Interferon α selectively affects expression of the human myeloid cell nuclear differentiation antigen in late stage cells in the monocytic but not the granulocytic lineage

The human myeloid cell nuclear differentiation antigen (MNDA) is expressed constitutively in cells of the myeloid lineage, appearing in myeloblast cells in some cases of acute myeloid leukemia and consistently being detected in promyelocyte stage cells as well as in all later stage cells including peripheral blood monocytes and granulocytes. The human myeloid leukemia cell lines, HL-60, U937, and THP-1, express similar levels of immunochemically detectable MNDA. Although, the level of MNDA mRNA in primary monocytes is very low it was up-regulated at 6 h following the addition of interferon α. The effect of interferon α on the MNDA mRNA is also observed in the cell lines HL-60, U937, and THP-1. The MNDA mRNA level in primary granulocytes was unaffected by addition of interferon α and other agents including interferon γ, endotoxin, poly (I) · poly (C), and FMLP. The MNDA mRNA level in the myeloid cell lines was also unaffected by the latter four agents. Induction of differentiation in the myeloid cell lines with phorbol ester induces monocyte differentiation which was accompanied by a decrease in MNDA mRNA level. This reduced level of mRNA could then be elevated with subsequent interferon α treatment. The effects of phorbol ester on MNDA mRNA appeared to be associated with induced differentiation since inhibiting cell proliferation did not alter the level of MNDA mRNA and cell cycle variation in MNDA mRNA levels were not observed. The ability of interferon α to up-regulate MNDA mRNA in phorbol ester treated myeloid cell lines is consistent with the observations made in primary monocytes. Granulocyte differentiation induced by retinoic acid treatment of HL-60 cells did not alter the MNDA mRNA level which was also unchanged following subsequent treatment with interferon α. The lack of interferon α effects on retinoic acid treated HL-60 cells is consistent with its inability to influence MNDA mRNA level in primary granulocytes.     

IFI 16 gene encodes a nuclear protein whose expression is induced by interferons in human myeloid leukaemia cell lines

We have characterized the induction of mRNA and protein products of the human IFI 16 gene in response to IFN-γ, IFN-α, and IFN-β₂ (IL-6). We demonstrate that the IFI 16 gene product is a novel nucleoprotein expressed in association with the differentiation of myeloid precursor cell lines. In Northern blots, IFI 16 mRNA was increased ～25-fold above barely detectable levels in unstimulated promyelocytic HL-60 cells, in response to IFN-γ. Other myeloid cell lines, U937 and K562, also demonstrated a marked IFN-γ-inducibility of IFI 16 mRNA. However, all three cell lines were far less responsive to IFN-α, and there was no response to IL-6. By comparison, a panel of T and B cell lines demonstrated high constitutive expression of IFI 16 mRNA that was not regulated by these cytokines. Culture of HL-60 cells in medium containing dimethylsulfoxide, retinoic acid, and 1,25 dihydroxyvitamin D₃, agents that stimulate the differentiation of HL-60 along myeloid pathways, also caused the induction of IFI 16 mRNA. To characterize the protein product of IFI 16, a monoclonal antibody was raised against a recombinant bacterial protein comprising the amino terminal 159 amino acids of IFI 16 fused to glutathione S-transferase. The antibody, designated 1G7, was used in Western blotting to demonstrate the strong induction of a cluster of proteins of 85–95 kDa in the nuclear extracts of IFN-γ-treated HL-60. The nuclear localization of IFI 16 antigen was confirmed by immunohistochemical staining of HL-60 cells treated with IFN-γ, dimethylsulfoxide, and retinoic acid. IFI 16 was also detected in the nuclei of monocytes, neutrophils, and lymphocytes in normal peripheral blood. Database comparisons of the IFI 16 amino acid sequence revealed 51% identity with the recently cloned myeloid cell nuclear differentiation antigen (MNDA), and extensive similarity to protein products of the Gene 200 cluster of IFN-inducible genes, Ifi 202 and Ifi 204. The amino terminal domain of IFI 16 encodes a putative nuclear localization signal, ¹²⁴PGAQKRKK, which is strongly conserved in MNDA and 204. Nuclear IFI 16 was able to bind double-stranded DNA in vitro and exhibited a similar elution profile from DNA-cellulose as previously observed for MNDA and 204. Therefore, IFI 16 and MNDA are members of a novel family of human DNA-binding proteins whose expression is associated with myeloid cell differentiation induced by cytokines and chemical agents.     

Cloning and expression of the human myeloid cell nuclear differentiation antigen: regulation by interferon alpha.

The human myeloid cell nuclear differentiation antigen (MNDA) is a protein of 406 amino acids that is expressed specifically in granulocytes, monocytes and earlier stage cells of these lineages. Degenerate oligonucleotides that could encode regions of MNDA amino acid sequence were used to amplify the MNDA cDNA sequence using the polymerase chain reaction. The amplified cDNA product was sequenced to confirm that it encoded the MNDA protein. It was then used as a probe to isolate five clones from a human bone marrow lambda gt10 cDNA library. A clone containing a 1,672 base pair cDNA insert was sequenced and found to encode the entire MNDA open reading frame, as well as 5' and 3' untranslated regions. The primary structure of the MNDA contains extensive regions of sequence similarity with the protein products of the interferon-inducible genes: 204 and interferon regulatory factor 2. In addition, a 12-base sequence matching the interferon-stimulated response element consensus sequence [GAAAN(N)GAAA] is located in the 5' untranslated region of the MNDA cDNA. The 1.8 kb MNDA mRNA was detected only in cells that express the antigen and the level of MNDA mRNA was elevated in cells treated with either recombinant or natural interferon alpha. The MNDA mRNA was not induced by interferon alpha in cells that do not exhibit a constitutive level of the MNDA mRNA. The MNDA contains sequence motifs found in gene regulatory proteins. The expression and the primary structure of the MNDA indicates that it plays a role in the granulocyte/monocyte cell-specific response to interferon.     

Expression of surface antigen and mRNA for the CD11c (alpha X, p150) subunit of the human leukocyte adherence receptor family in hematopoietic cells

We characterized the surface antigen and mRNA expression for the CD11c (alpha X, p150) subunit of the human leukocyte adherence receptor family during hematopoietic cell differentiation. The CD11c subunit antigen and mRNA are constitutively expressed in undifferentiated HL-60 promyelocytic leukemia cells, and levels increase markedly with differentiation along the monocyte/macrophage pathway using phorbol myristate acetate. Human monocyte-derived macrophages and human alveolar macrophages express elevated levels of the CD11c subunit antigen and mRNA, indicating that the changes observed in vitro are present in vivo. Dot blot analysis of immature and mature lymphoid and myeloid cells and cell lines demonstrate equivalent levels of CD11c mRNA expression. We conclude that CD11c gene expression is selectively increased during hematopoietic cell differentiation along the monocyte/macrophage pathway.     

Development of mature and functional human myeloid subsets in hematopoietic stem cell-engrafted NOD/SCID/IL2rγKO mice

Although physiological development of human lymphoid subsets has become well documented in humanized mice, in vivo development of human myeloid subsets in a xenotransplantation setting has remained unevaluated. Therefore, we investigated in vivo differentiation and function of human myeloid subsets in NOD/SCID/IL2rγ(null) (NSG) mouse recipients transplanted with purified lineage(-)CD34(+)CD38(-) cord blood hematopoietic stem cells. At 4-6 mo posttransplantation, we identified the development of human neutrophils, basophils, mast cells, monocytes, and conventional and plasmacytoid dendritic cells in the recipient hematopoietic organs. The tissue distribution and morphology of these human myeloid cells were similar to those identified in humans. After cytokine stimulation in vitro, phosphorylation of STAT molecules was observed in neutrophils and monocytes. In vivo administration of human G-CSF resulted in the recruitment of human myeloid cells into the recipient circulation. Flow cytometry and confocal imaging demonstrated that human bone marrow monocytes and alveolar macrophages in the recipients displayed intact phagocytic function. Human bone marrow-derived monocytes/macrophages were further confirmed to exhibit phagocytosis and killing of Salmonella typhimurium upon IFN-γ stimulation. These findings demonstrate the development of mature and functionally intact human myeloid subsets in vivo in the NSG recipients. In vivo human myelopoiesis established in the NSG humanized mouse system may facilitate the investigation of human myeloid cell biology including in vivo analyses of infectious diseases and therapeutic interventions.     

Effect of cellular differentiation on cytokine-induced expression of human immunodeficiency virus in chronically infected promonocytic cells: dissociation of cellular differentiation and viral expression.

Cellular differentiation is thought to play an important role in the susceptibility of monocytic lineage cells to human immunodeficiency virus (HIV) infection as well as in their ability to support virus replication. In addition, virus replication in monocytes/macrophages has been demonstrated in vitro to be strongly modulated by several cytokines such as tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor. The purpose of the present study was to investigate the interaction between cellular differentiation and cytokines in the regulation of HIV expression from chronically infected monocytic lineage cells. U1, a persistently HIV-infected promonocytic cell line, is characterized by low levels of virus expression which can be modulated by several cytokines. 1 alpha,-25-Dihydroxyvitamin D3 (Vit.D3), a well-known differentiating agent for myelomonocytic cells which has been previously reported to modulate HIV replication in other in vitro systems, induced maturation of U1 cells toward a macrophage-like phenotype, as demonstrated by the induction of the differentiation-associated cell surface markers CD14 and CD11b. Vit.D3-induced differentiation did not result in induction of HIV expression; however, when U1 cells were stimulated with tumor necrosis factor alpha in the presence of Vit.D3, a synergistic induction of cell differentiation and viral expression was demonstrated. In contrast, Vit.D3 suppressed the induction of HIV expression in U1 cells stimulated with gamma interferon, interleukin-6, and granulocyte-macrophage colony-stimulating factor, although synergy between Vit.D3 and these cytokines was observed in terms of cellular differentiation. These data suggest that differentiation of monocytic cells does not necessarily correlate with increased HIV expression.     

Lineage-specific expression of c-fos and c-fms in human hematopoietic cells: Discrepancies with the in vitro differentiation of leukemia cells

In vitro differentiation studies using the bipotential human leukemia cell line, HL60, have indicated that high levels of expression of two proto-oncogenes, c-fos and c-fms, are restricted to the myelomonocytic lineage. No such expression has been detected in induced granulocytic cells. In striking contrast to these observations, we found that c-fos mRNA levels are very high in purified human granulocytes, but barely detectable in blood monocytes and tissue macrophages. Human granulocytes contain, however, relatively low levels of c-fos protein, indicating that c-fos mRNA is inefficiently translated or that the protein is rapidly degraded in these cells. In closer agreement with the in vitro results, the level of the expression of c-fms is high in purified blood monocytes and undetectable in granulocytes. We found, however, that the evolution of monocytes into tissue macrophages is accompanied by a significant decrease in c-fms expression, suggesting that the function of c-fms is restricted to specific stages of monocytic differentiation. Our observations also show that results obtained using in vitro differentiation systems have to be regarded with caution, since they may not reflect the in vivo situation.     

Lineage-specific expression of c-fos and c-fms in human hematopoietic cells: Discrepancies with the in vitro differentiation of leukemia cells

Abstract. In vitro differentiation studies using the bipotential human leukemia cell line, HL60, have indicated that high levels of expression of two proto-oncogenes, c- fos and c- fms , are restricted to the myelomonocytic lineage. No such expression has been detected in induced granulocytic cells. In striking contrast to these observations, we found that c- fos mRNA levels are very high in purified human granulocytes, but barely detectable in blood monocytes and tissue macrophages. Human granulocytes contain, however, relatively low levels of c- fos protein, indicating that c- fos mRNA is inefficiently translated or that the protein is rapidly degraded in these cells. In closer agreement with the in vitro results, the level of the expression of c- fms is high in purified blood monocytes and undetectable in granulocytes. We found, however, that the evolution of monocytes into tissue macrophages is accompanied by a significant decrease in c- fms expression, suggesting that the function of c- fms is restricted to specific stages of monocytic differentiation. Our observations also show that results obtained using in vitro differentiation systems have to be regarded with caution, since they may not reflect the in vivo situation.     

Expression of protein kinase C genes in hemopoietic cells is cell-type- and B cell-differentiation stage specific

We have studied the expression of mRNA encoding all known protein kinase C (PKC) isozymes (alpha, beta, gamma, delta, epsilon, zeta, and eta) in murine tumor cell lines that exemplify hemopoietic cells arrested at different stages of development as well as in normal hemopoietic cells. We demonstrate that some of the isozymes, PKC-alpha, -beta, and -eta, are differentially expressed in different lineages. PKC-alpha and -beta generally are not detectable in myeloid cell lines, where PKC-delta is the predominant isoform. Both PKC-alpha and -beta are abundant in most T and B lymphocytic lines, but steady state levels of PKC-beta mRNA are lowest in plasma cell tumors, which exemplify the terminally differentiated B lymphocyte. In contrast, the levels of PKC-alpha mRNA remain high in plasma cell tumors, and a novel, 2.5-kb PKC-alpha mRNA gains prominence. PKC-eta mRNA is the major PKC isoform expressed in T lymphocytes, but it also is highly abundant in some myeloid lines. PKC-delta is expressed at high levels in all the lines we studied, whereas PKC-epsilon and -zeta are found in most cells but only at rather low levels. Analysis of myeloid clones derived from bipotential B lineage progenitor cell lines suggests that the B cell phenotype is associated with the expression of PKC-alpha. The close correlation of protein levels with mRNA levels indicates that PKC expression in hemopoietic cells is mainly regulated at the level of mRNA. The lineage- and differentiation stage-specific patterns of PKC-isozyme expression presented here suggest the involvement of specific PKC isozymes in differentiation as well as lineage determination of hemopoietic cells.     

Temporal relationships between induced changes in c-myc mRNA abundance, proliferation, and differentiation in HL60 cells

Changes in the relative abundances of c-myc mRNA have been related to changes in other parameters of differentiation (histochemical, clonogenic) during the course of the differentiation of HL60 cells to monocytes/macrophages or to granulocytes. Induction of differentiation to monocytes/macrophages was marked by a rapid rate of appearance of committed cells (80 to 90% in 24 hours) and a concomitant rapid loss of c-myc mRNA. Induction of granulocytic differentiation resulted in a much slower rate of appearance of committed cells (50% in 48 hours), and a much faster rate of loss of c-myc mRNA (tenfold in 1 hour). These data are consistent with there being a direct link between down-regulation of the expression of c-myc and the onset of proliferation arrest and monocytic differentiation, but show there is no such association of c-myc mRNA abundance and proliferation or differentiation during the maturation of HL60 granulocytes.     

Identification of the differentiation-associated p93 tyrosine protein kinase of HL-60 leukemia cells as the product of the human c-fes locus and its expression in myelomonocytic cells

A differentiation-associated 93-kDa tyrosine kinase (p93) was purified previously from the human promyelocytic leukemia cell line HL-60. The present study conclusively identifies p93 as the c-fes proto-oncogene product and shows that expression of p93c-fes and its associated tyrosine kinase activity are marked in mature granulocytes, monocytes, and human myeloid leukemia cell lines. Antisera to peptides obtained by expression of c-fes cDNA fragments in Escherichia coli reacted strongly with p93 purified from HL-60 cells. Western blots using one of these antisera demonstrated high levels of p93c-fes protein in normal human granulocytes and monocytes, as well as the cell lines KG-1, THP-1, HEL, and U-937, all of which can be induced to differentiate along the myelomonocytic pathway. Conversely, in cell lines resistant to myeloid differentiation, p93c-fes expression was either very low or absent. Expression of immunoreactive p93c-fes in these cell lines showed a strong positive correlation with p93c-fes tyrosine kinase activity, which was measured in cell extracts using a nondenaturing gel assay. Finally, the expression of p93c-fes, its tyrosine kinase activity, and the binding of 125I-granulocyte-macrophage colony-stimulating factor (GM-CSF) were all coordinately increased in HL-60 cells treated with the granulocytic differentiation inducer dimethyl sulfoxide, while all three parameters were low in untreated or differentiation-resistant HL-60 cells. These results suggest that expression of p93c-fes tyrosine kinase activity may be an essential component of myeloid differentiation and responsiveness to granulocyte-macrophage colony-stimulating factor.     

Triglyceride enhances susceptibility to TNF-α-induced cell death in THP-1 cells

Monocytes and macrophages play a major role in atherosclerosis development. Previously, we found that triglyceride (TG) promoted cell death of PMA-differentiated THP-1 macrophages. In this study, we compared the responsiveness of THP-1 monocytes and PMA-differentiated THP-1 macrophages to TNF-α-induced cell death. We found that, whereas THP-1 monocytes were TNF-α-resistant, THP-1 macrophages were sensitive to TNF-α-induced cell death. THP-1 monocytes treated with TG underwent cell death beginning at 24 h and addition of TNF-α further increased cell death. Based on these observations, we hypothesized that TG-induced differentiation of THP-1 monocytes into THP-1 macrophages, subsequently allowing sensitivity to TNF-α. To determine if TG could induce differentiation of THP-1 monocytes into THP-1 macrophages, we examined the mRNA expression levels of the macrophage-specific markers, CD11b, CD18, CD36 and CD68, by RT-PCR analysis. Our results show that expression of CD11b, CD36 and CD68 increased in TG-treated THP-1 monocytes in a dose- and time-dependent manner; furthermore, TNF-α expression was upregulated in TG-treated THP-1 monocytes. We have concluded that TG induces differentiation of THP-1 monocytes into macrophages concomitant with the production of TNF-α and increased sensitivity to TNF-α-dependent cell death.     

Membrane protein hMYADM preferentially expressed in myeloid cells is up-regulated during differentiation of stem cells and myeloid leukemia cells

We report here the molecular cloning and characterization of a novel human gene (hMYADM) derived from a human bone marrow stromal cell (BMSC) cDNA library, which shares high homology with mouse myeloid-associated differentiation marker (MYADM). hMYADM is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved MYADM-like family. hMYADM with 322-residue protein contains eight putative transmembrane segments and confocal microscopic analysis confirmed its membrane localization by using anti-hMYADM monoclonal antibody. hMYADM mRNA was selectively expressed in human monocytes, dendritic cells, promyeloid or monocytic leukemia cell lines, but not in CD4+, CD8+, CD19+ cells, nor in T cell leukemia or lymphocytic leukemia cell lines. hMYADM expression was also found in normal human bone marrow enriched for CD34+ stem cells, and the expression was up-regulated when these cells were induced to differentiate toward myeloid cells. The mRNA expression level of hMYADM significantly increased in acute promyelocytic leukemia HL-60 and chronic myelogenous leukemia K562 cell line after phorbol myristate acetate (PMA)-induced differentiation. Our study suggests that hMYADM is selectively expressed in myeloid cells, and involved in the myeloid differentiation process, indicating that hMYADM may be one useful membrane marker to monitor stem cell differentiation or myeloid leukemia differentiation.