatment with phorbol 12-myristate 13-acetate (PMA) led to rapid and sustained activation of MEK-1/-2, ERK-1/MAPK and ERK-2/MAPK, while induction of granulocytic differentiation by retinoic acid (RA) caused similar activation of MEK-1/-2 and ERK-2/MAPK, but not ERK-1/MAPK. The total levels of these kinases were not affected during the course of differentiation along either pathway. Pretreatment of cells with 5 microM of the MEK-1/-2-specific inhibitor U0126 abrogated PMA- or RA-induced activation of ERK-1/MAPK and ERK-2/MAPK. Importantly, pretreatment of HL-60 cells with U0126 was found to potently inhibit both monocytic and granulocytic differentiation, as assessed by cytochemical staining for non-specific esterase or nitroblue tetrazolium reduction, flow cytometric analysis of myeloid surface markers, and immunoblotting for the cell cycle inhibitor p21 WAF1/Cip1. Similar results were seen in U937 cells, where U0126 inhibited PMA-induced monocytic differentiation, and in 32D cells, where G-CSF-induced granulocytic differentiation was inhibited by U0126 pretreatment. Additional experiments revealed that inhibition of MEK-1/-2 in HL-60 cells resulted in nearly complete inhibition of differentiation-induced cell death during monocytic differentiation. By contrast, U0126 only partially inhibited cell death resulting from granulocytic differentiation. Taken together, our findings demonstrate that the MEK/ERK/MAP kinase signaling pathway is activated, and plays a critical role, during both monocytic and granulocytic differentiation of myeloid cell lines.

Activation of the MEK/MAPK Pathway Is Involved in Bryostatin1-Induced Monocytic Differenciation and Up-regulation of X-Linked Inhibitor of Apoptosis Protein

Induction of monocytic differentiation by bryostatin1 (bryo1) conferred on THP-1 leukemia cells the ability to resist Z-LLL-CHO-induced apoptosis. The mechanism of resistance developed during this process was investigated. Apoptosis resistance was associated with an enhanced expression of X-linked inhibitor of apoptosis protein (XIAP), an endogenous caspase inhibitor, in differentiated THP-1 cells. Bryo1 also increased the level of c-IAP-1, yet decreased the level of c-IAP-2 in THP-1 cells, indicating that distinct regulatory mechanisms are operative. In addition, treatment of THP-1 cells with bryo1 induced a rapid and sustained activation of MEK, prior to the upregulation of XIAP and monocytic differentiation. Pretreatment of THP-1 cells with MEK inhibitors (U0126 and PD98059) prior to bryo1 induction blocked the expression of both XIAP and the c-fms product (M-CSF receptor), a hallmark of monocytic differentiation, but not Bcl-2. In addition, the expression of XIAP in bryo1-treated cells was inhibited by CAPE, a NF-κB-specific inhibitor, indicating that its expression is under the transcriptional regulation of NF-κB downstream of the MEK/MAPK pathway. The importance of XIAP in mediating apoptosis resistance was illustrated in cells transiently transfected with XIAP, which conferred on THP-1 cells the ability to resist Z-LLL-CHO-induced apoptosis. These findings suggest that the expression of XIAP is linked to monocytic differentiation in bryo1-treated THP-1 cells and represents one of the potential antiapoptotic mechanisms acquired during this process.

Induction of monocyte differentiation and foam cell formation in vitro by 7-ketocholesterol

Oxidized LDL (OxLDL) is composed of many potentially proatherogenic molecules, including oxysterols. Of the oxysterols, 7-ketocholesterol (7-KC) is found in relatively large abundance in OxLDL, as well as in atherosclerotic plaque and foam cells in vivo. Although there is evidence that 7-KC activates endothelial cells, its effect on monocytes is unknown. We tested the hypothesis that 7-KC may induce monocyte differentiation and promote foam cell formation. THP-1 cells were used as a monocyte model system and were treated with 7-KC over a range of concentrations from 0.5 to 10 microg/ml. Changes in cell adhesion properties, cell morphology, and expression of antigens characteristic of differentiated macrophages were monitored over a 7-day period. 7-KC promoted cells to firmly adhere and display morphologic features of differentiated macrophages; this effect was time and dose dependent and was markedly more potent than cholesterol treatment (45% of cells became adherent after 7 days of treatment with 7-KC at 10 microg/ml vs. less then 5% for control cells, P < 0.01). Similar effects were obtained when LDL enriched with 7-KC or OxLDL were added to THP-1 cells. 7-KC-differentiated cells expressed CD11b, CD36, and CD68, phagocytized latex beads, and formed lipid-laden foam cells after exposure to acetylated LDL or OxLDL. In contrast to 7-KC, oxysterols with known cell regulatory effects such as 25-hydroxycholesterol, 7beta-hydroxycholesterol, and (22R)-hydroxycholesterol did not effectively promote THP-1 differentiation. In conclusion, these results demonstrate for the first time that 7-KC, a prominent oxysterol formed in OxLDL by peroxidation of cholesterol, may play an important role in promoting monocyte differentiation and foam cell formation. These studies also suggest that 7-KC induces monocyte differentiation through a sterol-mediated regulatory pathway that remains to be characterized.

Differentiation associated modulation of the cytokine and chemokine expression pattern in human myeloid cell lines

Hematopoietic progenitor cell differentiation is associated with the expression of different sets of genes including those encoding membrane bound molecules and cytokines. While expression of the former has meticulously been linked to both lineage specificity and maturation stages and is routinely used in the diagnosis of human leukemias, the production of cytokines has not systematically been analyzed in this respect. Secretion of cyto- and chemokines by HPC has been discussed as a key element of autocrine regulation of cell differentiation and proliferation in normal and malignant hematopoietic cells. Hematopoietic cell lines and their in vitro generated mature progeny were used as a model to investigate the cytokine and chemokine expression pattern prior to and after induction of differentiation. We show that a variety of cytokines are produced by these cells either constitutively or upon stimulation. Low levels of TNF-α and IL-8 were widely expressed by immature and mature cells, while peak values of TNF-α were detected in promyelocytic NB4 cells, as reported previously. Induction of monocytic differentiation by various agents was associated with upregulation of IL-1β and IL-1ra expression, while a differentiation shift to the granulocytic lineage in the presence of retinoic acid (RA) led to a marked increase of macrophage chemoattractant protein-1 (MCP-1) producing cells. These data indicate that lineage determination as well as maturation of hematopoietic cells may not only be associated with expression of specific surface molecules but also with a distinct cytokine expression pattern. Further studies are necessary to show if this holds true for primary leukemic and normal hematopoietic cells.

Interleukin‐8 Secretion of Human Epithelial and Monocytic Cell Lines Induced by Middle Ear Pathogens

Otitis media with effusion (OME) is one of the most common diseases in children. Alloiococcus otitidis, a new gram-positive bacterial species, was isolated from the middle ear fluid of children with OME; however, the pathogenic role of this bacteria is yet unknown. In this study, the ability of cultured epithelial cell lines (Hep-2 and Hela) and monocytic cell lines (THP-1 and U 937) to secrete chemokine interleukin-8 (IL-8) in response to the A. otitidis organism and three bacterial organisms mainly detected from middle ear fluid in OME, and bacterial cell components was investigated. When stimulated with four viable bacterial cells, epithelial cells and monocytes secreted IL-8 in a time-dependent manner. The monocytes produced significantly higher levels of IL-8 than the epithelial cells. Compared with that by viable bacterial cells, IL-8 secretion by stimulated epithelial cells and monocytes was reduced when the bacteria were heated and treated with glutaraldehyde. With bacterial stimulations, cell treatment of interferon-gamma caused monocytes to increase the induction of IL-8 production, however, the induction of monocyte differentiation caused monocytes to reduce the induction of IL-8 production. Furthermore, epithelial cells and monocytes stimulated by four viable bacterial organisms physically separated from cultured cells reduced the induction of IL-8 compared with directly stimulated cells, and monocytes stimulated with soluble extracts prepared from A. otitidis organisms produced IL-8 in a dose-dependent manner. These results suggest that part of the IL-8 stimulation of the A. otitidis organism may exist in a diffusable factor released by the bacteria or soluble components of the bacteria itself.

C-Maf Induces Monocytic Differentiation and Apoptosis in Bipotent Myeloid Progenitors

The transcriptional mechanisms that drive colony-forming unit granulocyte-macrophage (CFU-GM) myeloid progenitors to differentiate into cells of either the granulocytic or monocytic lineage are not fully understood. We have shown that the c-Maf and c-Myb transcription factors physically interact in myeloid cells to form inhibitory complexes that hinder transactivation of c-Myb target genes through direct binding to Myb consensus sites. These complexes arise in a developmentally regulated pattern, peaking at the promyelocyte stage, or in cell model systems, appearing soon after the induction of monocytic differentiation. We wished to determine if this developmentally related interaction is a consequence of myeloid differentiation or an intrinsic differentiating stimulus. Because the elevated Myb:Maf status seen in differentiating cells can be recapitulated by overexpression of c-Maf in myeloid cell lines, we inducibly expressed the c-Maf cDNA in 2 bipotent human myeloid progenitor cells. Elevated levels of c-Maf protein led to marked increases in Myb:Maf complexes and the accumulation of monocyte/macrophage cells, followed by eventual programmed cell death. Analysis of targets that could mediate these phenotypic changes indicated that c-Maf likely plays a key role in myeloid cell development through dual mechanisms; inhibition of a select set of c-Myb regulated targets, such as Bcl-2 and CD13/APN, coupled with the activation of as yet undefined differentiation-promoting genes.

C-Maf Induces Monocytic Differentiation and Apoptosis in Bipotent Myeloid Progenitors

AbstractThe transcriptional mechanisms that drive colony-forming unit granulocyte-macrophage (CFU-GM) myeloid progenitors to differentiate into cells of either the granulocytic or monocytic lineage are not fully understood. We have shown that the c-Maf and c-Myb transcription factors physically interact in myeloid cells to form inhibitory complexes that hinder transactivation of c-Myb target genes through direct binding to Myb consensus sites. These complexes arise in a developmentally regulated pattern, peaking at the promyelocyte stage, or in cell model systems, appearing soon after the induction of monocytic differentiation. We wished to determine if this developmentally related interaction is a consequence of myeloid differentiation or an intrinsic differentiating stimulus. Because the elevated Myb:Maf status seen in differentiating cells can be recapitulated by overexpression of c-Maf in myeloid cell lines, we inducibly expressed the c-Maf cDNA in 2 bipotent human myeloid progenitor cells. Elevated levels of c-Maf protein led to marked increases in Myb:Maf complexes and the accumulation of monocyte/macrophage cells, followed by eventual programmed cell death. Analysis of targets that could mediate these phenotypic changes indicated that c-Maf likely plays a key role in myeloid cell development through dual mechanisms; inhibition of a select set of c-Myb regulated targets, such as Bcl-2 and CD13/APN, coupled with the activation of as yet undefined differentiation-promoting genes.

Ubiquitin-proteasome system is involved in induction of LFA-1/ICAM-1-dependent adhesion of HL-60 cells.

Membrane-permeable proteasome inhibitors, lactacystin (LC) and N-acetyl-Leu-Leu-norleucinal (ALLN), but not calpain inhibitor Z-Leu-leucinal (ZLL), prevented LFA-1/ICAM-1-dependent cellular adhesion of TPA-stimulated HL-60 cells. These proteasome inhibitors affected neither the induction of monocytic differentiation nor the accompanying protein-tyrosine phosphorylation. They suppressed the increase in the avidity of LFA-1 to ICAM-1 without changing the expression of these molecules. Immunoblotting using monoclonal antibody FK-1, which reacts specifically with polyubiquitinated proteins, demonstrated that the proteasome inhibitors caused the drastic accumulation of the polyubiquitinated proteins in the membrane fraction of TPA-treated HL-60 cells. This indicates that accompanying activation of LFA-1, TPA induces the polyubiquitination of the membrane proteins, which are rapidly degraded by proteasomes. These data taken together show that proteolysis mediated by the ubiquitin-proteasome system is a prerequisite for the induction of LFA-1-dependent adhesion of HL-60 cells.

Regulation of MCL1 through a Serum Response Factor/Elk-1-mediated Mechanism Links Expression of a Viability-promoting Member of the BCL2 Family to the Induction of Hematopoietic Cell Differentiation

Proliferation, differentiation, and apoptosis are tightly regulated during hematopoiesis, allowing amplification along specific lineages while preventing excessive proliferation of immature cells. The MCL1 member of the BCL2 family is up-regulated during the induction of monocytic differentiation (approximately 10-fold with 12-O-tetradecanoylphorbol 13-acetate (TPA)). MCL1 has effects similar to those of BCL2, up-regulation promoting viability, but differs from BCL2 in its rapid inducibility and its pattern of expression. Nuclear factors that regulate MCL1 transcription have now been identified, extending the previous demonstration of signal transduction through mitogen-activated protein kinase. A 162-base pair segment of the human MCL1 5'-flank was found to direct luciferase reporter activity, allowing approximately 10-fold induction with TPA that was suppressible upon inhibition of the extracellular signal-regulated kinase (ERK) pathway. Serum response factor (SRF), Elk-1, and Sp1 bound to cognate sites within this segment, SRF and Elk-1 acting coordinately to affect both basal activity and TPA inducibility, whereas Sp1 affected basal activity only. Thus, the mechanism of the TPA-induced increase in MCL1 expression seen in myelomonocytic cells at early stages of differentiation involves signal transduction through ERKs and transcriptional activation through SRF/Elk-1. This finding provides a parallel to early response genes (e.g. c-FOS and EGR1) that affect maturation commitment in these cells and therefore suggests a means through which enhancement of cell viability may be linked to the induction of differentiation.