Development of specific functionally active receptors for platelet-activating factor in HL-60 cells following granulocytic differentiation

A human promyelocytic leukemia cell line (undifferentiated HL-60 cells) as well as a granulocyte form of HL-60 cells induced in vitro by exposure to dimethyl sulfoxide were examined for binding, metabolism, and biological responses to platelet-activating factor (PAF). Undifferentiated and differentiated HL-60 cells each exhibit a high capacity to incorporate and metabolize [3H]PAF at 37 degrees C; however, the amount of [3H]PAF that is assimilated by both cell populations is greatly reduced and its metabolism abolished at less than or equal to 4 degrees C. At 0 degrees C HL-60 granulocytes bind more [3H]PAF than their undifferentiated counterparts. Binding to differentiated cells reaches equilibrium within 80 min and is saturable, reversible and specific; PAF receptor antagonists WEB 2086, L-659,989, BN 52021, and kadsurenone abolish this specific [3H]PAF binding. In contrast, [3H]PAF uptake by undifferentiated HL-60 cells is neither saturable nor sensitive to specific receptor antagonists. Scatchard analyses reveal 5850 +/- 850 binding sites per differentiated HL-60 cell with a dissociation constant of 0.66 +/- 0.15 nM. In the presence of cytochalasin B, PAF (200 nM) induces degranulation only in differentiated cells and this response also is blocked by PAF receptor antagonists. Our results demonstrate that HL-60 cells develop specific and functionally active PAF receptors only after chemically induced differentiation into granulocytes.     

An arachidonoyl (polyenoic)-specific phospholipase A2 activity regulates the synthesis of platelet-activating factor in granulocytic HL-60 cells

Human promyelocytic leukemia cells (HL-60) were used as a cell model to determine how arachidonic acid stimulates the synthesis of platelet-activating factor (PAF) synthesized via the remodeling pathway. In these studies HL-60 cells were cultured over 30 passages in fatty acid-free medium to deplete them of arachidonic acid. Even though the phospholipid classes from these cells contained no arachidonate, they could still be differentiated into granulocytes by dimethyl sulfoxide (1.25%). When the differentiated HL-60 cells, depleted of arachidonic acid, were stimulated with calcium ionophore A23187 in the presence of Ca2+ and [3H]acetate, only minimal amounts of [3H]PAF were produced. In contrast, if the differentiated HL-60 cells were supplemented with 10 microM arachidonic acid for 24 h and then stimulated with the ionophore, there was a large amount of [3H]PAF formed. The increase in PAF synthesis depended on the length of time the cells were supplemented with arachidonic acid; only a small increase in PAF synthesis occurred during the early hours of supplementation whereas stimulation of PAF synthesis was maximal (3-5-fold) after a 24-h period of the 20:4 supplementation. Other polyenoic fatty acid supplements (20:5, 22:4, and 22:6 for 24 h) also stimulated PAF production in the ionophore-treated HL-60 cells depleted of 20:4, but the amount of PAF was significantly less than found for the supplements of 20:4 under identical experimental conditions. Also noteworthy is that undifferentiated cells supplemented with 20:4 or their unsupplemented controls could not be stimulated by the calcium ionophore to produce PAF. Addition of indomethacin (cyclooxygenase inhibitor), A63162 (5'-lipoxygenase inhibitor), or eicosatetraynoic acid (cyclooxygenase/lipoxygenase inhibitor) to the incubations caused little change in the production of [3H]PAF in the differentiated cells supplemented with 20:4 for 24 h. On the other hand, the addition of mepacrine, bromophenacyl bromide, or U26384 (phospholipase A2 inhibitors) resulted in very large decreases (80-90% lower than controls) in the amount of [3H]PAF produced under the same conditions. Analysis of the molecular species of [3H]alkylacyl-GroPCho (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, the precursor of PAF in the remodeling pathway) in 20:4-supplemented cells prelabeled with [3H]alkyl-lyso-GroPCho revealed that only the alkylarachidonoyl-GroPCho species were preferentially decreased after stimulation with the A23187 ionophore.These results demonstrate that arachidonate must be at the sn-2 position of alkylacyl-GroPCho in order for it to serve as a precursor of PAF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis of a novel acetylated neutral lipid related to platelet-activating factor by acyl-CoA:1-O-alkyl-2-acetyl-sn-glycerol acyltransferase in HL-60 cells

Acyl-CoA:1-O-hexadecyl-2-acetyl-sn-glycerol acyl-transferase, a newly detected enzyme related to platelet-activating factor metabolism, has been characterized in microsomes of a human leukemia cell line (HL-60 cells). It has a sharp pH optimum of 6.8, does not require divalent metal ions, is stable at preincubation temperatures up to 45 degrees C, and among a variety of acyl-CoA thioesters (8:0-20:4) tested, linoleoyl-CoA is the best substrate. Km and Vmax values for 1-O-hexadecyl-2-acetyl-sn-glycerol acyltransferase are 8.5 microM and 1.7 nmol/min/mg of protein, respectively. For comparative purposes acyl-CoA:1,2-dioleoyl-sn-glycerol acyltransferase was also characterized in HL-60 microsomes. It has a relatively broad pH optimum of 6.1, is stimulated 1.4-fold by Mg2+, is relatively labile at preincubation temperatures higher than 25 degrees C, and among the various acyl-CoA thioesters tested, myristoyl-CoA is the best substrate. In substrate competition experiments, we found 1-O-hexadecyl-2-oleoyl-sn-glycerol is a competitive inhibitor (Ki = 32 microM). Our findings indicate acyl-CoA:1-O-hexadecyl-2-acetyl-sn-glycerol acyltransferase in HL-60 cells is distinctly different from acyl-CoA:1,2-dioleoyl-sn-glycerol acyltransferase. Our experimental results demonstrate that the unique enzyme activity characterized in this report also is expressed in intact HL-60 cells.     

Tumor necrosis factor induced DNA fragmentation of HL-60 cells

Tumor necrosis factor (TNF) induces differentiation of HL-60 cells, with only slight effects upon proliferation and little or no cytotoxicity. TNF induced cytotoxicity of other target cell lines has been associated with DNA fragmentation. To assess whether TNF-induced DNA fragmentation might also contribute to HL-60 differentiation, studies were performed using a [3H]-dThd release assay. Between 1 and 2 hours of culture, significant [3H]-dThd release was induced by TNF at concentrations of 10 U/ml and greater. This response was blocked by inhibiting energy metabolism, but not by several inhibitors of cell surface signal transduction, protein or RNA synthesis, or free radical scavengers. DNA electrophoresis of the released DNA disclosed a wide range of low molecular weight fragments. It is possible that TNF-induced DNA fragmentation contributes to HL-60 differentiation.     

The endogenous differentiation factor of the HL-60 cells shows a nuclease activity

A structural homology between the endogenous differentiation factor of the HL-60 cell line of promyelocyte leukemia (HLDF) and several DNA/RNA-binding and DNA/RNA-hydrolyzing proteins was revealed, and expression of the hldf gene in prokaryotic systems was studied. On the basis of these experiments, the amino acid sequence of an 8-membered fragment of HLDF with potential nuclease activity was identified. The synthetic octapeptide RRWHRLKE was shown to be capable of the cleavage of RNA, linear DNA from phage lambda, and all forms of plasmid DNA. We established that treatment of the HL-60 cell culture with this peptide (10(-6) M) results in an increase in the number of apoptotic cells and suggested that HLDF is involved in processes of apoptosis.     

Intracellular localization of platelet-activating factor synthesis in human neutrophils

Human neutrophils were homogenized and fractionated on a continuous sucrose gradient to assess the subcellular location of acetyl-CoA: lyso-PAF acetyltransferase and of newly synthesized PAF (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Acetyltransferase activity showed two subcellular locations in resting neutrophils. One of them cofractionated with plasma membrane and endoplasmic reticulum markers, whereas another major location corresponded to a region of the gradient enriched in tertiary granules. No PAF was detected in resting neutrophils, but PAF synthesis was induced by cell stimulation with ionophore A23187. Most of the newly synthesized PAF was found cell-associated, showing a bimodal subcellular distribution similar to that found for acetyltransferase activity in activated cells. PAF and acetyltransferase were located in a light membrane fraction, enriched in plasma membrane and endoplasmic reticulum, and in an ill-defined region of the gradient between the specific and azurophilic granules in A23187-stimulated cells. These data support the involvement of the acetyltransferase pathway in the synthesis of PAF induced by ionophore A23187, and demonstrate the synthesis and accumulation of newly synthesized PAF in a light membrane fraction as well as in an intracellular dense organelle upon neutrophil activation.     

A novel CoA-independent transacetylase produces the ethanolamine plasmalogen and acyl analogs of platelet-activating factor (PAF) with PAF as the acetate donor in HL-60 cells.

In this study, we demonstrate the presence of a unique membrane-associated transacetylase that transfers the acetate group from platelet-activating factor (PAF) to lysoplasmalogen (in the presence of EDTA and sodium acetate) with the formation of 1-alk-1-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alk-1-enylacetyl-GPE). The identity of alk-1-enylacetyl-GPE was confirmed by acid hydrolysis, phospholipases A2 or C treatment and derivatization by fluorodinitrobenzene. The transacetylase has no requirement for Ca2+, Mg2+, or CoA and a broad pH optimum (7.0-8.0) with Km values of 12.0 microM for PAF and 106.4 microM for lysoplasmalogens. The enzyme activity from the isolated membrane fraction is not changed when whole cells are supplemented with 20:4, induced to differentiate into granulocytes, or treated with ionophore A23187. Radyllyso-sn-glycero-3-phosphocholine (GPC), radyllyso-GPE, acyllyso-sn-glycero-3-phosphoserine (GPS), acyllyso-sn-glycero-3-phosphoinositol (GPI), alkyllyso-sn-glycero-3-phosphate (GP), acyllyso-GP, or cis-9-octadecen-1-ol can also serve as acetate acceptors, whereas alkylglycerol, acylglycerol, or cholesterol are inactive. Differences in substrate acceptor specificity, sensitivity toward phenylmethylsulfonyl fluoride, and response to temperature suggest that the CoA-independent transacetylase and the CoA-independent transacylase that transfers long-chain acyl moieties are two separate enzymes. With intact differentiated HL-60 cells, [3H]acetate from [3H]PAF can be incorporated into alk-1-enylacetyl-GPE in the presence of ionophore A23187, but not in its absence. Moreover, phospholipase A2 inhibitors (p-bromophenacyl bromide and mepacrine) block the transacetylation process in whole cell system. These results indicate the production of alk-1-enyllyso-GPE is a rate-limiting factor for the subsequent transacetylation step during cell activation. We conclude that the transacetylase may participate in the biosynthesis of ethanolamine plasmalogen and acyl analogs of PAF, in vivo, fine-tuning of PAF biological responses, and cross-talk between de novo and remodeling pathways of PAF biosynthesis.     

Immunofluorescent localization of platelet-activating factor (PAF) in the rat

Summary An immunofluorescent staining method for detecting platelet-activating factor (PAF) is described. This method employs a polyclonal anti-PAF rabbit antibody. When rat brain, heart, lung, liver or kidney tissue was stained using this method, the heart, lung and kidney exhibited PAF-specific staining. Analysis of the amount of PAF in different organs, either by immunofluorescence or by bioassay, showed that kidney tissue contains the greatest amount of PAF.     

EMF induces differentiation in HL-60 cells

This investigation provides evidence that a 60-Hz electromagnetic field (EMF) at 1 gauss (G) can drive differentiation of cultured hematopoietic progenitor cells. HL-60 cells are known to differentiate from a nonphagocytic suspension culture to an attached fibroblast-like culture with high phagocytic activity in the presence of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbal-13-acetate (TPA). The effect of 60-Hz EMF at 1 G on differentiation is approximately equivalent to treatment of the cells with 250-500 pg/ml TPA. Furthermore, the effect of both EMF and TPA treatment on differentiation is additive at low TPA concentrations. The results strongly suggest similarities between the effects of TPA treatment and EMF exposure and thus provide an approach for tracing the origins of the molecular effects of EMF exposure, as many transduction pathways in the differentiative process are defined.     

Bone marrow extracellular matrix induces HL-60 cells to produce an autonomous differentiation factor.

Conditioned medium from cultures of HL-60 myeloid leukemia cells grown on extracellular bone marrow matrix induces macrophage-like differentiation of fresh HL-60 cells. The active medium component is sensitive to protease treatment, indicating that it is a protein, but it is heat stable. Conditioned medium from HL-60 cells grown on protease-treated bone marrow matrix still contains the active component. Thus, it appears that the differentiation-inducing protein is produced by HL-60 cells and is not released from the bone marrow matrix. To identify this differentiation factor, RNA was isolated from HL-60 cells grown on bone marrow matrix and assayed by Northern analysis for expression of mRNA for human differentiation factor, tumor necrosis factor, and macrophage colony-stimulating factor, all inducers of monocyte/macrophage differentiation. Expression of differentiation factor, tumor necrosis factor, or macrophage colony-stimulating factor mRNA was not enhanced in HL-60 cells grown on matrix compared to cells grown on uncoated plastic flasks. Thus, the maturation factor does not appear to be differentiation factor, tumor necrosis factor, or macrophage colony-stimulating factor within the limits of detection of Northern analysis. Elution of the active conditioned medium fraction on a Sephacryl S-200 column revealed a molecular weight of approximately 40,000. The active protein eluted on a DEAE-cellulose ion-exchange column at an ionic strength of 0.3 M NaCl, indicating that it is fairly anionic. Thus, bone marrow matrix is able to induce HL-60 cells to produce a maturation-inducing 40 kilodalton protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamine promotes colony formation in bone marrow and HL-60 cells; accelerates myeloid differentiation in induced HL-60 cells

Several studies indicate that glutamine is a critical requirement for growth of cultured cells. The present studies describe the effect of deprivation of glucose or glutamine on mouse bone marrow cell or HL-60 cell colony formation in soft agar. The mouse bone marrow cells were induced to undergo granulocyte/macrophage type differentiation by colony-stimulating factor. Glutamine, but not glucose, was found to be an indispensable metabolite for the cloning of HL-60 cells or differentiated mouse bone marrow cells. In addition, the effect of glucose or glutamine on the rate of differentiation of dimethylsulfoxide (DMSO)-induced HL-60 cells in liquid culture was studied. Glutamine was found to be superior to glucose in its ability to support the proliferation and myeloid differentiation of HL-60 cells. When an optimal concentration of DMSO was used, the rate of differentiation of induced HL-60 cells was found to be a function of the concentration of glutamine. In addition to these studies glutamine utilization and product formation was studied in induced and uninduced HL-60 cells after 60 min incubation with 1 mM initial glutamine concentration. The fractional distribution of the glutamine carbon into its metabolic products remained unchanged in induced versus uninduced HL-60 cells. However, the rate of utilization of glutamine and product formation by terminally differentiated HL-60 cells was less than the rate of utilization of glutamine by undifferentiated HL-60 cells. The data do not explain the role of glutamine in the complex process of differentiation but establish the critical requirements for glutamine, but not glucose, in myelopoiesis.     

Glutamine promotes colony formation in bone marrow and HL-60 cells; Accelerates myeloid differentiation in induced HL-60 cells

Summary Several studies indicate that glutamine is a critical requirement for growth of cultured cells. The present studies describe the effect of deprivation of glucose or glutamine on mouse bone marrow cell or HL-60 cell colony formation in soft agar. The mouse bone marrow cells were induced to undergo granulocyte/macrophage type differentiation by colony-stimulating factor. Glutamine, but not glucose, was found to be an indispensable metabolite for the cloning of HL-60 cells or differentiated mouse bone marrow cells. In addition, the effect of glucose or glutamine on the rate of differentiation of dimethylsulfoxide (DMSO)-induced HL-60 cells in liquid culture was studied. Glutamine was found to be superior to glucose in its ability to support the proliferation and myeloid differentiation of HL-60 cells. When an optimal concentration of DMSO was used, the rate of differentiation of induced HL-60 cells was found to be a function of the concentration of glutamine. In addition to these studies glutamine utilization and product formation was studied in induced and uninduced HL-60 cells after 60 min incubation with 1 mM initial glutamine concentration. The fractional distribution of the glutamine carbon into its metabolic products remained unchanged in induced versus uninduced HL-60 cells. However, the rate of utilization of glutamine and product formation by terminally differentiated HL-60 cells was less than the rate of utilization of glutamine by undifferentiated HL-60 cells. The data do not explain the role of glutamine in the complex process of differentiation but establish the critical requirements for glutamine, but not glucose, in myelopoiesis. This work has been supported by USPHS Grants AM 31624 and CA 00859 and a Faculty Research Grant from Texas College of Osteopathic Medicine.     

Dictyostelium cells produce platelet-activating factor in response to cAMP.

Evidence is provided that Dictyostelium discoideum cells produce 1-O-alkyl-2-delta-acetyl-O-sn-glycero-3-phosphocholine (platelet-activating factor, PAF). D. discoideum PAF has been characterized as being identical with mammalian platelet-activating factor, based on its stimulation of rabbit platelet aggregation, its physicochemical properties and mass spectrum. The basal activity of PAF increases after starvation and during aggregation and declines at the slug stage. PAF is not detected in the extracellular space. Cell treatment with cAMP pulses stimulates a transient accumulation of PAF, probably via activation of a cAMP-dependent acetyltransferase, suggesting a possible involvement of PAF in cAMP-regulated processes in Dictyostelium.     

Antimycin A-induced apoptosis of HL-60 cells

BACKGROUND: Previous experiments in our laboratory investigating apoptosis induced in HL-60 cells by camptothecin (CAM) have revealed that the sequence and rapidity of the apoptotic phenomena in an individual cell depend on the proliferative state of that cell when it encounters CAM. The role of mitochondria in HL-60 apoptosis was explored using an inhibitor of oxidative phosphorylation, antimycin A (AMA). METHODS: Changes in cell light scatter, binding of annexin V-fluorescein isothiocyanate (FITC), uptake of propidium iodide (PI), and DNA content after membrane fixation/permeabilization were monitored by flow cytometry. Z-VAD-FMK was used to inhibit caspases. Fluorescence microscopy was used to examine cell morphology. RESULTS: Cells in the G1 phase of the cell cycle were the first to exhibit signs of apoptosis in response to 100 microM AMA and some of these cells disintegrated without exposing to phosphatidylserine (PS). Caspase inhibition prevented fragmentation of DNA, the nucleus, and the cell, but only delayed PS exposure and loss of plasma membrane integrity. CONCLUSIONS: The highly active mitochondria of G1-phase HL-60 cells make them particularly sensitive to AMA. PS exposure and plasma membrane damage are mediated by noncaspase molecules released from mitochondria. We hypothesize that if mitochondria are subjected to a sufficiently severe insult, whether indirectly as a result of extensive CAM-induced DNA damage or directly by the effect of AMA on electron transport, the nature and quantities of the proapoptotic molecules released are such that apoptosis proceeds to the point of cell disintegration before the PS exposure pathway is complete.     

Inducible gene expression of activin A/erythroid differentiation factor in HL-60 cells.

Treatment of HL-60 cells with 12-O-tetradecanoyl-phorbol 13-acetate (TPA) for 48 h induced expression of mRNA of beta A chain of activin A/erythroid differentiation factor. Under the same condition, interferon-gamma caused a slight increase in beta A chain mRNA, whereas 1 alpha, 25-dihydroxyvitamin D3, dimethylsulfoxide and all-trans-retinoic acid failed to induce this mRNA in HL-60 cells. Furthermore, 4 h-treatment with TPA or lipopolysaccharide (LPS) induced a marked increase in beta A chain mRNA levels in interferon-gamma-pretreated HL-60 cells. In the cells pretreated with 1 alpha, 25-dihydroxyvitamin D3, TPA and LPS induced as little increase in beta A chain mRNA as in the control cells. Neither alpha nor beta B chain mRNA was detected in any sample. These results indicate that interferon-gamma has a priming effect on the activation of activin A/erythroid differentiation factor gene by TPA or LPS in HL-60 cells.     

Identification of platelet-activating factor as the inflammatory lipid mediator in CCl4-metabolizing rat liver

Unmitigated oxidative stress is deleterious, as epitomized by CCl4 intoxication. In this well-characterized model of free radical-initiated damage, liver metabolism of CCl4 to CCl3. causes lipid peroxidation, F-ring isoprostane formation, and pathologic leukocyte activation. The nature of the mediator that couples oxidation to the hepatotoxic inflammatory response is uncharacterized. We found that oxidatively modified phosphatidylcholines were present in the livers of CCl4-exposed rats and not in livers from control animals, that CCl4 metabolism generated lipids that activated 293 cells stably transfected with the human platelet-activating factor (PAF) receptor, and that this PAF-like activity was formed as rapidly as isoprostane-containing phosphatidylcholine (iPC) during oxidation. iPC and the PAF-like activity also had similar chromatographic properties. The potential for iPC activation of the PAF receptor has been unexplored, but we conclude that iPC themselves did not activate the PAF receptor, as phospholipase A1 hydrolysis completely destroyed iPC, but none of the PAF-like bioactivity. Oxidatively fragmented phospholipids are potent agonists of the PAF receptor, but mass spectrometry characterized PAF as the major inflammatory component coeluting with iPC. Oxidatively fragmented phospholipids and iPC are markers of free radical generation in CCl4-intoxicated liver, but PAF generation by activated hepatic cells generated the inflammatory agent.     

The expression and localization of plasma platelet-activating factor acetylhydrolase in endotoxemic rats

The production of platelet-activating factor (PAF) and PAF-like phospholipids that also bind the PAF receptor are implicated in numerous pathological situations including bacterial endotoxemia and injury-induced oxidative damage. PAF and PAF-like phospholipids are hydrolyzed and inactivated by the enzyme PAF acetylhydrolase. In the intact rat, infusion of lipopolysaccharide (LPS) into a mesenteric vein served as an acute, liver-focused model of endotoxemia. We determined that the liver responds to LPS exposure with the production of plasma-type PAF acetylhydrolase mRNA and protein expression specifically in the resident macrophages of the liver. Liver macrophages, defined immunohistochemically using antibodies against ED1, present in livers from saline-treated animals contained no detectable PAF acetylhydrolase. Twenty-four hours following in vivo LPS administration, immunohistochemistry detected a slight increase in the number of ED1 staining cells and the ED1-positive cells now contained an abundance of PAF acetylhydrolase. The systemic administration of LPS resulted in increased expression of PAF acetylhydrolase in several tissues. Of the tissues examined, the greatest increase in PAF acetylhydrolase expression was observed in lung followed by increases in spleen, liver, kidney, and thymus. Additionally, the expression of PAF acetylhydrolase mRNA increased in circulating leukocytes and in peritoneal macrophages in response to systemic exposure to LPS. We examined the regulation of PAF acetylhydrolase expression and demonstrated the administration of the PAF receptor antagonists, BN 50739 and WEB 2170, inhibited by 50% the increase in PAF acetylhydrolase expression in response to LPS. The up-regulation of the plasma-type PAF acetylhydrolase expression constitutes an important mechanism for elevating the local and systemic ability to inactivate PAF and oxidized phospholipids in order to minimize PAF-mediated pathophysiology consequent from exposure to endotoxin. The abundance of PAF acetylhydrolase production in the liver lobule likely limits endotoxin-mediated tissue damage due to PAF synthesis.     

Geranylgeranylacetone induces apoptosis in HL-60 cells.

Geranylgeranylacetone (GGA) induces apoptosis in human leukemia HL-60 cells in a dose- and time-dependent manner. This effect was completely prevented by the pan-caspase inhibitor z-Val-Ala-Asp(OMe) fluoromethylketone, thereby implicating the caspase cascade in the process. Prior to DNA fragmentation, GGA treatment markedly activated caspase-3(-like) proteases, which might be responsible for the observed apoptosis. In addition, GGA treatment interfered with the processing and membrane localization of Rap1 and Ras, and these changes may be a result of apoptosis. Moreover, nitric oxide donors significantly accentuated the GGA-induced apoptosis, suggesting that the apoptotic pathway induced by GGA might be regulated by a redox-sensitive mechanism. Taken together, these data suggest that the isoprenoid, GGA, is an effective inducer of apoptotic cell death in HL-60 cells.     

Ethanol induced apoptosis in human HL-60 cells

In this study flow cytometric and morphologic methods of apoptosis detection in human promyelocytic leukemia cell line HL-60 were compared. HL-60 cells were harvested at 4, 7, 16, 24 a 48 hours after induction of apoptosis by 3 % ethanol. Little changes were observed both by flow cytometry (decrease of forward scatter, increase of unprocessed cells staining with APO2.7 antibody) and viability determination by Trypan-blue staining until after 7 hours. However, after 4 hours morphologic changes were observed in the nuclear and cytoplasmic structures using Diff-Quik stained cytospin preparations and standard light microscopic techniques (50% apoptotic cells). The same results were obtained by flow cytometric measurement of sub-diploid DNA content (sub-G1 cells), and an increase of staining with APO2.7 antibody in cells permeabilised by digitonin prior to staining. After 7 hours almost all cells exhibited apoptotic morphology. After 16 hours the cell size (forward scatter) decreased significantly, and 54% of unprocessed cells were APO2.7 positive. After 24 hours only 6% of cells were alive (high forward scatter) and these cells were APO2.7 negative. The HL-60 cells did not proliferate during the cultivation in 3% ethanol, and after 48 hours all stained by Trypan blue. HL-60 leukemic cells were CD34-/AC133-, CD33+/CD15+, and only 2% of the cells were CD95+. Induction of apoptosis by ethanol did not enhance CD95 antigen expression.     

Regulation of platelet-activating factor receptor and platelet-activating factor receptor-mediated biological responses by cAMP in rat Kupffer cells

Treatment of cultured Kupffer cells with the beta-adrenergic agonist isoproterenol (10 microM) for a short period of time (30 min) attenuated the subsequent platelet-activating factor (PAF)-induced arachidonic acid release and cyclooxygenase-derived eicosanoid (e.g. thromboxane B2 and prostaglandin E2) production. This effect of isoproterenol was highly specific since the alpha-adrenergic agonist phenylephrine and the beta-adrenergic antagonist propranolol had no effect on the stimulatory effect of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC). The inhibitory effect of isoproterenol on the AGEPC-induced arachidonic acid release was demonstrated through the use of a specific beta-adrenergic subtype agonist and antagonist to be mediated by beta 2-adrenergic receptors on Kupffer cells. These inhibitory effects of isoproterenol can be mimicked by dibutyryl cAMP but not by dibutyryl cGMP, suggesting that a cAMP-dependent mechanism is likely involved in the regulatory action of isoproterenol. Ligand binding studies indicated that short term (i.e. 30 min) treatment of the cultured Kupffer cells with either isoproterenol or dibutyryl cAMP had no effect on the specific [3H]PAF binding. However, long term incubation (9-24 h) with dibutyryl cAMP caused down-regulation of the PAF receptors in rat Kupffer cells. Forskolin (0.1 mM), an adenylyl cyclase activator, down-regulated the surface expression of the AGEPC receptors more rapidly, decreasing the specific [3H]AGEPC binding by approximately 40% within 2 h. The receptor regulatory effect of dibutyryl cAMP and forskolin was time- and concentration-dependent. These observations suggest that a cAMP-dependent mechanism coupled with beta 2-adrenergic receptors may have important regulatory effects on the PAF receptor and post-receptor signal transducing mechanisms for PAF in hepatic Kupffer cells.