Retinoids in myelopoiesis.

The vitamin A derivative retinoic acid plays a critical role during the differentiation of myeloid progenitors towards the neutrophil lineage. This role is primarily mediated by binding of retinoic acid to retinoic acid receptor alpha (RARalpha), a nuclear receptor that modulates the expression of multiple downstream targets via retinoic acid response elements. The importance of this signalling pathway in myelopoiesis is evidenced by the recurrent disruption of the RARalpha gene by chromosomal rearrangements in all cases of acute promyelocytic leukemia (APL). Biochemical evidence suggests RARalpha performs two opposing functions, one as a repressor of gene expression in the absence of ligand, the second as a transcriptional activator in the presence of ligand, each controlled by multimeric complexes of transcription corepressors and coactivators, respectively. Here the molecular mechanisms activated by retinoic acid during myelopoiesis in the context of neutrophil development will be reviewed, together with some of the more recently identified targets of the retinoic signalling pathway.

Erythropoietin mediates terminal granulocytic differentiation of committed myeloid cells with ectopic erythropoietin receptor expression.

OBJECTIVES: The precise role of hematopoietic cytokine/cytokine receptor interactions in lineage-restricted hematopoietic differentiation giving rise to mature blood cells of diverse function is incompletely defined. To study lineage-specific effects of cytokines during terminal hematopoietic differentiation, we examined the ability of erythropoietin (Epo) to mediate terminal granulocytic differentiation and induction of myeloid gene expression in committed myeloid cells, engineered to ectopically express Epo receptor (EpoR). METHODS: A cell culture model for granulocyte-macrophage colony stimulating factor (GM-CSF)-mediated granulocytic differentiation was used. EpoR was introduced by retrovirus-mediated gene transfer into multipotential, hematopoietic murine cell line EML, from which GM-CSF-responsive, promyelocytic EPRO cells were generated. In EPRO cells ectopically expressing EpoR, we examined the ability of Epo to mediate granulocytic differentiation and determined whether Epo-mediated neutrophil differentiation is associated with a pattern of myeloid gene expression comparable to that induced by GM-CSF. RESULTS: Studies of EpoR function in myeloid EPRO cells revealed that Epo/EpoR interaction can mediate terminal granulocytic differentiation of committed myeloid cells. In EPRO cells expressing EpoR, Epo-mediated neutrophil differentiation was associated with surface CD11b/CD18 (Mac-1) expression and induction of mRNA expression of specific myeloid genes including lactoferrin, gelatinase and C/EBPepsilon, in a manner similar to GM-CSF-mediated differentiation. CONCLUSIONS: These results indicate that Epo can deliver differentiative signals along a non-erythroid lineage, providing evidence for interchangeable cytokine receptor signals that mediate terminal differentiation of committed myeloid cells.

C/EBP epsilon mediates myeloid differentiation and is regulated by the CCAAT displacement protein (CDP/cut).

Neutrophils from CCAAT enhancer binding protein epsilon (C/EBP epsilon) knockout mice have morphological and biochemical features similar to those observed in patients with an extremely rare congenital disorder called neutrophil-specific secondary granule deficiency (SGD). SGD is characterized by frequent bacterial infections attributed, in part, to the lack of neutrophil secondary granule proteins (SGP). A mutation that results in loss of functional C/EBP epsilon activity has recently been described in an SGD patient, and has been postulated to be the cause of the disease in this patient. We have previously demonstrated that overexpression of CCAAT displacement protein (CDP/cut), a highly conserved transcriptional repressor of developmentally regulated genes, suppresses expression of SGP genes in 32Dcl3 cells. This phenotype resembles that observed in both C/EBP epsilon(-/-) mice and in SGD patients. Based on these observations we investigated potential interactions between C/EBP epsilon and CDP/cut during neutrophil maturation. In this study, we demonstrate that inducible expression of C/EBP epsilon in 32Dcl3/tet cells results in granulocytic differentiation. Furthermore, Northern blot analysis of G-CSF-induced CDP/cut overexpressing 32Dcl3 cells revealed absence of C/EBP epsilon mRNA. We therefore hypothesize that C/EBP epsilon positively regulates SGP gene expression, and that C/EBP epsilon is itself negatively regulated by CDP/cut during neutrophil maturation. We further demonstrate that the C/EBP epsilon promoter is regulated by CDP/cut during myeloid differentiation.

Genomic and proteomic analysis of the myeloid differentiation program.

Although the mature neutrophil is one of the better characterized mammalian cell types, the mechanisms of myeloid differentiation are incompletely understood at the molecular level. A mouse promyelocytic cell line (MPRO), derived from murine bone marrow cells and arrested developmentally by a dominant-negative retinoic acid receptor, morphologically differentiates to mature neutrophils in the presence of 10 microM retinoic acid. An extensive catalog was prepared of the gene expression changes that occur during morphologic maturation. To do this, 3'-end differential display, oligonucleotide chip array hybridization, and 2-dimensional protein electrophoresis were used. A large number of genes whose mRNA levels are modulated during differentiation of MPRO cells were identified. The results suggest the involvement of several transcription regulatory factors not previously implicated in this process, but they also emphasize the importance of events other than the production of new transcription factors. Furthermore, gene expression patterns were compared at the level of mRNA and protein, and the correlation between 2 parameters was studied. (Blood. 2001;98:513-524)

Rapid reconstitution of Epstein-Barr virus-specific T lymphocytes following allogeneic stem cell transplantation.

Epstein-Barr virus (EBV)-specific CD8 T lymphocytes are present at remarkably high frequencies in healthy EBV(+) individuals and provide protection from EBV-associated lymphoproliferative diseases. Allogeneic peripheral blood stem cell transplantation (allo-PBSCT) is a commonly used therapy in which T-cell surveillance for EBV is temporarily disrupted. Herein, human leukocyte antigen (HLA) class I tetramers were used to investigate the reestablishment of the EBV-specific CD8 T-cell repertoire in patients following allo-PBSCT. CD8(+) T cells specific for lytic and latent cycle-derived EBV peptides rapidly repopulate the periphery of matched sibling allo-PBSCT patients. The relative frequencies of T cells specific for different EBV peptides in transplantation recipients closely reflect those of their respective donors. Investigation of patients at monthly intervals following unmanipulated allo-PBSCT demonstrated that the frequency of EBV-specific T cells correlates with the number of EBV genome copies in the peripheral blood and that expansion of EBV-specific T-cell populations occurs even in the setting of immunosuppressive therapy. In contrast, patients undergoing T-cell-depleted or unrelated cord blood transplantation have undetectable EBV-specific T cells, even in the presence of Epstein-Barr viremia. The protective shield provided by EBV-specific CD8 T cells is rapidly established following unmanipulated matched sibling allo-PBSCT and demonstrates that HLA class I tetramers complexed with viral peptides can provide direct and rapid assessment of pathogen-specific immunity in this and other vulnerable patient populations. (Blood. 2000;96:2814-2821)

Sp1 and C/EBP are necessary to activate the lactoferrin gene promoter during myeloid differentiation.

In this study, we sought to identify factors responsible for the positive modulation of lactoferrin (LF), a neutrophil-specific, secondary-granule protein gene. Initial reporter gene transfection assays indicated that the first 89 base pairs of the LF promoter are capable of directing myeloid-specific LF gene expression. The presence of a C/EBP site flanked by 2 Sp1 sites within this segment of the LF promoter prompted us to investigate the possible role of these sites in LF expression. Cotransfection studies of LF-89luc plasmid with increasing concentrations of a C/EBPalpha expression vector in myeloid cells resulted in a linear transactivation of luciferase reporter activity. Electrophoretic mobility shift assays found that the C/EBP site is recognized by C/EBPalpha and that both LF Sp1 binding sites bind the Sp1 transcription factor specifically in myeloid cells. Mutation of either Sp1 site markedly reduced activity of the LF-89luc plasmid in myeloid cells, and neither Sp1 mutant plasmid was transactivated by a C/EBPalpha expression plasmid to the same extent as wild-type LF-89luc. We also transfected LF-89luc into Drosophila Schneider cells, which do not express endogenous Sp1, and demonstrated up-regulation of luciferase activity in response to a cotransfected Sp1 expression plasmid, as well as to a C/EBPalpha expression plasmid. Furthermore, cotransfection of LF-89luc plasmid simultaneously with C/EBPalpha and Sp1 expression plasmids resulted in an increase in luciferase activity greater than that induced by either factor alone. Taken together, these observations indicate a functional interaction between C/EBP and Sp1 in mediating LF expression. (Blood. 2000;95:3734-3741)

Modulation of a calcium/calmodulin-dependent protein kinase cascade by retinoic acid during neutrophil maturation.

Retinoic acid is a lipophilic derivative of vitamin A that can cause differentiation in a variety of cell types. A large body of evidence has shown that normal retinoid signaling is required for proper neutrophil maturation in vitro and in vivo. In this study, we have found that calcium/calmodulin dependent (CaM) protein kinase kinase alpha (CaMKKalpha) is upregulated in an immediate early fashion during retinoic acid induced neutrophil maturation. Furthermore, we describe the expression and modulation of various components of the CaM kinase cascade during neutrophil maturation. We have confirmed upregulation of CaMKKalpha protein by Western analysis and further show that CaMKKbeta is expressed, although its protein levels are constant throughout induction. We also find that neutrophil progenitor cells express both CaMKI and CaMKIV transcripts. RNase protection and Western analysis show that CaMKIV is downregulated during neutrophil maturation. In contrast, CaMKI transcript and protein is expressed in uninduced cells and is induced by all-trans retinoic acid. These data represent the first report of a CaM kinase cascade in myeloid cells and suggests that this cascade may mediate some of the well-characterized effects of calcium on neutrophil function. These observations also support the idea that the retinoic acid receptors play a major role in mediating neutrophil specific gene expression and differentiation.

Introduction: molecular diagnostics in hematology.

Advances in molecular biology of the last 30 tears have transformed the field of hematology. Molecular analysis has clarified the pathogenesis of a host of hematologic disorders, including hemoglobinopathies, coagulation disorders, hypercoaguable states, and hematologic malignancies. This volume is aimed at bringing these compelling advances in molecular biology research to the bedside. Through progress in the molecular understanding of hematology, great strides have been made in our ability to diagnose and treat patients with hematologic disease. These advances in turn have given rise to more questions that will guide future studies of the biology of hematopoietic abnormalities and continue the exciting interaction of basic science and clinical medicine.

Neutrophil maturation and the role of retinoic acid.

Neutrophil maturation occurs in well defined morphological stages that correlate with the acquisition of molecular markers associated with neutrophil function. A variety of factors are known to play a role in terminal neutrophil maturation, including the vitamin A derivative, retinoic acid. Retinoic acid can directly modulate gene expression via binding to its nuclear receptors, which can, in turn, activate transcription of target genes. A role for retinoic acid during neutrophil maturation has been suggested from a variety of sources. Here we present a review of the mechanism of retinoic acid receptor action and the major evidence showing that normal retinoid signaling is required for neutrophil maturation.

Normal neutrophil differentiation and secondary granule gene expression in the EML and MPRO cell lines.

The EML and MPRO cell lines express a dominant negative retinoic acid receptor alpha that causes a block at specific stages of myelopoiesis. The EML cell line is multipotent and gives rise to erythroid, lymphoid, and myeloid lineages depending on the presence of appropriate cytokines. The MPRO cell line is promyelocytic and undergoes neutrophilic differentiation when induced with all-trans retinoic acid in the presence of granulocyte/macrophage colony-stimulating factor. Previous studies have shown that both of these cell lines undergo morphological differentiation into neutrophils. In this study, we show that unlike other models of neutrophil differentiation such as NB4 and HL60, both EML and MPRO cell lines undergo complete, normal granulocytic differentiation programs. Similar to HL60, MPRO and EML induce expression of CD11b/CD18 and also exhibit downregulation of CD34 on differentiation. In contrast to HL60 and NB4, EML and MPRO cell lines coordinately upregulate secondary granule transcripts for lactoferrin and neutrophil gelatinase. Furthermore, we have confirmed previous observations that serum can induce a low level of differentiation in MPRO cells and that it is possible to grow these cells in serum-free medium, thereby eliminating this effect. Based on these studies, it appears that these lines can serve as a model for normal retinoic acid-induced neutrophil differentiation and provide insight into the role of the retinoic acid-responsive pathway in normal and leukemic myelopoiesis.

Representational difference analysis of a committed myeloid progenitor cell line reveals evidence for bilineage potential.

In this study we have sought to characterize a committed myeloid progenitor cell line in an attempt to isolate general factors that may promote differentiation. We used cDNA representational difference analysis (RDA), which allows analysis of differential gene expression, to compare EML and EPRO cells. We have isolated nine differentially expressed cDNA fragments as confirmed by slot blot, Northern, and PCR analysis. Three of nine sequences appear to be novel whereas the identity of the remaining fragments suggested that the EPRO cell line is multipotent. Among the isolated sequences were eosinophilic, monocytic, and neutrophilic specific genes. Therefore, we tested the ability of EPRO cells to differentiate along multiple myeloid lineages and found that EPRO cells exhibited morphologic maturation into either monocyte/macrophages or neutrophils, but not eosinophils. Furthermore, when EPRO cells were exposed to ATRA, neutrophil specific genes were induced, whereas monocytic markers were induced by phorbol ester treatment. This study highlights the use of cDNA RDA in conjunction with the EML/EPRO cell line to isolate markers associated with macrophage and neutrophil differentiation and establishes the usefulness of this system in the search for factors involved in myeloid commitment.

Isolation and characterization of the cDNA for mouse neutrophil collagenase: demonstration of shared negative regulatory pathways for neutrophil secondary granule protein gene expression.

A characteristic of normal neutrophil maturation is the induction of secondary granule protein (SGP) mRNA expression. Several leukemic human cell lines mimic normal morphologic neutrophil differentiation but fail to express SGPs, such as lactoferrin (LF) and neutrophil gelatinase (NG). In contrast, two murine cell lines (32D C13 and MPRO) are able to differentiate into neutrophils and induce expression of LF and NG. Therefore, to study the normal regulation and function of these genes, the corresponding murine homologs must be isolated. Using cDNA representational difference analysis (RDA) to compare a committed myeloid progenitor cell line (EPRO) with the multipotent stem cell line from which it was derived (EML), we isolated a fragment bearing homology to human neutrophil collagenase (hNC). Here, we describe the cloning and characterization of a full-length ( approximately 2 kb) clone that exhibits nearly 65% nucleotide and 73% amino acid identity to hNC. Ribonuclease protection analysis (RPA) of the tissues and cell lines shows that mouse NC (mNC) is expressed only in cell lines exhibiting neutrophilic characteristics, further confirming its identity as the mouse homolog of hNC. Furthermore, we have demonstrated a shared negative regulatory pathway for this and other SGP genes. We have previously shown that CCAAT displacement protein (CDP/cut) binds to a specific region of the LF promoter, and overexpression of CDP blocks G-CSF-induced upregulation of LF gene expression in 32D C13 cells. We show here that in these cells, upregulation of both NC and NG is also blocked. CDP is thus the first identified transcription factor that is a candidate for mediating the shared regulation of neutrophil SGP protein genes.

Molecular biology of neutrophil differentiation.

Myeloid precursors undergo striking morphologic and functional changes during the process of granulocytic maturation. These changes are associated with significant changes in cell size and nuclear shape, and with the development of stage-specific organelles which contain proteins necessary for the highly specialized roles of neutrophils in phagocytosis, in bacterial killing, and in mediating the inflammatory response. This complex process reflects a carefully regulated and sequential pattern of gene expression, which only recently has begun to be understood at the molecular level. The critical signals for the neutrophil differentiation program are postulated to derive from cytokines, and these cytokines are thought to induce a series of maturational events primarily by transcriptional regulation of sequentially expressed genes. Recent studies have identified a large number of transcriptional regulators, both positive and negative, that appear to act in concert in the developing neutrophil. These genes drive the complex and delicately timed sequence of genetic events that takes the cell from a progenitor to a functionally active neutrophil. Many of these genes are expressed throughout neutrophil differentiation, however, and the ongoing challenge is to elucidate how the function of these factors is modulated to allow the induction of sequential gene expression.

CCAAT displacement protein (CDP/cut) recognizes a silencer element within the lactoferrin gene promoter.

Expression of neutrophil secondary granule protein (SGP) genes is coordinately regulated at the transcriptional level, and is disrupted in specific granule deficiency and leukemia. We analyzed the regulation of SGP gene expression by luciferase reporter gene assays using the lactoferrin (LF) promoter. Reporter plasmids were transiently transfected into non-LF-expressing hematopoietic cell lines. Luciferase activity was detected from reporter plasmids containing base-pair (bp) -387 to bp -726 of the LF promoter, but not in a -916-bp plasmid. Transfection of a -916-bp plasmid into a LF-expressing cell line resulted in abrogation of the silencing effect. Sequence analysis of this region revealed three eight-bp repetitive elements, the deletion of which restored wild-type levels of luciferase activity to the -916-bp reporter plasmid. Electrophoretic mobility shift assay and UV cross-linking analysis identified a protein of approximately 180 kD that binds to this region in non-LF-expressing cells but not in LF-expressing cells. This protein was identified to be the CCAAT displacement protein (CDP/cut). CDP/cut has been shown to downregulate expression of gp91-phox, a gene expressed relatively early in the myeloid lineage. Our observations suggest that the binding of CDP/cut to the LF silencer element serves to suppress basal promoter activity of the LF gene in non-LF-expressing cells. Furthermore, overexpression of CDP/cut in cultured myeloid stem cells blocks LF expression upon granulocyte colony-stimulating factor-induced neutrophil maturation without blocking phenotypic maturation. This block in LF expression may be due, in part, to the persistence of CDP/cut binding to the LF silencer element.

Control of late neutrophil-specific gene expression: insights into regulation of myeloid differentiation.

During myeloid differentiation, the pluripotent hematopoietic stem cell passes through several well-defined morphologic stages within the bone marrow. These changes include progressive nuclear segmentation and the acquisition of stage-specific granules. Primary granules appear at the myeloblast stage, and are found in both neutrophils and monocytes. At the myelocyte stage, neutrophil precursors acquire specific granules, a marker of commitment to terminal neutrophil differentiation. This complex developmental pathway is just beginning to be elucidated. Current evidence suggests that myeloid differentiation is regulated primarily by transcriptional regulatory proteins, and that dysfunction of those regulators is involved in most disorders of neutrophil maturation. Furthermore, there is evidence that study of late gene expression may provide insights into more proximal events in granulocytic maturation. In this review, we provide a brief overview of myeloid differentiation with emphasis on the culture systems available for the study of granulopoiesis and the insights they provide into the regulation of late neutrophil-specific gene expression. We discuss the relevance of these observations to our understanding of the pathogenesis of defects in neutrophil differentiation.

Laminin promotes differentiation of NB4 promyelocytic leukemia cells with all-trans retinoic acid.

The promyelocytic leukemia cell line, NB4, carries the t(15; 17) translocation and undergoes limited maturation in response to differentiation agents. Growth on laminin enhanced the ability of all-trans retinoic acid (ATRA) to promote morphologic maturation of these cells. Although exposure to ATRA in suspension yielded minimal maturation beyond the myelocyte stage, after 72 hours of exposure to ATRA on laminin the cells acquired the histologic appearance of metamyelocytes, band forms, and segmented neutrophils. After 96 hours, some cells acquired a spindle shape and became tightly adherent. Growth on collagen types I, III, IV, or fibronectin did not have this effect, although some cells did adhere to fibronectin. NB4 cells treated with ATRA in suspension or on laminin acquired the equivalent ability to reduce nitroblue tetrazolium or cytochrome C. Despite the improved morphologic maturation on laminin, the cells did not express secondary granule proteins such as lactoferrin or neutrophil collagenase. In addition, growth on laminin abolished cell proliferation in the presence of ionomycin. Growth on laminin and/or with ATRA induced new expression of alpha 6 integrin, a laminin receptor, as assessed by reverse transcription-polymerase chain reaction. Different conditions of growth (laminin or differentiation agent) resulted in specific patterns of expression of the alpha 6A and alpha 6B isoforms. Treatment with ATRA also resulted in the acquisition of high-level surface expression of alpha 6 integrin, as assessed by flow cytometry. Thus, treatment of NB4 promyelocytic leukemia cells with ATRA induced expression of alpha 6 integrin (a laminin receptor alpha-chain) and enabled more advanced maturation when the cells were grown on the extracellular matrix component, laminin, compared with tissue culture plastic.

Retroviral insertional activation of the EVI1 oncogene does not prevent G-CSF-induced maturation of the murine pluripotent myeloid cell line 32Dcl3.

Evi1 is a myeloid-specific protooncogene that encodes 145 kDa and 88 kDa proteins via alternative splicing. Overexpression of the gene via retroviral insertion in murine tumors or chromosomal rearrangement in human tumors is associated with myeloid leukemias and myelodysplasias; however, the mechanism by which such overexpression leads to transformation is not clear. It has been postulated that overexpression of evi1 acts to block normal myelopoiesis. In attempts to assess the effect of overexpression of evi1 on myelopoiesis, we chose to utilize the IL-3-dependent murine 32Dcl3 cell line, which has been shown to differentiate in culture in response to G-CSF. Previous experiments with this cell line, which we have confirmed, showed that overexpression of evi1, mediated by retroviral vector transfer, caused a block to G-CSF-induced cell survival and differentiation. We report here that the naive 32Dcl3 cell line contains a rearrangement of the evi1 locus and constitutively overexpresses evi1 mRNA and protein; this expression is downregulated only slightly during G-CSF-induced myeloid maturation. The steady state levels, molecular weight and DNA binding characteristics of the EVI1 protein in these cells is comparable to that seen in NFS 58, a myeloid leukemia cell line with retroviral insertion at evi1. The observed ability of the murine 32Dcl3 cells to fully differentiate in the presence of G-CSF while evi1 continues to be expressed indicates that, at the levels expressed in naive 32Dcl3, evi1 does not block G-CSF-induced survival and differentiation. Thus, retroviral insertions at evi1 may have been selected for in 32Dcl3 cells due to effects other than that on G-CSF-induced cell survival.