Lipopolysaccharide and phorbol 12-myristate 13-acetate both impair monocyte differentiation, relating cellular function to virus susceptibility

Both lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) impeded monocyte to macrophage differentiation with respect to typical phenotypic modulation and certain phagocyte-related processes. The down-regulation of the porcine monocyte marker SWC1, and up-regulation of the SWC9 macrophage marker were retarded, but not inhibited, as was the differentiation-associated down-regulation of p53 and myeloperoxidase. Despite this clear impairment of macrophage differentiation, not all cellular functions were equally susceptible. Both agents inhibited phagocytosis, but not low-density lipoprotein receptor-associated endocytosis. Only LPS inhibited tartrate-resistant acid phosphatase up-regulation. In contrast, increase of vacuolar acidification rates was more susceptible to PMA. The activity of certain endosomal/lysosomal enzymes - esterase, nucleotidase, peroxidase and cathepsins - was generally enhanced by both LPS and PMA. This contrasted with autophagosomal activity, detected through the induction of an antiviral state. Disruption of autophagosomes and lysosomes (methionine-O-methyl ester), but not lysosomes alone (glycyl-L-phenylalanine) reversed LPS-induced inhibition of virus replication, without influencing the PMA-induced antiviral effect. Thus, PMA is similar to LPS in inhibiting monocyte to macrophage differentiation, when primary blood monocytes are employed, but not all pathways are equally susceptible. The analyses demonstrate that the pathways modulated during monocyte differentiation function somewhat independently. Moreover, certain functions of monocytic cells are more important with respect to the outcome of virus infection, with autophagosomal activities in particular favouring cell survival.     

Function and stimulus-specific effects of phorbol 12-myristate 13-acetate on human polymorphonuclear neutrophils: Autoregulatory role for protein kinase C in signal transduction

Exposure of polymorphonuclear neutrophils (PMNs) to phorbol 12-myristate 13-acetate (PMA) resulted in a concentration-dependent (1–10 ng/ml) inhibition of granule exocytosis induced with the receptor-specific ligands,N-formylmethionyl-leucyl-phenylalanine (FMLP), pepstatin A, 5(S),12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (LTB₄), and acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), PMA exerted a marginal inhibitory effect on calcium ionophore A23187-induced PMN degranulation, and the PMA analog, 4-phorbol 12, 13-didecanoate (4-PDD), was inactive. However, PMA potentiated AGEPC, pepstatin A, FMLP, LTB₄, and A23187-stimulated Superoxide anion (O ₂ ^– ) production. The mobilization of intracellular sequestered calcium (Ca²⁺) by the receptor-specific ligands, as reflected by a rise in the cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) in PMNs loaded with the CA²⁺-sensitive dye, Fura-2, was suppressed by PMA. A protein kinase C (PKC) inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) reversed the PMA-mediated inhibition of PMN degranulation and intracellular CA²⁺ mobilization. However, another, but less potent PKC inhibitor,N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), had no effect on the inhibition of PMN activation by PMA.     

Trilinolein potentiates the pro-aggregating effect of phorbol-12-myristate 13-acetate in human polymorphonuclear leukocytes

Trilinolein, a triacylglycerol with linoleic acid as the only type of fatty acid residue in all three of the glycerol esterified positions, was recently reported to have an antiplatelet effect, mediated through stimulating nitric oxide and cyclic guanosine monophosphate (GMP) formation. In our study, trilinolein induced aggregation of human polymorphonuclear neutrophils (PMNs) and, pretreatment with 0.1 nM trilinolein enhanced phorbol-12-myristate 13-acetate (PMA) induced aggregation. Further investigation showed that trilinolein at concentrations ranging from 0.1 nM to 10 microM increased cyclic GMP formation after 10 min of incubation with PMNs. Pretreatment of trilinolein with 10 microM d-sphingosine, before being incubated with PMNs, attenuated the stimulatory effect of trilinolein on cyclic GMP formation, and pretreatment of 10 microM d-sphingosine also attenuated the aggregation induced by PMA and trilinolein. We conclude that trilinolein can induce the aggregation of human PMNs, and enhance the aggregation induced by PMA.     

Effects of thymidine and phorbol 12-myristate 13-acetate on erythroid differentiation of K562 cells and their sensitivity to nonspecific lysis by rat splenocytes

Induction of hemoglobin synthesis in K562 cells by thymidine (2 mM)in vitro did not significantly enhance major histocompatibility complex antigen-unrestricted lysis of splenocyte-exposed K562 cells. Inhibition of thymidine-induced hemoglobin synthesis by simultaneous incubation of cells with thymidine and phorbol 12-myristate 13-acetate (100 nM) decreased cytolytic activity of splenocytes against K562 cells. Preincubation of tumor cells with phorbol ester alone did not affect major histocompatibility complex antigen-unrestricted lysis induced by rat splenocytes but decreased the basal level of hemoglobin synthesis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 11, pp. 521–524, November, 1999     

Rapid,transient effects of the protein kinase C activator phorbol 12-myristate 13-acetate on activity and trafficking of the rat high-affinity choline transporter

Cholinergic neurons rely on the sodium-dependent choline transporter CHT to provide choline for synthesis of acetylcholine. CHT cycles between cell surface and subcellular organelles, but little is known about regulation of this trafficking. We hypothesized that activation of protein kinase C with phorbol ester modulates choline uptake by altering the rate of CHT internalization from or delivery to the plasma membrane. Using SH-SY5Y cells that stably express rat CHT, we found that exposure of cells to phorbol ester for 2 or 5 min significantly increased choline uptake, whereas longer treatment had no effect. Kinetic analysis revealed that 5 min phorbol ester treatment significantly enhanced V_max of choline uptake, but had no effect on K_m for solute binding. Cell-surface biotinylation assays showed that plasma membrane levels of CHT protein were enhanced following 5 min phorbol ester treatment; this was blocked by protein kinase C inhibitor bisindolylmaleimide-I. Moreover, CHT internalization was decreased and delivery of CHT to plasma membrane was increased by phorbol ester. Our results suggest that treatment of neural cells with the protein kinase C activator phorbol ester rapidly and transiently increases cell surface CHT levels and this corresponds with enhanced choline uptake activity which may play an important role in replenishing acetylcholine stores following its release by depolarization.     

Dermal carcinogenicity in transgenic mice: effect of vehicle on responsiveness of hemizygous Tg.AC mice to phorbol 12-myristate 13-acetate (TPA).

The Tg.AC mouse is being evaluated for use in short-term carcinogenicity bioassays. Because the dermal test protocol necessitates dissolving test agents we determined the effects of several solvents on responsiveness of hemizygous mice to dermal applications of the classical skin tumor promoter. phorbol 12-myristate 13-acetate (TPA). Mice of both sexes received dermal applications of either acetone (negative control) or TPA in various vehicles [acetone, 100% methanol, 70% and 100% ethanol, DMSO and mixtures of acetone and ethanol (1:1), acetone and DMSO (4:1 and 1: 1). and acetone and olive oil (4:1)]. Negative control animals did not exhibit papillomas. When administered in acetone. ethanolic or methanolic vehicles TPA caused prompt and robust papillomatous responses. TPA was also tumorigenic in all nonalcoholic vehicles, except the acetone-olive oil mixture. Papilloma responses were generally delayed when TPA was applied in the nonalcoholic solvents but the distinction between TPA-dosed and negative control groups was unequivocal. These results show that choice of vehicle may affect the quantitative and qualitative nature of the response of Tg.AC mice to TPA, but 8 of 9 vehicles proved satisfactory for delivery of TPA.     

Measles virus induces apoptotic cell death in lymphocytes activated with phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore

Peripheral blood mononuclear cells (PBMC) and T lymphocytes were infected with measles virus (MV) and cultured with a protein kinase C (PKC) activator, PMA and a calcium ionophore, ionomycin. After stimulation, cell viability and incorporation of 5-bromo-2′-deoxyuridine (BrdU) were decreased in MV-infected cells compared with mock-infected cells. DNA content analysis and terminal deoxytransferase (TdT)-mediated dUTP nick end labelling demonstrated that the hypodiploid fraction and DNA fragmentation were increased in MV-infected, T lymphocytes activated with PMA plus ionomycin. These data suggest that MV induces apoptotic cell death in cells activated by PMA plus ionomycin. In contrast to stimulation with PMA plus ionomycin, mitogenic stimulation with phytohaemagglutinin (PHA) did not induce apoptotic cell death in MV-infected cells, although cell proliferation was suppressed. Apoptosis induced in stimulated, MV-infected cells may be one mechanism of immunosuppression.     

Induction of CD3δεω) by phorbol 12-myristate 13-acetate

The effect of phorbol 12-myristate 13-acetate (PMA) on the synthesis, assembly and processing of the components of the T cell receptor (TcR) was studied with special focus on the CD3ω chain. Treatment of the human leukemic T cell line Jurkat with PMA increased the synthesis of the Tiα, CD3γ and CD3ζ chains two-to threefold and the synthesis of Tiβ and CD35δεω complexes five- to sevenfold as assessed by metabolic labeling, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by scanning densitometry. The amount of total assembled TcR complexes increased approximately threefold and the maturation of the TcR was not affected as determined by analysis of oligosaccharide side chain processing in the Golgi apparatus. Activation of Jurkat cells with anti-CD3 monoclonal antibody, calcium ionophore, or mitogenic lectins did not affect the synthesis of the TcR components. In other cells studied (the human leukemic T cell line CEM, a panel of variants of the Jurkat T cell line and peripheral blood mononuclear cells) PMA also increased the synthesis of the TcR components. However, for all cell lines studied the amount of TcR complexes expressed on the cell surface was decreased after 16 h of PMA treatment. Based on these results we propose a role of CD3ω in retention of TcR complexes. From PMA-treated CEM cells more than 50-fold the amount of CD3δεω complexes was immunoprecipitated as compared to the amount obtained from untreated Jurkat cells, and these observations indicate that the CEM cell line may be a qualified candidate for purification of CD3ω.     

Stimulation of protein phosphorylation in mixed glial cell primary cultures and subcultures by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate

Glial cell primary cultures consisting of protoplasmic and fibrous astrocytes, oligodendrocytes and progenitor glial cells incubated in medium containing 0.5% foetal calf serum and treated with 25 nM 12-o-tetradecanoylphorbol-13-acetate (TPA) for periods between 15 and 60 min showed a stimulation of protein phosphorylation which was most prominent in a polypeptide with a molecular weight of about 80,000 Da. Glial subcultures consisting mainly of Type 2 astrocytes, oligodendrocytes and progenitor glia showed a similar TPA stimulation of 80,000 Da protein phosphorylation detectable within 1 min of phorbol ester addition. TPA treatment of primary glial cultures led to an enhancement of phospholipid turnover but exposure of primary glial cultures to concentrations of TPA up to 250 nM caused no morphological change in protoplasmic astrocytes. 4-Phorbol (4-PH) or dimethylsulfoxide (DMSO) was without effect on protein phosphorylation or lipid turnover in glial cultures.     

Effect of 12-O-tetradecanoyl phorbol 13-acetate on solute transport and production of cAMP in isolated frog skin

In the present study we have examined the action of the phorbol diester tetradecanoyl phorbol acetate, an activator of protein kinase C, on the transepithelial transport of sodium, chloride and water and the production of cAMP in the isolated frog skin epithelium (Rana esculenta). Addition of tetradecanoyl phorbol acetate to the mucosal solution resulted initially in an increase in the short-circuit current, which was followed by a progressive decrease. If the short-circuit current was first activated by addition of the antidiuretic hormone, arginine vasotocin, then the addition of tetradecanoyl phorbol acetate resulted only in a pronounced inhibition. The changes in the short-circuit current were the result of changes in the active influx of Na+. The effect of tetradecanoyl phorbol acetate on the intracellular potential measured under short-circuited conditions (Vscc) was time-dependent. Just after addition of tetradecanoyl phorbol acetate to the mucosal solution, Vscc depolarized; this was followed by a slight hyperpolarization, after which Vscc continued to decline. The inhibition of the Na+ transport by tetradecanoyl phorbol acetate was associated with a decline in the response to the antidiuretic hormone (arginine vasotocin), but the ability of arginine vasotocin to increase the cellular level of cAMP and to stimulate the osmotic water flow was not affected by the presence of tetradecanoyl phorbol acetate. In skin halves in which the short-circuit current was stimulated with arginine vasotocin, addition of tetradecanoyl phorbol acetate resulted in a dose-dependent inhibition of the short-circuit current, but only minor changes in Vscc were observed. The results presented suggest that the addition of tetradecanoyl phorbol acetate to the isolated frog skin first increases and then decreases the arginine vasotocin-sensitive sodium permeability of the apical membrane. This might be due to a stimulating effect of tetradecanoyl phorbol acetate on both the activation and deactivation (turnover) of the sodium channels.     

Mitogenic effect of 12-0 tetradecanoyl phorbol 13-acetate on non-human primate mononuclear cells andin vitro interaction with Epstein-Barr virus transformation

Summary 12-0 tetradecanoyl phorbol 13-acetate (TPA), known to promote tumors in mice and also to enhance viral transformation as well as induction of viral antigens, was demonstrated to be mitogenic to peripheral blood mononuclear cells from rhesus monkeys and three species of marmosets. Even though mitogenic response varied between species and within species, the mitogenic dose response due to TPA was comparable to the response of phytohemagglutinin (PHA-P). A significant synergistic effect of PHA-P and TPA on mononuclear cells from marmosets was evident when they were used together at optimal doses. TPA also increased the efficiency ofin vitro transformation of marmoset lymphocytes by Epstein-Barr virus.With 5 FiguresThis study was supported partly by National Cancer Institute, Division of Cancer Cause and Prevention, NIH, Program Resource Contracts No. 1-CP-8-1023, 1-CP-VO-81039-66 and Grant No. R-01-CA-21665 to M.N.     

Actions of the protease inhibitor phenylmethylsulfonyl fluoride on neutrophil granule enzyme secretion and superoxide production induced by fMet-Leu-Phe and phorbol 12-myristate-13-acetate

The protease inhibitor, phenylmethylsulfonyl fluoride inhibits granule enzyme release and, above 1 mM, superoxide production from rabbit peritoneal neutrophils induced by the chemotactic peptide, fMet-Leu-Phe. At concentrations below 1 mM, it enhances superoxide production. Superoxide generation stimulated by phorbol 12-myristate-13-acetate is increased by phenylmethylsulfonyl fluoride at all concentrations studied. Phenylmethylsulfonyl fluoride has no effect on the rise in intracellular calcium or the depolarization induced by fMet-Leu-Phe but does decrease the extent of repolarization and abolishes hyperpolarization. It depresses actin polymerization and abolishes cytoplasmic alkalinization caused by fMet-Leu-Phe. The increased phosphorylation induced by phorbol 12-myristate-13-acetate in four of the five proteins studied was not affected by phenylmethylsulfonyl fluoride, but the increased phosphorylation of the fifth, a 21-kD protein was enhanced. We conclude that phenylmethylsulfonyl fluoride acts on inhibitory and enhancing processes or steps induced by fMet-Leu-Phe which are subsequent to or independent of calcium mobilization and protein kinase C activity.     

TPA (12-O-tetradecanoyl-phorbol-13-acetate) activation and differentiation of human peripheral B lymphocytes

The effect of the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate) on normal human peripheral blood and tonsil B lymphocytes was investigated. A strong DNA-synthesis response with the maximum at day 4 was detected. This response was, however, inhibited by increasing concentrations of serum in the medium. The membrane Ig expression was changed with a rapid decrease in IgD expression and a slower decrease in IgM and IgG expression. TPA-induced Ig secretion was detected in 12 out of 22 tested donors and the response was found to be independent of T cells and macrophages. The expression of four monoclonal antibody-detected B cell activation and differentiation markers, B1, B2, LB1 and BB1, was followed. The results indicate activation and differentiation of the B cells.     

Modulation of human T-cell differentiation markers by 12-O-tetradecanoylphorbol-13-acetate

The analysis of seven differentiation markers following incubation with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined in the human leukemic T-cell line MOLT-3. Significant changes were observed in the activity of the markers terminal deoxynucleotidyl transferase (TdT). spontaneous proliferation and the ability of these cells to bind sheep erythrocytes. Levels of human thymus-leukemia-associated antigen (HThy-L) recently identified as a low molecular weight form of adenosine deaminase (ADA), were reduced by about 50%. No significant changes were observed in ecto-5'-nucleotidase [5'-NT) activities, in the proliferative response to PHA, or in the expression of IA-like antigens. These data and the time kinetics of the changes suggest that following incubation of these T-lymphoblasts with TPA there is a sequential loss of TdT, loss of the capacity for spontaneous proliferation, and the appearance of receptors for sheep erythrocytes. Subsequently there is a decrease in the level of HThy-L/ADA. This sequence appears to follow that proposed for prethymic precursor T-cell differentiation following activation with thymic epithelium.     

The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate,enhances the evoked quanta release of acetylcholine at the frog neuromuscular junction

The possible role of protein kinase C in the regulation of quantal transmitter release was studied at the frog neuromuscular junction by using the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a compound known to mimic the effects of the physiological activator of the enzyme, endogenous diacylglycerol. The main effect of the phorbol ester was to increase the quantal content,m, of the endplate potential. The initial values ofm were adjusted over a wide range by changing the Ca²⁺ concentration of the extracellular medium, and the TPA-induced fractional increase inm was significantly greater at junctions with a lower initial quantal content. On the other hand, the absolute increases inm induced by the phorbol ester were positively correlated with the square root of the initial quantal content. The possible physiological significance of this correlation is discussed in view of the well known relationship between extracellular Ca²⁺ concentration and the quantal content of the end plate potential.     

Colonic inflammation in the rabbit induced by phorbol-12-myristate-13-acetate

Neutrophil (PMNL) infiltration of inflamed colonic tissue is a prominent feature of human inflammatory bowel disease (IBD). Colitis was established in New Zealand white rabbits by the intrarectal instillation of 1.5 mg/kg (in 10 ml 20% ethanol) phorbol-12-myristate-13-acetate (PMA) and assessed by visual grading of colonic inflammation, levels of the neutrophil marker enzyme myeloperoxidase (MPO), and histological examination. After 24 h there was a significant (P<0.001) increase in MPO levels in the PMA-treated colons compared to ethanol control. There was also increased inflammation based on visual scoring. Histologically, PMA-treated colons were necrotic with focal ulceration, heavy PMNL infiltration and edema at 24 h; by 96 h colitis was sustained with mild edema, crypt abscesses, and a staining pattern suggesting altered mucus quality. These results suggest that PMA-induced colitis in rabbits may be a new model of IBD in which to evaluate drugs known to mitigate the inflammatory process.     

Neurite-promoting effects of 12-O-tetradecanoylphorbol-13-acetate on chick embryo neurons

The addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to defined growth medium enhanced the survival of ciliary neurons in dissociated cultures and promoted the rapid development of neurites from sympathetic and parasympathetic ganglion explants. Explants of the central nervous system including the cerebral cortex and spinal cord of chick embryos were, however, unresponsive to either 10 or 100 ng/ml of TPA.