Regulation of human eosinophil viability, density, and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fibroblasts.

Normodense human peripheral blood eosinophils were isolated under sterile conditions from the 22/23 and 23/24% interfaces and the cell pellet of metrizamide gradients. After culture for 7 d in RPMI media in the presence of 50 pM biosynthetic (recombinant) human granulocyte/macrophage colony-stimulating factor (rH GM-CSF), 43 +/- 7% (mean +/- SEM, n = 8) of the cells were viable; in the absence of rH GM-CSF, no eosinophils survived. The rH GM-CSF-mediated viability was concentration dependent; increased survival began at a concentration of 1 pM, a 50% maximal response was attained at approximately 3 pM, and a maximal effect was reached at concentrations of greater than or equal to 10 pM rH GM-CSF. In the presence of rH GM-CSF and mouse 3T3 fibroblasts, 67 +/- 6% (mean +/- SEM, n = 8) of the eosinophils survived for 7 d. In a comparative analysis, there was no difference in eosinophil viability after 7 and 14 d (n = 3) in the presence of 50 pM GM-CSF and fibroblasts. Culture with fibroblasts alone did not support eosinophil survival. The addition of fibroblast-conditioned media to rH GM-CSF did not further improve eosinophil viability, indicating a primary role for GM-CSF in supporting these eosinophil cell suspensions ex vivo and a supplementary role for 3T3 fibroblasts. Eosinophils cultured for 7 d localized on density gradient sedimentation at the medium/18, 18/20, and 20/21 interfaces of metrizamide gradients, indicating a change to the hypodense phenotype from their original normodense condition. In addition, the cultured eosinophils generated approximately 2.5-fold more LTC4 than freshly isolated cells when stimulated with the calcium ionophore A23187 and manifested sevenfold greater antibody-dependent killing of S. mansoni larvae than the freshly isolated, normodense cells from the same donor. Thus we demonstrate the rH GM-CSF dependent conversion in vitro of normodense human eosinophils to hypodense cells possessing the augmented biochemical and biological properties characteristic of the hypodense eosinophils associated with a variety of hypereosinophilic syndromes. In addition, these studies provide a culture model of at least 14 d suitable for the further characterization of hypodense eosinophils.

Genetic analysis of the human tumor necrosis factor alpha/cachectin promoter region in a macrophage cell line.

The 615-bp 5' flanking region of the human TNF-alpha/cachectin gene was isolated and ligated to the luciferase reporter gene. In addition, a series of truncated promoter constructs was generated by exonuclease III digestion. The promoter activity of these constructs was studied in a transient transfection system using the TNF-alpha-producing U937 cell line. Full-length and truncated TNF promoter constructions extending from -615 to -95 bp relative to the transcription start site (TSS) could be induced by phorbol esters. A construct truncated to within 36 bp of the TSS (and within 11 bp of the TATAA box) was inactive. Therefore, the phorbol ester responsive is localized in the TNF/cachectin promoter to a relatively short region proximal to the TATAA box.

A new determinant of glucocorticoid sensitivity in lymphoid cell lines.

The SAK cell line, derived from a spontaneous thymic lymphoma in an AKR mouse, is resistant to lysis by glucocorticoids in spite of the presence of functional glucocorticoid receptor. Receptor function was determined by hormone binding analyses, as well as characterization of hormonal effects on cell growth and on the accumulation of murine leukemia virus and metallothionein mRNAs. SAK cells were fused with a receptor-defective (and therefore resistant) variant of a well-characterized murine thymoma line, W7. The resulting hybrids are glucocorticoid sensitive, demonstrating complementation of the receptor defect in W7 cells by the functional glucocorticoid receptor of SAK. This fusion shows that SAK cells are resistant to the hormone due to the absence of another function designated "I" for lysis. SAK cells were also fused with glucocorticoid-sensitive W7 cells (containing wild-type receptor), generating glucocorticoid-sensitive hybrids, which demonstrate that the dexamethasone-resistant phenotype of the SAK cells is recessive. Resistant derivatives of this hybrid were found which still contain the full amount of receptor. Chromosome analysis revealed that, on the average, the resistant derivatives had lost two chromosomes, suggesting segregation of chromosomes carrying genetic material necessary for the "lysis" function. The drug 5-azacytidine (a known inhibitor of DNA methylation) has been shown to cause heritable changes in gene expression. Treatment of SAK cells with 5-azacytidine generated glucocorticoid-sensitive clones at high frequency, suggesting that the gene(s) involved in the "lysis" function are intact and have been inactivated through a process such as differentiation.

Cytokines alter production of HIV-1 from primary mononuclear phagocytes.

Some strains of human immunodeficiency virus type 1 (HIV-1) can infect primary monocytes and monocyte-derived macrophages in vitro. In this report, the effect of cytokines on the production of one of these strains that shows a tropism for mononuclear phagocytes, designated HIV-1JR-FL, was studied. Primary peripheral blood mononuclear phagocytes infected with HIV-1JR-FL were treated with the hematopoietic factors: granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), macrophage colony-stimulating factor (M-CSF), and gamma-interferon (gamma-IFN). The M-CSF, GM-CSF, IL-3, and gamma-IFN were able to alter HIV-1 production under different conditions.

The human gene encoding GM-CSF is at 5q21-q32, the chromosome region deleted in the 5q- anomaly.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 22,000-dalton glycoprotein that stimulates the growth of myeloid progenitor cells and acts directly on mature neutrophils. A full-length complementary DNA clone encoding human GM-CSF was used as a probe to screen a human genomic library and isolate the gene encoding human GM-CSF. The human GM-CSF gene is approximately 2.5 kilobase pairs in length with at least three intervening sequences. The GM-CSF gene was localized by somatic cell hybrid analysis and in situ hybridization to human chromosome region 5q21-5q32, which is involved in interstitial deletions in the 5q- syndrome and acute myelogenous leukemia. An established, human promyelocytic leukemia cell line, HL60, contains a rearranged, partially deleted GM-CSF allele and a candidate 5q- marker chromosome, indicating that the truncated GM-CSF allele may reside at the rejoining point for the interstitial deletion on the HL60 marker chromosome.

Evidence for the involvement of GM-CSF and FMS in the deletion (5q) in myeloid disorders.

By in situ chromosomal hybridization, the GM-CSF and FMS genes were localized to human chromosome 5 at bands q23 to q31, and at band 5q33, respectively. These genes encode proteins involved in the regulation of hematopoiesis, and are located within a chromosome region frequently deleted in patients with neoplastic myeloid disorders. Both genes were deleted in the 5q-chromosome from bone marrow cells of two patients with refractory anemia and a del(5)(q15q33.3). The GM-CSF gene alone was deleted in a third patient with acute nonlymphocytic leukemia (ANLL) who has a smaller deletion, del(5)(q22q33.1). Leukemia cells from a fourth patient who has ANLL and does not have a del(5q), but who has a rearranged chromosome 5 that is missing bands q31.3 to q33.1 [ins(21;5)(q22;q31.3q33.1)] were used to sublocalize these genes; both genes were present on the rearranged chromosome 5. Thus, the deletion of one or both of these genes may be important in the pathogenesis of myelodysplastic syndromes or of ANLL.

Purified human granulocyte-macrophage colony-stimulating factor: direct action on neutrophils.

Neutrophil migration inhibition factor from T lymphocytes (NIF-T) is a lymphokine that acts to localize granulocytes. Medium conditioned by the Mo human T-lymphoblast cell line was used to purify NIF-T, a glycoprotein with a molecular weight of 22,000. The NIF-T was found to potently stimulate the growth of granulocyte and macrophage colonies from human bone marrow and colony formation by the KG-1 myeloid leukemia cell line. Thus a human lymphokine (NIF-T) that modulates the activities of mature neutrophilic granulocytes is also a colony-stimulating factor acting on precursors to induce growth and differentiation of new effector cells.

Studies of the putative transforming protein of the type I human T-cell leukemia virus.

The putative transforming protein of the type I human T-cell leukemia virus (HTLV-1) is a 40-kilodalton protein encoded by the X region and is termed p40XI. On the basis of both subcellular fractionation techniques and immunocytochemical analysis, it is now shown that p40XI is a nuclear protein with a relatively short half-life (120 minutes). It is synthesized de novo in considerable quantities in a human T-cell line infected with and transformed by the virus in vitro, and it is not packaged in detectable amounts in the extracellular virus.

Granulocyte-macrophage colony-stimulating factor and interleukin-3 signaling pathways converge on the CREB-binding site in the human egr-1 promoter.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates myeloid progenitor cell proliferation and enhances the function of terminally differentiated effector cells. Interleukin-3 (IL-3) stimulation results in the proliferation and maturation of early bone marrow progenitor cells. These activities are mediated by non-tyrosine kinase-containing receptors which consist of ligand-specific alpha subunits that complex with a common beta subunit required for signal transduction. Both GM-CSF and IL-3 rapidly and transiently induce expression of early growth response gene 1 (egr-1) in the human factor-dependent cell line TF-1. To define the mechanism of early response gene induction by GM-CSF and IL-3, growth factor- and serum-starved TF-1 cells transfected with recombinant constructs containing sequences of the human egr-1 promoter were stimulated with GM-CSF or IL-3. A 116-nucleotide (nt) region of the egr-1 promoter which contains sequences inducible by GM-CSF and IL-3 was defined. DNase I footprint analysis identified a 20-nt region, including nt -57 to -76, which contains a potential cyclic AMP (cAMP) response element (CRE). Electrophoretic mobility shift assays performed with CREB antibody confirmed the presence of CREB in the DNA-binding complex. Mutational analysis of the cytokine-responsive region of the egr-1 promoter revealed that both the cAMP response and serum response elements are required for induction by GM-CSF and IL-3. Nuclear extracts from GM-CSF- or IL-3-stimulated but not unstimulated TF-1 cells contain factors which specifically bind to the Egr-1-binding site in the nt -600 to -480 region of the promoter. Electrophoretic mobility shift assays were performed with antibodies against the Egr-1 protein to demonstrate the presence of the protein product in the shifted complex. Our studies suggest that the Egr-1 protein may further stimulate transcription of the egr-1 gene in response to GM-CSF as a secondary event.

Characterization of a cell-type-restricted negative regulatory activity of the human granulocyte-macrophage colony-stimulating factor gene.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of normal myeloid progenitor cells and can also stimulate the growth of acute myelogenous leukemia (AML) blasts. GM-CSF is not normally produced by resting cells but is expressed by a variety of activated cells including T lymphocytes, macrophages, and certain cytokine-stimulated fibroblasts and endothelial cells. Production of GM-CSF by cultured AML cells has been demonstrated, and GM-CSF expression by normal myeloid progenitors has been postulated to play a role in myelopoiesis. We have investigated the regulation of expression of GM-CSF in AML cell lines, and our results demonstrate the presence of a strong constitutive promoter element contained within 53 bp upstream of the cap site. We have also identified a negative regulatory element located immediately upstream of the positive regulatory element (within 69 bp of the cap site) that is active in AML cell lines but not T cells or K562 CML cells. Competition transfection and mobility shift studies demonstrate that this activity correlates with binding of a 45-kDa protein.

Characterization of the human granulocyte-macrophage colony-stimulating factor promoter region by genetic analysis: correlation with DNase I footprinting.

T-cell activation induces expression of the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). To define the molecular events involved in the induction of GM-CSF gene expression more clearly, we prepared and analyzed deletion mutants of GM-CSF promoter recombinant constructs. The results localized inducible expression to a 90-base-pair region (-53 to +37) which is active in uninfected and human T-cell leukemia virus-infected T-cell lines but not in resting or mitogen-stimulated B cells. DNase I footprinting experiments revealed protection of sequences contained within this region, including a repeated nucleotide sequence, CATT(A/T), which could serve as a core recognition sequence for a cellular transcription factor. Upstream of these GM-CSF promoter sequences is a 15-base-pair region (-193 to -179) which has negative regulatory activity in human T-cell leukemia virus-infected T cells. These studies revealed a complex pattern of regulation of GM-CSF expression in T cells; positive and negative regulatory sequences may play critical roles in controlling the expression of this potent granulopoietin in the bone marrow microenvironment and in localized inflammatory responses.

Granulocyte-macrophage colony-stimulating factor and tetradecanoyl phorbol acetate induce a distinct, restricted subset of primary-response TIS genes in both proliferating and terminally differentiated myeloid cells.

Induction of early-response genes (tetradecanoyl phorbol acetate [TPA]-induced sequences, or TIS genes; R.W. Lim, B.C. Varnum, and H.R. Herschman, Oncogene 1:263-270, 1987) by granulocyte-macrophage colony-stimulating factor (GM-CSF) and TPA was examined both in a factor-dependent murine cell line, 32D clone 3, and in mature human neutrophils. When GM-CSF-deprived 32D clone 3 cells were exposed to GM-CSF or to TPA, four TIS mRNAs (TIS7, TIS8, TIS10, and TIS11) were rapidly and transiently induced. However, neither GM-CSF nor TPA could induce accumulation of TIS1 mRNA in 32D clone 3 cells, even under superinducing conditions. Both GM-CSF and TPA also elicited rapid, transient expression of TIS8 and TIS11 mRNA in postmitotic human neutrophils. However, neither agent could induce accumulation of TIS1 mRNA in human neutrophils. TIS1 is a member of the nuclear receptor supergene family that codes for ligand-dependent transcription factors. Cell-type restriction of inducible transcription factors may contribute to developmental specification.

Induction of glucocorticoid-resistant variants in a murine thymoma line by antitumor drugs.

Several antitumor drugs are shown to be mutagenic in murine thymoma lines: mitomycin C, bleomycin, streptonigrin, Colcemid, and BD40, an analog of ellipticine. Using conditions yielding 3 to 40% cell survival, all five drugs tested increase the frequency of glucocorticoid-resistant variants. Mitomycin C is as efficient as the classical alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine and ethyl methanesulfonate. The other drugs, previously untested for mutagenic activity on mammalian cells, are weak mutagens yielding variants at frequencies 1 to 2 orders of magnitude lower than the alkylating agents. All 152 variants obtained result from defects in the glucocorticoid receptor. Variants induced by mitomycin C, streptonigrin, Colcemid, and BD40 have very reduced receptor activity, as measured by dexamethasone binding. In contrast, bleomycin or the combination of mitomycin C and dexamethasone induce a majority of variants having dexamethasone-binding activity comparable to the parental line. However, assays of nuclear transfer capacity and genetic complementation show that these receptors are nonfunctional and may result from point mutations in the gene encoding the glucocorticoid receptor. This study suggests that, in combination therapies, antitumor drugs might induce glucocorticoid-resistant lymphoid cell variants that could be selected by the hormone.

Differential gene expression between normal and tumor-derived ovarian epithelial cells.

The majority of ovarian tumors arise from the transformation of the ovarian surface epithelial cells, a single layer of cells surrounding the ovary. To identify genes that may contribute to the malignant phenotype of ovarian cancers, cDNA representational difference analysis was used to compare expressed genes in primary cultures of normal human ovarian surface epithelium (HOSE) and ovarian tumor-derived epithelial cells from the Cedars-Sinai Ovarian Cancer (CSOC) repository. A total of 255 differentially expressed genes were identified, of which 160 and 95 were specifically expressed in HOSE and CSOC cells, respectively. Using cDNA array hybridization, the expression profiles of the genes identified by cDNA-representational difference analysis were examined in an additional 5 HOSE and 10 CSOC lines. The comparison of average signal of each gene revealed 44 HOSE-specific and 16 CSOC-specific genes that exhibited at least a 2.5-fold difference in expression. A large number of genes identified in this study encode membrane-associated or secreted proteins and, hence, may be useful as targets in the development of serum-based diagnostic markers for ovarian cancer. Very few genes associated with protein synthesis or metabolism were identified in this study, reflecting the lack of observable differences in phenotypic or growth characteristics between HOSE and CSOC cells. Northern blot analysis on a subset of these genes demonstrated comparable levels of gene expression as observed in the cDNA array hybridization.

HTLV-I and HTLV-II tax trans-activate the human EGR-1 promoter through different cis-acting sequences.

The immediate-early response gene, EGR-1, encodes a zinc finger-containing transcription factor that is involved in growth and differentiation of a variety of cell types. EGR-1 is induced in normal T cells following mitogenic stimulation and has recently been shown to be constitutively expressed in human T-cell leukemia virus type I (HTLV-I)- and type II (HTLV-II)-transformed T-cell lines. The trans-activating protein of HTLV-I, Tax, has been demonstrated to trans-activate promoters of a number of cellular genes, some of which may be critical in regulating T-cell proliferation. In this study, we examine the effect of Tax on expression of EGR-1 in three T-cell lines and demonstrate that both HTLV-I and -II Tax are capable of trans-activating human EGR-1 recombinant promoter constructs. Interestingly, HTLV-I and -II Tax trans-activate the human EGR-1 promoter through different promoter regions in the Jurkat cell line, suggesting that HTLV-I and -II Tax may lead to constitutive expression of EGR-1 through different signaling pathways. Deregulated expression of EGR-1 may contribute to uncontrolled cell growth and transformation during early stages of T-cell activation in HTLV-I and -II-infected cells.

Human c-FES is a nuclear tyrosine kinase.

FES is a non-receptor protein tyrosine kinase expressed in hematopoietic progenitors and differentiated myeloid cells. It has recently been implicated in granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and erythropoietin signal transduction. To better understand the role played by FES in normal and neoplastic hematopoiesis, we used cell fractionation techniques to examine the subcellular localization of FES in myeloid cells and cell lines. FES was observed in the nuclear, granular and plasma membrane fractions of primary human neutrophils and the myeloid leukemia cell line, HL-60. The nuclear localization was confirmed by immunocytochemistry of neutrophils.

5' upstream sequence and genomic structure of the human primary response gene, EGR-1/TIS8.

EGR-1/TIS8 is a primary response gene that encodes a zinc finger containing protein and is induced in a number of cell types by a variety of ligands. We have isolated and mapped the human EGR-1/TIS8 gene and sequenced the 5' upstream flanking region. A 'TATA' homology and several putative regulatory elements, including two Sp1 sites, five serum response-like elements, two cAMP response-like elements, an EGR-1 binding site (EBS), and a tetra-decanoyl phorbol acetate (TPA)-responsive element have been identified within 700 nucleotides of the upstream region. We demonstrated that a 500-base pair fragment, which includes several of these possible regulatory sequences, is functional and responsive to TPA in transient transfection assays. A further understanding of the regulation and function of human EGR-1/TIS8 gene expression may provide insight into the mechanisms that control normal and neoplastic proliferation of human cells.

Activation of the GM-CSF promoter by HTLV-I and -II tax proteins.

Production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by normal T lymphocytes requires activation by antigen, mitogen or lectin, whereas T-cell lines transformed by human T-cell leukemia virus type I (HTLV-I) or type II (HTLV-II) constitutively produce high levels of GM-CSF. Using transient cotransfection assays, we demonstrate that introduction of the tax gene of either HTLV-I or HTLV-II is sufficient to activate GM-CSF promoter constructs in an unstimulated T-cell line. The GM-CSF 5' flanking sequences previously shown to be sufficient for GM-CSF induction following T-cell activation are also sufficient for activation by the HTLV tax proteins. The sequences required for trans-activation of GM-CSF are distinct from those required for the activation of other T-cell-inducible genes (IL-2R alpha, IL-2) by tax, suggesting that tax can have pleiotropic effects on gene expression in T cells. Constitutive GM-CSF production by HTLV-infected T cells may therefore be due to trans-activation of its promoter by tax. Expression of GM-CSF by HTLV-I infected lymphocytes may be important in the granulocytosis and eosinophilia frequently seen in patients with HTLV-I-induced adult T-cell leukemia/lymphoma.

Transactivation of cellular genes by human retroviruses.

We have focused this chapter on interactions with two of the best characterized transregulatory genes, tax for HTLV-I/II and Tat for HIV-1. Both genes illustrate the complex interplay between retroviral regulatory genes and cellular gene regulation. In both instances a viral gene of relatively straightforward function in the viral context appears to cause extensive dysregulation of cellular genes, either directly or as a consequence of altered cellular differentiation. Understanding this viral/cellular gene cross-talk may elucidate mechanisms leading to malignant transformation autoimmune disease and to neurologic and paraneoplastic complications such as hypercalcemia for HTLV-I/II, as well as the pathogenesis of immune dysfunction and opportunistic malignancy in HIV-I/II-infected individuals. An understanding of functional mechanisms of these transregulatory viral genes will undoubtedly afford better explanations for the myriad manifestations of retroviral infection.

Relationship between human T cell leukemia virus-II and atypical hairy cell leukemia: a serologic study of hairy cell leukemia patients.

Human T cell leukemia virus (HTLV-II) is an infrequently encountered human T cell leukemia virus first isolated from a patient with atypical hairy cell leukemia. Recently, we identified a second patient infected with HTLV-II who had a similar clinical syndrome of atypical hairy cell leukemia associated with peripheral T cell lymphocytosis. HTLV-II was detected by molecular hybridization studies, and more recently, by electron microscopy, in cell lines derived from the patient. Both patients came from the Los Angeles area and had spent several years in Alaska. As opposed to our two patients, 21 patients with more typical cases of hairy cell leukemia were seronegative for HTLV-II. Two additional cases of unusual T cell malignancy linked to HTLV-II have been described by other investigators and bear limited similarity to our index cases. Further studies are necessary to define the spectrum of malignancies linked to HTLV-II and to identify infected individuals for prospective study.