interleukin-8 and Leukemia--Promyelocytic--Acute

To evaluate the expression and clinical significance of interleukin 6, soluble glycoprotein 130 (sgp 130), interleukin 8 and type A interleukin 8 receptor (IL-8RA) in acute promyelocytic leukemia (APL) patients during all-trans retinoic acid (ATRA) treatment.. Serum and bone marrow mononuclear cell (MNC) culture supernatant IL-6, sgp 130, IL-8 concentrations of 18 cases APL patients were measured (ELISA). Bone marrow MNC IL-8RA was measured by flow cytometry after cultured with ATRA (10(-6) mmol/L).. Serum IL-6, sgp130, IL-8 levels were higher than normal (P < 0.05), IL-6, sgp130 levels correlated with white blood cell (WBC) counts (P < 0.05) while IL-8 level correlated with body temperature (P < 0.05) at initial diagnosis after 72-hour incubation with ATRA, concentration of IL-6 of bone marrow MNC culture supenatant did not change, that of sgp130 mildly decreased, and IL-8 significantly decreased while the positive rate of IL-8RA on bone marrow MNC increased. During ATRA treatment, serum IL-6 changes were correlated with WBC changes. Peak level of IL-6 and WBC was lower in patients received intermittent therapy than continuous therapy. Serum IL-6 and IL-8 increased when complicated with infection and increase in IL-8 seemed more sensitive.. Serum levels of IL-6, sgp130, IL-8 may reflect patient's responsiveness to ATRA treatment, predict hyperleukocytosis and intercurrent infection. ATRA induces APL cell differentiation possibly via gp130 signal transduction.

Topics: Adolescent; Adult; Antigens, CD; Female; Glycoproteins; Humans; Interleukin-6; Interleukin-8; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Receptors, Interleukin; Receptors, Interleukin-8A; Signal Transduction; Tretinoin

In patients with acute promyelocytic leukemia (APL), intracranial hemorrhage (ICH), if not identified promptly, could be fatal. It is the leading cause of failure of induction and early death. Thus, biomarkers that could promptly predict severe complications are critical. Here, cytokine differences between patients with APL with and without ICH were investigated to develop predictive models for this complication. The initial cytokine profiling using plasma samples from 39 patients and 18 healthy donors found a series of cytokines that were remarkedly different between patients with APL and healthy controls. The APL patients were subsequently divided into high and low white blood cell count groups. Results showed that tumor necrosis factor a and interleukin 8 (IL-8) were vital in distinguishing patients with APL who did or did not develop ICH. In addition, verification in 81 patients with APL demonstrated that the two cytokines were positively correlated with the cumulative incidence of ICH. Finally, in-vitro and in-vivo experimental evidence were provided to show that IL-8 influenced the migration of APL-derived NB4 cells and impaired the blood-brain barrier in PML/RARα positive blast-transplanted FVB/NJ mice. These assessments may facilitate the early warning of ICH and reduce future mortality levels in APL.

Topics: Animals; Cytokines; Interleukin-8; Intracranial Hemorrhages; Leukemia, Promyelocytic, Acute; Mice; Oncogene Proteins, Fusion; Tretinoin; Tumor Necrosis Factor-alpha

Differentiation syndrome (DS) is the main life-threatening adverse event that occurs in acute promyelocytic leukemia (APL) patients treated with all-trans retinoic acid (ATRA). Cytokine imbalances have been reported to play role during the developing of acute promyelocytic leukemia differentiation syndrome (APL-DS). However, the relationship between the plasma cytokine levels and their prognostic value for the prediction of DS developing in patients with APL during the treatment with ATRA and anthracyclines has not been previously reported.. In this study, we followed an APL cohort (n = 17) over 7 days of ATRA therapy in DS (n = 6) and non-DS groups (n = 11). Interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF-α were measured in the peripheral blood plasma from 17 patients with APL and 11 healthy adult controls by using the cytometric bead array method.. In non-DS patients, IL-8 plasma levels were significantly reduced in the seventh day of ATRA treatment (34.16; 6.99 to 147.11 pg mL. We demonstrated that the modulation of IL-8 following ATRA treatment may occur regardless of the development of DS and, therefore, does not appear to be a predictive biomarker to monitor the APL-DS.

Topics: Adult; Aged; Antineoplastic Agents; Biomarkers, Tumor; Cell Differentiation; Female; Humans; Interleukin-6; Interleukin-8; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Retrospective Studies; Syndrome; Treatment Outcome; Tretinoin; Young Adult

The bone marrow microenvironment is physiologically hypoxic with areas being as low as 1% O2, e.g. the stem cell niche. Acute myeloid leukaemia (AML) blasts misuse these bone marrow niches for protection by the local microenvironment, but also might create their own microenvironment. Here we identify IL-8 as a hypoxia-regulated cytokine in both AML cell lines and primary AML samples that is induced within 48 hours of severe hypoxia (1% O2). IL-8 lacked effects on AML cells but induced migration in mesenchymal stromal cells (MSC), an integral part of the bone marrow. Accordingly, MSC were significantly increased in AML bone marrow as compared to healthy bone marrow. Interestingly, mononuclear cells obtained from healthy bone marrow displayed both significantly lower endogenous and hypoxia-induced production of IL-8. IL-8 mRNA expression in AML blasts from 533 patients differed between genetic subgroups with significantly lower expression of IL-8 in acute promyelocytic leukaemia (APL), while in non APL-AML patients with FLT ITD had the highest IL-8 expression. In this subgroup, high IL-8 expression was also prognostically unfavourable. In conclusion, hypoxia as encountered in the bone marrow specifically increases IL-8 expression of AML, which in turn impacts niche formation. High IL-8 expression might be correlated with poor prognosis in certain AML subsets.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bone Marrow; Bone Marrow Cells; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation, Leukemic; Humans; Hypoxia; Immunohistochemistry; Interleukin-8; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Mesenchymal Stem Cells; Middle Aged; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Stem Cell Niche; Survival Analysis; Young Adult

PDGFR inhibitors are successfully used in a number of cancer treatments. The standard treatment for acute promyelocytic leukemia (APL) involves differentiation therapy with retinoic acid (RA). However, the relapse rates are significant. In the present work we evaluated the effects of RA therapy in the presence of PDGFR inhibitor, AG1296. Adding AG1296 with RA increased secretion of TNF-alpha, IL-8, and MMP-9 expression. This treatment induced higher levels of ICAM-1 endothelial cell expression, and increased cellular mobility. Inhibiting PDGFR enhanced RA-induced expression of integrin. Integrin ligand increased differentiation markers CD11b, inducible oxidative metabolism and PDGFR-beta phosphorylation. While the neutrophil-endothelial cell interactions are strengthened by the combined treatment, the endothelium-substratum interactions are weakened, a situation common in RAS.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; CD18 Antigens; Cell Differentiation; Cell Movement; Contraindications; Drug Resistance, Neoplasm; Endothelial Cells; HL-60 Cells; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; Leukemia, Promyelocytic, Acute; Ligands; Macrophage-1 Antigen; Matrix Metalloproteinase 9; Protein Kinase Inhibitors; Receptor, Platelet-Derived Growth Factor beta; Receptors, Platelet-Derived Growth Factor; Syndrome; Tretinoin; Tumor Necrosis Factor-alpha; Tyrphostins

Disease relapse sometimes occurs after acute promyelocytic leukemia (APL) therapy with all-trans retinoic acid (ATRA). Among the diagnostic parameters predicting relapse, heterogeneity in the in vitro differentiation rate of blasts is an independent factor. To identify biologic networks involved in resistance, we conducted pharmacogenomic studies in APL blasts displaying distinct ATRA sensitivities. Although the expression profiles of genes invested in differentiation were similarly modulated in low- and high-sensitive blasts, low-sensitive cells showed higher levels of transcription of ATRA-target genes, transcriptional regulators, chromatin remodelers, and transcription factors. In opposition, only high-sensitive blasts expressed the CYP26A1 gene, encoding the p450 cytochrome which is known to be involved in retinoic acid catabolism. In NB4 cells, ATRA treatment activates a novel signaling pathway, whereby interleukin-8 stimulates the expression of the homeobox transcription factor HOXA10v2, an effective enhancer of CYP26A1 transcription. These data were corroborated in primary APL cells, as maturation levels correlated with CYP26A1 expression. Treatment with a retinoic acid metabolism blocking agent (RAMBA) results in high-nucleoplasmic concentrations of retinoid and growth of NB4-resistant subclones. Hence, for APL blasts associated with poor prognosis, the low CYP26A1 expression may explain high risk of resistance installation, by increased retinoid pressure. Pharmacogenomic profiles of genes involved in retinoid acid metabolism may help to optimize anticancer therapies, including retinoids.

Topics: Cell Proliferation; Cytochrome P-450 Enzyme System; Disease Progression; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Gene Library; Homeobox A10 Proteins; Homeodomain Proteins; Humans; Interleukin-8; Leukemia, Promyelocytic, Acute; Models, Biological; Pharmacogenetics; Retinoic Acid 4-Hydroxylase; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Although all-trans retinoic acid (ATRA) can treat acute promyelocytic leukemia (APL), it also causes retinoic acid syndrome with presentations similar to acute respiratory distress syndrome. We investigated the role of interleukin (IL)-8 and growth-regulated oncogene (GRO)-alpha in the chemotactic transmigration of ATRA-treated NB4 (ATRA-NB4) APL cells toward A549 alveolar epithelial cells.. An in vitro human cell culture study.. University hospital research laboratories.. NB4 and A549 cells.. NB4 and A549 cells were separately cultured with ATRA and/or dexamethasone for 1-3 days. NB4 or ATRA-NB4 cells were then placed in an upper insert and co-incubated with A549 cells or their conditioned medium located in a lower plate.. ATRA stimulated NB4 cells to transmigrate toward the A549 cells in a time- and dose-dependent manner. Replacement of A459 condition medium by its original medium abrogated this transmigration. Only A549 cells constitutively secreted GRO-alpha, and both A549 and NB4 cells constitutively secreted IL-8, which was enhanced by ATRA. Exogenous administration of IL-8 or GRO-alpha also promoted the ATRA-NB4 transmigration. The binding assay demonstrated that ATRA-NB4 cells bound IL-8, but not GRO-alpha, more avidly. Pretreatment with antibodies directed against IL-8 and GRO-alpha receptors reduced ATRA-NB4 transmigration by about 60%. Dexamethasone did not suppress their IL-8 secretion and transmigration in ATRA-NB4 cells, but when applied to A549 cells, IL-8 secretion was suppressed but not GRO-alpha secretion, and there was attenuation of ATRA-NB4 transmigration.. IL-8 and GRO-alpha secreted from alveolar epithelial cells play an important role in the cell-cell interaction involved in the chemotactic transmigration of ATRA-treated APL cells toward alveolar epithelial cells.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Chemokine CXCL1; Chemokines, CXC; Chemotaxis, Leukocyte; Epithelial Cells; Flow Cytometry; Humans; In Vitro Techniques; Interleukin-8; Leukemia, Promyelocytic, Acute; Pulmonary Alveoli; Respiratory Distress Syndrome; Tretinoin

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Chemokine CXCL1; Chemokines, CXC; Chemotaxis, Leukocyte; Epithelial Cells; Flow Cytometry; Humans; In Vitro Techniques; Interleukin-8; Leukemia, Promyelocytic, Acute; Pulmonary Alveoli; Respiratory Distress Syndrome; Syndrome; Tretinoin

We previously reported the induction of interleukin-8 (IL-8), one of the CXC chemokines, by all-trans retinoic acid (ATRA) in PL-21 and NB4 human myeloid leukemia cells, which may be implicated in APL differentiation syndrome that is a relatively frequent complication in patients with acute promyelocytic leukemia (APL) during treatment with ATRA. We, therefore, further investigated the effects of ATRA on the expression of chemokine family in NB4 cells and APL cells prepared from two APL patients. The RNase protection assay using a multi-probe template set for human chemokines revealed that ATRA induced gene expressions of a number of CC chemokines, such as monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1alpha and MIP-1beta in NB4 cells. Their antigen levels were also increased in the cultured media. APL cells prepared from two APL patients showed gene expression of chemokines, such as IL-8, MCP-1, MIP-1alpha, and MIP-1beta when stimulated with ATRA in vitro. Furthermore, serum levels of IL-8, MIP-1beta and RANTES were increased during the course of ATRA treatment in both APL patients who developed APL differentiation syndrome. These chemokines are all chemoattractants of particular inflammatory cell types, including neutrophils, monocytes and lymphocytes; therefore, the simultaneous induction of these chemokines after stimulation with ATRA may exacerbate the hyper-inflammation observed in ATRA-induced APL differentiation syndrome.

Topics: Antineoplastic Agents; Base Sequence; Blotting, Northern; Cell Line, Tumor; Chemokines, CC; Chemokines, CXC; DNA Primers; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Leukemia, Promyelocytic, Acute; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin

The protein kinase inhibitor staurosporine elicits multiple responses in various systems. We evaluated nine naturally occurring staurosporine derivatives as modulators of chemokine production by monitoring the secretion of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in the cell line NB-4. Several staurosporines increased, dose- and time-dependently, the IL-8 and MCP-1 concentration in the cell culture supernatants and three derivatives strongly inhibited proliferation of the NB-4 cells. By comparing the efficiency of these analogues at the same concentration, the lead compound staurosporine (STS-1) was the best inducer of chemokine secretion, whereas 3-hydroxystaurosporine (STS-3) was the most potent growth inhibitor. Besides the staurosporines, also 12-O-tetradecanoyl phorbol acetate (TPA) and tumor necrosis factor-alpha (TNFalpha) strongly increased the IL-8 and MCP-1 secretion of NB-4 cells. Several staurosporine analogues clearly inhibited the TPA-induced but enhanced the TNFalpha-mediated chemokine increase. These effects, namely the increase of chemokines in untreated or TNFalpha-treated cells and the inhibition of chemokine release in TPA-treated cells, cannot be explained by the exclusive inhibition of protein kinase C (PKC). It may indicate that staurosporines are additionally involved in activation of the PKC-triggered chemokine production.

Topics: Cell Division; Cell Survival; Chemokine CCL2; Chemokines; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Humans; Interleukin-8; Leukemia, Promyelocytic, Acute; Protein Kinase C; Staurosporine; Tumor Necrosis Factor-alpha

To detect the expression of cytokines by acute promyelocytic leukemia (APL) cells before and after exposure to arsenic trioxide.. Diagnoses were performed according to the FAB cytological classification criteria and cytogenetic criteria. Bone marrow or blood samples from APL patients were collected in heparinized tubes, then primary APL cells were separated by traditional Ficoll-Hypaque density centrifugation and purified after adherence to plastic surfaces. IL-1(beta), IL-6, IL-8, TNF alpha and G-CSF levels in the leukemia cell culture supernatants were detected by ELISA. At the same time, nitro blue tetrazolium (NBT) reduction test was used to detect the differentiation of APL cells.. After 96 hours exposure to arsenic trioxide, 10 - 6 mol/L in vitro or 10 mg/d in vivo, APL cells showed a significant increase of IL-1(beta) (P < 0.05) and G-CSF (P < 0.05) production, and a significant decrease of IL-6 (P < 0.05) and IL-8 (P < 0.05). However, there was no obvious variation of TNF alpha when compared with APL cells without exposure to arsenic trioxide. On the other hand, the proliferation ratio of APL cells in vitro was statistically correlated to the IL-1(beta) secretion ratio or G-CSF secretion ratio. The cell number ratio in patients with detectable IL-1(beta) or G-CSF was higher than that without detectable IL-1(beta) or G-CSF.. IL-1(beta) and G-CSF secretion may play an important role in the proliferation of APL cells after exposure to arsenic trioxide.

Topics: Arsenic Trioxide; Arsenicals; Cells, Cultured; Cytokines; Granulocyte Colony-Stimulating Factor; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Leukemia, Promyelocytic, Acute; Oxides; Tumor Necrosis Factor-alpha

Differentiation therapy with all-trans retinoic acid (ATRA) represents a landmark approach in the treatment of acute promyelocytic leukemia (APL). However, a potentially fatal complication of retinoic acid (RA) syndrome occurs in about a quarter of patients and its pathophysiology is still unclear. In order to investigate whether or not the treatment with ATRA leads to increased elaboration of inflammatory cytokines and adhesion molecules by the APL cells, the expression of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-8, L-selectin and intercellular adhesion molecule-1 (ICAM-1) was examined in the APL cells after induction of differentiation with ATRA in the presence or absence of granulocyte-colony stimulating factor (G-CSF) or IL-3 in the present study. Cytokine elaboration by the treated cells was detected using both Northern blotting and enzyme-linked immunosorbent assay. Our results have shown that ATRA induces an increased expression of IL-8, IL-1beta, TNF-alpha and ICAM-1 in APL cells, which can be amplified by the addition of G-CSF. These data imply that the induction of inflammatory cytokines in APL cells may play an important role in the pathogenesis of RA syndrome. Furthermore, G-CSF, through its potent differentiating activity, may increase the risk of such complications during ATRA treatment.

Topics: Antineoplastic Agents; Cell Adhesion Molecules; Cell Differentiation; Cell Division; Cytokines; Granulocyte Colony-Stimulating Factor; Hematopoietic Cell Growth Factors; Humans; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-8; L-Selectin; Leukemia, Promyelocytic, Acute; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

To evaluate the clinical significance of expression of interleukin-(IL-8) and its type A receptor(IL-8RA) in acute promyelocytic leukemia(APL) patients under all trans-retinoic acid(ATRA) induction.. Serum IL-8 level of 18 APL patients were dynamically studied(ELISA). Fresh APL cells from 3 patients were cultured with ATRA(10(-6) mmol/L). Supernatant IL-8 level and IL-8RA expression on APL cells were measured by FACS.. In vitro, IL-8 concentrations decreased 72 hours after incubation, while IL-8RA increased. In vivo, IL-8 increased more rapidly and markedly than temperature and WBC counts did before retinoic acid syndrome(RA-S) occurred. Serum IL-6 and IL-8 levels significantly increased when the patients suffered infection, and IL-8 increased even before fever. Both IL-8 and D-dimer increased while DIC progressed.. ATRA inhibited IL-8 secretion of APL cells while increased the expression of IL-8RA. Monitoring serum IL-8 concentrations could predict the development of RA-S and infection. Increase of both IL-8 and D-dimer concentrations suggested DIC progression.

Topics: Adolescent; Adult; Antineoplastic Agents; Female; Humans; Interleukin-8; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Receptors, Interleukin-8A; Tretinoin

All-trans retinoic acid (ATRA) is a differentiating agent that has been successfully used in the treatment of patients with acute promyelocytic leukemia (APL). Functional properties of peripheral blood neutrophils from a patient with APL during treatment with ATRA have been studied. Wright stain of patient neutrophils showed hypogranulation and loose nuclear chromatin when compared with normal neutrophils. These cells were of lower density than normal neutrophils and separated on density gradient centrifugation with mononuclear cells. Surface antigen expression by FACS distinguished these cells from lymphocytes. The histograms showed a population of larger cells expressing CD18 and CD11b, distinct from the smaller cells which did not express CD11b. fMLP caused an increase in intracellular calcium (measured spectrophotometrically) that was inhibited by the calcium chelator BAPTA. Actin polymerization following cell activation was measured using NBD-phallacidin staining and FACS. Both IL-8 and fMLP caused rapid increases using F-actin content (2.5-3.0 fold), which were of greater magnitude than generally seen with normal neutrophils. Treatment with BAPTA before activation with fMLP did not blunt the actin responses, despite complete inhibition of an intracellular calcium increase. In summary, neutrophils derives from differentiated APL cells express CD18/CD11b, and exhibit a similar degree of actin polymerization in response to fMLP and IL-8, independent of an increase in intracellular calcium. Although the actin responses are greater than normal neutrophils, most properties are similar, supporting the contention that these cells can protect the host. The exaggerated actin response to inflammatory mediators, however, may play a role in the 'retinoic acid syndrome'.

Topics: Actins; Antigens, Surface; Calcium; Cell Adhesion Molecules; Cell Size; Granulocytes; Humans; Immunophenotyping; In Vitro Techniques; Integrins; Interleukin-8; L-Selectin; Leukemia, Promyelocytic, Acute; Male; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Tretinoin

In HL-60 cells, retinoic acid (RA) and 9 cis-RA induce granulocytic differentiation, and calcitriol and sodium butyrate induce monocytic differentiation. To study the role of retinoid resistance on the response to these agents, we investigated their effects in HL-60 cells, retinoid-resistant HL-60R cells, and HL-60R+ cells in which retinoid sensitivity has been restored. In HL-60 cells, cathepsin D (ctsd) mRNA levels are increased by these agents and by cholera toxin after pretreatment with each agent. Calcitriol, 9 cis-RA, and sodium butyrate increase interleukin-8 (IL-8) mRNA expression, and pretreatment with these agents or RA potentiates the stimulation of IL-8 by phorbol ester (TPA). Pretreatment of HL-60 cells with all of the agents confers inducibility of cathepsin L (ctsl) mRNA by TPA in previously unresponsive cells. In HL-60R cells, none of the agents alone or in combination significantly enhances the expression of the ctsd, IL-8, or ctsl mRNAs. Retinoid stimulation (either alone or in combination with the other agents) of the three mRNAs is partially restored in the HL-60R+ cells. Calcitriol does not alter the expression of any of these mRNAs, and only the stimulation of IL-8 mRNA by sodium butyrate is recovered. Treatment with all of the agents inhibits proliferation and stimulates differentiation of the HL-60 cells. RA and calcitriol are unable to inhibit proliferation of the HL-60R cells, whereas only calcitriol fails to inhibit proliferation of the HL-60R+ cells. None of the agents induces differentiation in either the HL-60R or HL-60R+ cells. Therefore, the mutation of the RA receptor alpha is insufficient to account for the altered responses of the HL-60R cells, and there are likely defects in other signaling pathways in these cells. These cells may prove useful in examining the mechanism of cross-resistance between various differentiating agents.

Topics: Butyrates; Butyric Acid; Calcitriol; Cathepsin D; Cathepsin L; Cathepsins; Cell Differentiation; Cysteine Endopeptidases; Drug Resistance; Endopeptidases; Gene Expression; Humans; Interleukin-8; Leukemia, Promyelocytic, Acute; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Urinary trypsin inhibitor (UTI) is one of the Kunitz-type protease inhibitors in human. Little is known about its anti-inflammatory functions other than protease inhibition. We studied the effect of UTI on gene expression of interleukin-8 (IL-8), an inflammatory cytokine. UTI inhibited IL-8 gene expression induced by lipopolysaccharide (LPS) in HL60 cells. The IL-8 concentrations in the cells and medium after LPS stimulation increased time-dependently in the absence of UTI, but did not increase in the presence of UTI. On the other hand, UTI did not inhibit either the synthesis or the release of IL-8 induced by the calcium ionophore A23187. UTI inhibited increase of cytosolic Ca2+ stimulated by LPS but not by A23187. Our results suggest that the inhibition by UTI is due to its effect on the cell membrane involved in regulating Ca2+ influx.

Topics: Calcimycin; Calcium; Cell Membrane; Culture Media, Conditioned; Cytosol; Gene Expression Regulation; Humans; Interleukin-8; Leukemia, Promyelocytic, Acute; Lipopolysaccharides; RNA, Messenger; Trypsin Inhibitor, Kunitz Soybean; Tumor Cells, Cultured

All-trans-retinoic acid (ATRA) causes granulocyte differentiation in patients with acute promyelocytic leukemia. HL60 cells are frequently used as an in vitro model for studying granulocytes during maturation. We have previously studied actin polymerization in response to fMLP in HL60 cells undergoing DMSO induced maturation, and reported that IL-8 causes actin polymerization in neutrophils in a manner similar to fMLP. We now compare chemotaxis and actin polymerization in response to IL-8 and fMLP, and nitroblue tetrazolium (NBT) reduction in HL60 cells matured with ATRA and DMSO. Cells cultured for 4 days with ATRA and DMSO showed morphologic evidence of maturation. NBD-phallacidin staining and flow cytometry were used to measure changes in F-actin content in response to IL-8 and fMLP. Uninduced cells were not capable of actin polymerization or chemotaxis. Cells matured with ATRA exhibited a 2.6-fold increase in F-actin content in response to IL-8, but only a 1.2-fold increase in response to fMLP. Cells matured with DMSO responded to both IL-8 and fMLP in an equal manner with 1.6-fold increases in F-actin. The 2 h migration for ATRA induced cells was 124 microns in response to IL-8, 107 microns with fMLP, and 105 microns in buffer. DMSO induced cells migrated 89 microns in response to IL-8, 106 microns with fMLP, and 66 microns in buffer. With maturation, 65% of the ATRA induced cells reduced NBT compared with only 15% of the DMSO induced cells. In summary, HL60 cells cultured in ATRA develop greater functional maturity than those cultured in DMSO, and a greater responsiveness to IL-8 than fMLP, a finding distinct from previously reported work in neutrophils.

Topics: Actins; Cell Differentiation; Chemotaxis, Leukocyte; Dimethyl Sulfoxide; Granulocytes; Humans; Interleukin-8; Leukemia, Promyelocytic, Acute; N-Formylmethionine Leucyl-Phenylalanine; Nitroblue Tetrazolium; Oxidation-Reduction; Tretinoin; Tumor Cells, Cultured

Acute promyelocytic leukemia (APL) is a homogeneous subgroup of acute myeloid leukemias (AML) characterized by the presence of the t(15;17) translocation and the resulting PML/RAR alpha fusion proteins. To date APL is the only AML which is sufficiently sensitive to all-trans retinoic acid (ATRA) differentiating effect. We have recently reported that APL express and secrete hematopoietic growth factors (HGF) such as IL-1 beta, TNF alpha, and IL-6. In vivo ATRA alone allows achievement of complete remission in APL patients. One of ATRA therapy's drawbacks is the increase of peripheral blast cells often associated with the ATRA leukocyte activation syndrome. To determine if this specific side-effect was linked to an increase of HGF release by APL cells, we studied the modulation of cytokine production by APL cells, we studied the modulation of cytokine production by APL samples (n = 12) before and after incubation with ATRA. ATRA failed to modulate TNF alpha, IL-6 or GM-CSF secretion levels; however, IL-8 levels decreased in 11 cases, and in four cases up-regulation of IL-1 beta and G-CSF protein expression was observed. These modulations were found to be linked to ATRA sensitivity as ATRA failed to modulate cytokine production in non-APL cells (n = 8). Interestingly, the increase of IL-1 beta and G-CSF production in the presence of ATRA was highly correlated to an increase in APL cell count in vitro and in vivo hyperleukocytosis, resulting in fatal outcome. IL-1 beta, TNF alpha, IL-6, and IL-8 are known to be implicated in leukocyte activation. The results of this study suggest that ATRA-induced hyperleukocytosis and ATRA leukocyte activation syndrome in APL may be inherent to the secretion of specific hematopoietic growth factors by the APL cells.

Topics: Blotting, Northern; Blotting, Southern; Cell Differentiation; Granulocyte Colony-Stimulating Factor; Humans; Interleukin-1; Interleukin-8; Leukemia, Promyelocytic, Acute; Leukocytosis; Polymerase Chain Reaction; RNA-Directed DNA Polymerase; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Purified leukemic cells from 30 acute myeloid leukemia (AML) cases at diagnosis were investigated for the presence of interleukin 8 (IL-8) mRNA by Northern blot analysis. IL-8 specific transcripts were detected in uncultured blasts in 14/30 cases, 10/14 from patients with M4-M5 and 4/14 from cases with M0-M3 morphology. The transcript expression was associated with the detection of IL-8 molecule in blast cells by immunostaining performed on cytospin preparations. After 24-hour culture, a strong up-regulation or the appearance in cases negative before culture of IL-8 mRNA was observed in all cases tested, and culture supernatants contained high amounts of IL-8. Our data demonstrate that leukemic cells in AML are equipped with the functional apparatus for IL-8 production. Since IL-8 displays a wide range of biological activities, including the regulation of some membrane molecules relevant to adhesion and migration processes, its production by AML blasts might be of relevance for the pattern of leukemic growth.

Topics: Acute Disease; Cell Division; Gene Expression; Humans; Interleukin-8; Leukemia, Monocytic, Acute; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Leukemia, Promyelocytic, Acute; RNA, Messenger; Tumor Cells, Cultured

Specific receptors for a recently purified and cloned monocyte-derived neutrophil chemotactic factor (MDNCF) have been identified on the surface of normal human peripheral blood neutrophils using 125I-labeled recombinant human MDNCF (125I-MDNCF). Competitive binding of 125I-MDNCF to human neutrophils reached a maximal level at 1-3 h at 4 degrees C. The Scatchard analysis showed that there are approximately 20,000 receptors per cell with a single type of high affinity binding (Kd, 8 x 10(-10) M). The receptors for MDNCF are clearly distinct from the receptors for other cytokines and chemotactic agents, e.g., IL-1 alpha, TNF-alpha, and FMLP, C5a, leukotriene B4, and platelet activating factor. Based on the SDS-PAGE analysis of chemically crosslinked 125I-MDNCF receptor complex, there are two polypeptides that bind MDNCF; the molecular weight of these two MDNCF receptors were estimated to be 67,000 and 59,000. Treatment of a promyelocytic cell line, HL60, with 1.25% DMSO for 5 d in vitro increased the number of receptors up to 7,000 receptors/cell with a Kd of 1.2 x 10(-9) M.

Topics: Chemotactic Factors; Cross-Linking Reagents; Dimethyl Sulfoxide; Electrophoresis, Polyacrylamide Gel; Humans; Interleukin-8; Iodine Radioisotopes; Kinetics; Leukemia, Promyelocytic, Acute; Molecular Weight; Monocytes; Neutrophils; Receptors, Immunologic; Receptors, Interleukin-8A; Recombinant Proteins; Succinimides; Tumor Cells, Cultured

A cDNA library was constructed from HL60 human promyelocyte poly(A)+ RNA harvested 3 h after induction of macrophage differentiation with 12-O-tetradecanoyl phorbol-13-acetate in the presence of cycloheximide. We isolated from this library a 1.6-kilobase full-length clone designated b4 whose corresponding mRNA was greatly increased in abundance in cytoplasmic RNA under these conditions. Dideoxy sequencing revealed that this mRNA encoded MONAP (monocyte-derived neutrophil-activating peptide), a 10-kilodalton monokine with neutrophil-specific chemotactic and enzyme-releasing activities. The 3' untranslated region of this mRNA was found to be 1.2 kilobases long and possessed nine copies of the AUUUA sequence known to be associated with regulation of mRNA stability. Actinomycin D chase experiments yielded evidence that cytoplasmic stabilization was one of the means of regulation of MONAP expression. Analysis of cytoplasmic poly(A)- RNA revealed the presence of several discrete truncated species that shared a common 5' end and appeared to be intermediates of degradation. S1 mapping showed that the 3' ends of these molecules were distributed throughout the 3' untranslated region, preferentially in A + U-rich regions, broadly correlating with the distribution of AUUUA sites. Nuclear run-on experiments indicated that transcriptional induction accounted for less than 15% of the accumulation of MONAP mRNA. This mRNA was induced in HL60 cells by treatment with several differentiation-inducing agents: 12-O-tetradecanoyl phorbol-13-myristate alone, sodium butyrate, vitamin D3, and dimethyl sulfoxide. It was also induced in quiescent diploid lung fibroblasts stimulated to divide by serum, and it was constitutively overexpressed by some human tumor lines.

Topics: Amino Acid Sequence; Base Sequence; Cell Differentiation; Chemotactic Factors; DNA; Gene Expression Regulation; Granulocytes; Humans; Interleukin-8; Leukemia, Promyelocytic, Acute; Molecular Sequence Data; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured