Retinoic Acid-differentiated HL-60 Cells Research Articles

Immunomodulatory effects of testosterone evaluated in all-trans retinoic acid differentiated HL-60 cells, granulocytes, and monocytes

The sex hormones are known to affect innate immunity in humans. In this study we evaluated the immunomodulatory effects of testosterone in a model system comprising of all-trans retinoic acid differentiated HL-60 cells, and confirmed the results in human granulocytes and monocytes. Results showed that testosterone at pharmacological doses reduced the production of interleukin-8 and reactive oxygen species from differentiated HL-60 cells in a concentration dependent manner without affecting phagocytosis. The cells were stimulated with zymosan, lipopolysaccharide, or Bacillus subtilis. At the highest concentration of testosterone (120 μM), interleukin-8 secretion was reduced 42-80%, and production of reactive oxygen species was reduced 32-46%. Flutamide, an antagonist of the classical intracellular androgen receptor, was unable to antagonize the immunosuppressive effect of testosterone. We further demonstrated that the suppressive effect of testosterone has a short onset time. Our results suggest that testosterone affects the fast operating membrane bound androgen receptor or a rapid acting enzyme system. Testosterone, at pharmacological doses, was also shown to suppress generation of reactive oxygen species and interleukin-8 in human granulocytes and monocytes, respectively, to a similar extent as observed in differentiated HL-60 cells.

Phagocytosis of Streptococcus pyogenes by All-Trans Retinoic Acid-Differentiated HL-60 Cells: Roles of Azurophilic Granules and NADPH Oxidase

BackgroundNew experimental approaches to the study of the neutrophil phagosome and bacterial killing prompted a reassessment of the usefulness of all-trans retinoic acid (ATRA)-differentiated HL-60 cells as a neutrophil model. HL-60 cells are special in that they possess azurophilic granules while lacking the specific granules with their associated oxidase components. The resulting inability to mount an effective intracellular respiratory burst makes these cells more dependent on other mechanisms when killing internalized bacteria.Methodology/Principal FindingsIn this work phagocytosis and phagosome-related responses of ATRA-differentiated HL-60 cells were compared to those earlier described in human neutrophils. We show that intracellular survival of wild-type S. pyogenes bacteria in HL-60 cells is accompanied by inhibition of azurophilic granule–phagosome fusion. A mutant S. pyogenes bacterium, deficient in M-protein expression, is, on the other hand, rapidly killed in phagosomes that avidly fuse with azurophilic granules.Conclusions/SignificanceThe current data extend our previous findings by showing that a system lacking in oxidase involvement also indicates a link between inhibition of azurophilic granule fusion and the intraphagosomal fate of S. pyogenes bacteria. We propose that differentiated HL-60 cells can be a useful tool to study certain aspects of neutrophil phagosome maturation, such as azurophilic granule fusion.

Interleukin-1β mediates LPS-induced inhibition of apoptosis in retinoic acid-differentiated HL-60 cells

Promyelocytic HL-60 cells differentiated to a neutrophilic phenotype by incubation with all-trans retinoic acid become constitutively apoptotic. Exposure to either LPS or IL-1β inhibited the apoptosis of differentiated HL-60 cells. LPS induced the expression of pro-IL-1β message, upregulated the activity of the interleukin-1β converting enzyme (caspase-1), and increased the release of IL-1β into the culture medium. Prevention of IL-1β translation with an anti-sense oligonucleotide, or prevention of IL-1β cellular binding with a blocking antibody, accelerated rates of spontaneous apoptosis, and abrogated the inhibitory effects of LPS. However inhibition of caspase-1 activity further inhibited constitutive apoptosis of mature HL-60 cells. These studies provide further evidence of a complex regulatory pathway that modulates the expression of granulocyte apoptosis during inflammation, and point to a specific role for IL-1β as an autocrine survival factor.

Modulation by cADPr of Ca 2+ mobilization and oxidative response in dimethylsulfoxide- or retinoic acid-differentiated HL-60 cells

In human phagocytic cells, reactive oxygen species (ROS) generation in response to N-formyl- l-Methionyl- l-Leucyl- l-Phenylalanine (fMLF) is largely dependent on cytosolic free calcium concentration ([Ca 2+] i). Cyclic ADP-ribose (cADPr) is able to regulate Ca 2+ release from intracellular stores through the ryanodine receptor but its potential role in biological responses has so far not been determined. In this study, we examined whether extracellular and intracellular cADPr is required in fMLF-induced [Ca 2+] i rise and consequently in the oxidative response in human neutrophil-like HL-60 cells differentiated with dimethylsulfoxide or all-trans-retinoic acid (ATRA). We establish that extracellular cADPr cannot elicit [Ca 2+] i elevation. Furthermore, we demonstrate that 8-Br-cADPr, a functional antagonist of cADPr, inhibits Ca 2+ entry into HL-60 cells differentiated with ATRA and stimulated with fMLF (95 ± 4 and 148 ± 5 nM respectively, n = 3). Finally, we show that this partial inhibition of Ca 2+ mobilization is unrelated to ROS production (10.0 ± 0.3 vs. 9.6 ± 0.5 A.U., n = 3). In conclusion, we showed that cADPr can control fMLF-induced Ca 2+ influx but is unable to regulate a Ca 2+-dependent biological response, i.e. H 2O 2 production.

Repression ofrac2mRNA Expression byAnaplasma phagocytophilaIs Essential to the Inhibition of Superoxide Production and Bacterial Proliferation

Anaplasma phagocytophila, the etiologic agent of human granulocytic ehrlichiosis, is an emerging bacterial pathogen that invades neutrophils and can be cultivated in HL-60 cells. Infected neutrophils and HL-60 cells fail to produce superoxide anion (O(2)(-)), which is partially attributable to the fact that A. phagocytophila inhibits transcription of gp91(phox), an integral component of NADPH oxidase. cDNA microarray and RT-PCR analyses demonstrated that transcription of the gene encoding Rac2, a key component in NADPH oxidase activation, was down-regulated in infected HL-60 cells. Quantitative RT-PCR demonstrated that rac2 mRNA expression was reduced 7-fold in retinoic acid-differentiated HL-60 cells and 50-fold in neutrophils following A. phagocytophila infection. Rac2 protein expression was absent in infected HL-60 cells. Rac1 and Rac2 are interchangeable in their abilities to activate NADPH oxidase. HL-60 cells transfected to express myc-tagged rac1 and gp91(phox) from the CMV immediate early promoter maintained the ability to generate O(2)(-) 120 h postinfection. A. phagocytophila proliferation was severely inhibited in these cells. These results directly attribute the inhibition of rac2 and gp91(phox) transcription to the loss of NADPH oxidase activity in A. phagocytophila-infected cells and demonstrate its importance to bacterial intracellular survival.

Association of FcgammaRII with low-density detergent-resistant membranes is important for cross-linking-dependent initiation of the tyrosine phosphorylation pathway and superoxide generation.

IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcgammaRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcgammaR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcgammaRs resulted in a marked increase in the tyrosine phosphorylation of FcgammaRIIa, p58(lyn), and p120(c-cbl), which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcgammaRs and tyrosine-phosphorylated proteins including p120(c-cbl) were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcgammaRs as well as p120(c-cbl) with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-beta-cyclodextrin, the cross-linking did not induce the association of FcgammaRs as well as p120(c-cbl) with DRMs. In addition, although the physical association between FcgammaRIIa and p58(lyn) was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-beta-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcgammaRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcgammaRII to lipid rafts is important for the activation of FcgammaRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.

Molecular Cloning and Characterization of Another Leukotriene B4 Receptor

Molecular Cloning and Characterization of Another Leukotriene B4 Receptor

ヒト好中球ＭＡＰキナーゼサブタイプのサイトカイン（Ｇ‐ＣＳＦ，ＧＭ‐ＣＳＦ，ＴＮＦ）特異的活性化と活性酸素産生

In order to clarify the differences of the signaling pathways utilized by G-CSF, GM-CSF and TNF, we investigated activation of MAPK subtype cascades in human neutrophils stimulated by these cytokines. G-CSF exclusively activates the MEK-ERK cascade. GM-CSF activates the MEK-ERK cascade strongly and the MKK 3/ 6-p 38 MAPK cascade weakly, whereas TNF activates the MKK 3/6-p 38 MAPK cascade strongly and the MEK-ERK cascade weakly. The potency of these cytokines to activate the MEK-ERK cascade was GM-CSF>G-CSF>TNF, whereas that to activate the MKK 3/6-p 38 MAPK cascade was TNF>GM-CSF. JNK was not tyrosine-phosphorylated by any cytokine in spite of the existence of JNK proteins in human neutrophils, whereas it was tyrosine-phosphorylated by TNF in undifferentiated and all-trans retinoic acid-differentiated HL-60 cells. Increased phosphorylation of ERK or p 38 MAPK was detected within 1-5 minutes after stimulation with each cytokine and was dependent on the concentrations of cytokines used. GM-CSF-or TNF-induced superoxide (O2-) release was inhibited by p 38 MAPK inhibitor (SB203580) in a dose-dependent manner. The results indicate that G-CSF, GM-CSF and TNF activate the overlapping but distinct MAPK subtype cascades in human neutrophils and p 38 MAPK may be involved in GM -CSF-or TNF-induced O2- release.

Cytokine-Specific Activation of Distinct Mitogen-Activated Protein Kinase Subtype Cascades in Human Neutrophils Stimulated by Granulocyte Colony-Stimulating Factor, Granulocyte-Macrophage Colony-Stimulating Factor, and Tumor Necrosis Factor-

To clarify the differences of the signaling pathways used by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor- (TNF), we investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by these cytokines. G-CSF exclusively tyrosine-phosphorylated extracellular signal-regulated kinase (ERK). GM-CSF tyrosine-phosphorylated ERK strongly and p38 MAPK weakly, whereas TNF tyrosine-phosphorylated p38 MAPK strongly and ERK weakly. Consistent with these findings, MEK, an upstream kinase of ERK, was phosphorylated by G-CSF, GM-CSF, and TNF, whereas MKK3/MKK6, an upstream kinase of p38 MAPK, was phosphorylated by GM-CSF and TNF, but not by G-CSF. The potency of these cytokines to phosphorylate ERK and MEK was GM-CSF > G-CSF > TNF, whereas that to phosphorylate p38 MAPK and MKK3/MKK6 was TNF > GM-CSF. C-Jun amino-terminal kinase (JNK) was not tyrosine-phosphorylated by any cytokine despite the existence of JNK proteins in human neutrophils, whereas it was tyrosine-phosphorylated by TNF in undifferentiated and all-trans retinoic acid-differentiated HL-60 cells. Increased phosphorylation of ERK or p38 MAPK was detected within 1 to 5 minutes after stimulation with each cytokine and was dependent on the concentrations of cytokines used. MEK inhibitor (PD98059) reduced tyrosine phosphorylation of ERK, but not p38 MAPK, induced by G-CSF, GM-CSF, or TNF. GM-CSF– or TNF-induced superoxide (O2−) release was inhibited by p38 MAPK inhibitor (SB203580) in a dose-dependent manner, suggesting the possible involvement of p38 MAPK in GM-CSF– or TNF-induced O2− release. The results indicate that G-CSF, GM-CSF, and TNF activate the overlapping but distinct MAPK subtype cascades in human neutrophils and suggest that the differential activation of ERK and p38 MAPK cascades may explain the differences of the effects of these cytokines on human neutrophil functions.

Cytokine-Specific Activation of Distinct Mitogen-Activated Protein Kinase Subtype Cascades in Human Neutrophils Stimulated by Granulocyte Colony-Stimulating Factor, Granulocyte-Macrophage Colony-Stimulating Factor, and Tumor Necrosis Factor-α

AbstractTo clarify the differences of the signaling pathways used by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF), we investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by these cytokines. G-CSF exclusively tyrosine-phosphorylated extracellular signal-regulated kinase (ERK). GM-CSF tyrosine-phosphorylated ERK strongly and p38 MAPK weakly, whereas TNF tyrosine-phosphorylated p38 MAPK strongly and ERK weakly. Consistent with these findings, MEK, an upstream kinase of ERK, was phosphorylated by G-CSF, GM-CSF, and TNF, whereas MKK3/MKK6, an upstream kinase of p38 MAPK, was phosphorylated by GM-CSF and TNF, but not by G-CSF. The potency of these cytokines to phosphorylate ERK and MEK was GM-CSF > G-CSF > TNF, whereas that to phosphorylate p38 MAPK and MKK3/MKK6 was TNF > GM-CSF. C-Jun amino-terminal kinase (JNK) was not tyrosine-phosphorylated by any cytokine despite the existence of JNK proteins in human neutrophils, whereas it was tyrosine-phosphorylated by TNF in undifferentiated and all-trans retinoic acid-differentiated HL-60 cells. Increased phosphorylation of ERK or p38 MAPK was detected within 1 to 5 minutes after stimulation with each cytokine and was dependent on the concentrations of cytokines used. MEK inhibitor (PD98059) reduced tyrosine phosphorylation of ERK, but not p38 MAPK, induced by G-CSF, GM-CSF, or TNF. GM-CSF– or TNF-induced superoxide (O2−) release was inhibited by p38 MAPK inhibitor (SB203580) in a dose-dependent manner, suggesting the possible involvement of p38 MAPK in GM-CSF– or TNF-induced O2− release. The results indicate that G-CSF, GM-CSF, and TNF activate the overlapping but distinct MAPK subtype cascades in human neutrophils and suggest that the differential activation of ERK and p38 MAPK cascades may explain the differences of the effects of these cytokines on human neutrophil functions.

A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis.

Leukotriene B4 (LTB4) is a potent chemoattractant that is primarily involved in inflammation, immune responses and host defence against infection. LTB4 activates inflammatory cells by binding to its cell-surface receptor (BLTR). LTB4 can also bind and activate the intranudear transcription factor PPAR alpha, resulting in the activation of genes that terminate inflammatory processes. Here we report the cloning of the complementary DNA encoding a cell-surface LTB4 receptor that is highly expressed in human leukocytes. Using a subtraction strategy, we isolated two cDNA clones (HL-1 and HL-5) from retinoic acid-differentiated HL-60 cells. These two clones contain identical open reading frames encoding a protein of 352 amino acids and predicted to contain seven membrane-spanning domains, but different 5'-untranslated regions. Membrane fractions of Cos-7 cells transfected with an expression construct containing the open reading frame of HL-5 showed specific LTB4 binding, with a K(d) (0.154nM) comparable to that observed in retinoic acid-differentiated HL-60 cells. In CHO cells stably expressing this receptor, LTB4 induced increases in intracellular calcium, D-myo-inositol-1,4,5-triphosphate (InsP3) accumulation, and inhibition of adenylyl cyclase. Furthermore, CHO cells expressing exogenous BLTR showed marked chemotactic responses towards low concentrations of LTB4 in a pertussis-toxin-sensitive manner. Our findings, together with previous reports, show that LTB4 is a unique lipid mediator that interacts with both cell-surface and nuclear receptors.

Signal transduction via the CD38/NAD+ glycohydrolase.

The human cell surface CD38 molecule is a 46-kDa type-II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long Cys-rich C-terminal extracellular one. We previously demonstrated that an ecto-form NAD+ glycohydrolase (NADase) activity induced by all-trans retinoic acid in HL-60 cells is due to the extracellular domain of CD38. In the present study, we investigated a possible signal transduction mediated through CD38 in the retinoic acid-differentiated HL-60 cells with anti-CD38 monoclonal antibodies (mAbs). The addition of selected anti-CD38 mAbs to the cells induced rapid tyrosine phosphorylation of the cellular proteins with the molecular weights of 120,000, 87,000 and 77,000; the phosphorylated 120-kDa protein was identified as the c-cbl proto-oncogene product, p120c-cbl. Furthermore, the phosphorylated p 120c-cbl associated with the 85-kDa subunit of phosphatidylinositol 3-kinase. To determine the relationship between the amino acid sequence responsible for the NADase activity and epitopes recognized by the stimulatory mAbs, we produced its carboxy-terminal deletion mutants in COS-7 cells. The mutants with less than 15 amino acids deleted from the carboxyl terminus of the 300-amino acid wild-type molecule still maintained NADase activity, but those with more than 27 amino acids deleted did not. Introduction of site-directed mutation of a cysteine residue (Cys275), located in the 273-285 sequence, completely abolished the NADase activity. These CD38 mutants were also used for an epitope mapping of anti-CD38 mAbs. All the epitopes recognized by the mAbs inducing the tyrosine phosphorylation were mapped on the same Cys275-containing sequence of 273-285. Thus, the discrete carboxy-terminal sequence not only plays a key role in its ecto-NADase activity, but also contains the epitopes of the agonistic anti-CD38 mAbs for the transmembrane signaling. We also found that the agonistic mAbs markedly potentiate superoxide generation induced by the stimulation of G protein-coupled chemotactic receptors. Our results suggested that the stimulation of CD38 might generate an accessory signal(s) to enhance the G protein-mediated signaling, probably though the protein-tyrosine phosphorylation.

Tyrosine Phosphorylation of the c-cbl Proto-oncogene Product Mediated by Cell Surface Antigen CD38 in HL-60 Cells

The human cell surface antigen CD38 is a 46-kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long Cys-rich C-terminal extracellular one. We demonstrated previously that the extracellular domain of CD38 has NAD+ glycohydrolase (NADase) activity and that the ecto-form NADase activity induced in HL-60 cells during cell differentiation by retinoic acid is due to CD38. In the present study, we investigated the intracellular signaling mediated by CD38 in retinoic acid-differentiated HL-60 cells with an anti-CD38 monoclonal antibody. The addition of anti-CD38 monoclonal antibody to the cells induced rapid tyrosine phosphorylation of the cellular proteins with molecular weights of 120,000, 87,000, and 77,000. An increase in tyrosine kinase activity in the anti-phosphotyrosine immunoprecipitates of the cells was also observed after the addition of anti-CD38 monoclonal antibody. Moreover, one of the prominent tyrosine-phosphorylated proteins stimulated by the anti-CD38 monoclonal antibody was identified as the c-cbl proto-oncogene product, p120c-cbl. These results indicated that tyrosine phosphorylation of cellular proteins, including p120c-cbl, is possibly involved in transmembrane signaling mediated by CD38.

Rapid Degradation of NAD by Retinoic Acid-Differentiated HL-60 Granulocyte Membranes Prevents ADP Ribosylation

Pertussis and cholera toxins failed to ADP-ribosylate Gα s and Gα i in membranes isolated from retinoic acid-differentiated HL-60 granulocytes, although Gα i subunits were present. NAD was rapidly degraded in the presence of these membranes, primarily to ADP-ribose, to less than 10% of initial activity by 5 min. Metabolism of NAD was heat labile and inhibited by NADP, but not by imidazole or isonicotinic acid hydrazine. Pertussis toxin uncoupled LTB 4 receptors from G proteins in intact retinoic acid-differentiated HL-60 cells. Retinoic acid differentiation stimulates expression of a unique NAD-glycohydrolase activity in HL-60 granulocytes which prevents ADP-ribosylation in isolated membranes, but not intact cells.

Distinct patterns of sulfated proteoglycan biosynthesis in human monocytes, granulocytes and myeloid leukemic cells

The production of sulfated proteoglycans was compared in mature peripheral blood granulocytes and monocytes and HL-60 promyelocytic leukemia cells. We found that HL-60 cells synthesized 5–10 times more proteoglycans than peripheral blood leukocytes. Differentiation of HL 60 cells toward mature monocytes by treatment with 12- O-tetradecanoyl-phorbol-13-acetate (TPA) or towards granulocytes by treatment with retinoic acid or dibutyryl cyclic adenosine-3′,5′-monophosphate (dbcAMP) resulted in a small (20–30%) decrease in sulfated proteoglycan biosynthesis. Chondroitin sulfate was found to be the predominant proteoglycan produced by monocytes, PMN and undifferentiated HL-60 cells. Differentiated HL-60 cells produced chondroitin sulfate as well as sulfated proteoglycans sensitive to nitrous acid degradation. Similar results were observed in TPA, dbcAMP and retinoic acid differentiated HL-60 cells indicating that the changes in proteoglycan biosynthesis observed were independent of the developmental pathway. Using specific monoclonal antibodies and flow cytometry, we also found that HL-60 cells and monocytes produced chondroitin 4-sulfate, chondroitin-6-sulfate and chondroitin- O-sulfate while PMN only produced chondroitin-4 sulfate. In addition, although there were no significant differences in antibody binding to undifferentiated and differentiated HL-60 cells, the tumor cells bound 5–20 times more of the antibodies than the peripheral blood leukocytes. Our data demonstrate that sulfated proteoglycan production by HL-60 cells is distinct from PMN and monocytes. In addition, the fact that differentiated HL-60 cells continue to synthesize larger amounts of the proteoglycans than the peripheral blood leukocytes indicates these cells have not completely matured.

Transcriptional regulation of the leukocyte adherence protein beta subunit during human myeloid cell differentiation.

Adherence reactions involving human leukocytes are mediated by a family of glycoprotein surface antigens composed of three different alpha subunits designated alpha L, alpha M, and alpha X, each of which is associated with a single beta subunit in an alpha 1 beta 1 heterodimer structure. We cloned the cDNA for the common beta subunit and investigated beta subunit mRNA expression in HL-60 promyelocytic leukemia cells and human granulocytic cells. Leukocyte adherence receptor beta subunit mRNA transcripts were present in low levels in HL-60 myeloblasts and promyelocytes and increased 10-fold or greater with chemically induced differentiation to more mature granulocytes (using retinoic acid and dimethylformamide) or monocyte/macrophages (using phorbol myristate acetate). Levels of beta subunit mRNA expression were also increased both in normal human peripheral blood granulocytes and in granulocytes from patients with chronic myelogenous leukemia. Nuclear run-off assays indicated that the increased steady state level of the beta subunit mRNA in retinoic acid-differentiated HL-60 cells was secondary to enhanced beta subunit gene transcription. We conclude that mRNA levels for the beta subunit of the receptor on human leukocytes that mediates cellular adherence are increased in more mature granulocytic cells compared to immature myeloid precursors and that this enhanced mRNA expression is transcriptionally regulated.