Stimulation and inhibition of secretion by phorbol myristate acetate in different cell types

In washed human platelets and in HL60 granulocytes phorbol myristate acetate (PMA, 1-2000nM) synergised with threshold concentrations of secretogogues to induce a sustained maximum secretory response. Likewise, superoxide production from HL60 cells maintained a maximal response at PMA concentrations between 30-300nM. At concentrations up to 10nM PMA also augmented calcium ionophore, A23187, stimulated histamine release from rat peritoneal mast cells. However, in the mast cell PMA concentrations above 10nM reduced maximum histamine release in a dose-dependent manner.     

Macrophage plasminogen activator: induction by concanavalin A and phorbol myristate acetate

The synthesis and secretion of plasminogen activator by cultured macrophages can be induced and stimulated by concanavalin A and by phorbol myristate acetate, and inhibited by such agents as glucocorticoids, mitotic inhibitors and compounds affecting cAMP metabolism. By the manipulation of stimulatory and inhibitory influences, enzyme production can be modulated continuously over a 200 fold range. In the same way, the proportion of cells that secrete detectable levels of enzyme can be varied from 1–90%. No comparable modulation of lysozyme or acid hydrolase production is observed under the same conditions. These results suggest that the physiological control of macrophage plasminogen activator production is achieved by the interacting effects of mutually antagonistic stimuli; this emphasizes the utility of this enzyme for the study of regulatory phenomena, including those relating to inflammation.     

Modulation of human neutrophil LTA hydrolase activity by phorbol myristate acetate

The phorbol ester, phorbol 12-myristate 13-acetate enhanced leukotriene B4 production stimulated by formyl-methionyl-leucyl-phenylalanine and arachidonic acid and reduced the production of the all-trans isomers of LTB4 by human neutrophils. Production of 5-hydroxyeicosatetraenoic acid was unaffected. These observations are consistent with a stimulatory effect of phorbol ester on LTA hydrolase, the enzyme which catalyses the conversion of LTA4 to LTB4. We demonstrate that a protein of the same molecular weight as LTA hydrolase is phosphorylated upon stimulation of neutrophils with PMA. These data suggest that the activity of LTA hydrolase may be regulated by protein kinase C-dependent phosphorylation.     

Mechanism of action of phorbol myristate acetate on human natural killer cell activity

We have investigated the kinetics of inhibition and regeneration of human natural killer (NK) cell-mediated lysis of K562, a human erythroleukemia cell line, by the potent tumor-promoting agent phorbol-12-myristate-13-acetate (PMA). It is shown that PMA inhibits NK cell-mediated cytotoxicity (CMC) in a dose-dependent manner whether the compound is present throughout the 4-hr cytotoxic assay or the effector cells (EC) are pretreated with PMA. Pretreatment of the target cells (TC) with PMA produced a different profile of NK activity suggesting that PMA inhibition of NK-CMC is primarily due to the inactivation of EC. PMA-induced inhibition of NK-CMC does not affect TC binding and is not circumvented by compounds that enhance intracellular levels of cyclic guanosine monophosphate (cGMP) or calcium. Furthermore, and contrary to a recent report, PMA-induced inhibition of NK-CMC is independent of monocytes. Finally, kinetic studies revealed that PMA-induced inhibition of NK-CMC occurs rapidly and is fully reversible provided that “regenerated EC” are thoroughly washed, prior to the cytotoxic assay, to rid the cell suspension of residual PMA. The potential implications of these results to the currently accepted theory of TC destruction by NK cells, the stimulus-secretion model, are discussed.     

Effects of phorbol ester on cell growth inhibition by transforming growth factor beta 1 in human hepatoma cell lines

The effects of phorbol ester on cell growth inhibition by transforming growth factor beta 1 (TGF-beta 1) in human hepatoma cell lines, Mahlavu and PLC/PRF/5, were investigated. TGF-beta 1 (2.5 to 10 pM) alone could not inhibit the growth of Mahlavu cells, whereas in the presence of 12-O-tetradecanoyl phorbol 13-acetate (TPA) at 1 ng/ml, TGF-beta 1 could suppress their growth in a dose-dependent manner. The growth of PLC/PRF/5 cells could be inhibited by addition of TGF-beta 1 (2.5 to 10 pM) alone in a dose-dependent manner, and this action was not affected by TPA (1 ng/ml). The TGF-beta 1 inhibition induced by TPA in Mahlavu cells could not be cancelled by addition of protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) (10 microM) or staurosporin (1 nM). Thus, TPA could induce TGF-beta 1 inhibition of cell growth in Mahlavu cells which did not respond to TGF-beta 1 alone, and activation of protein kinase C does not seem to be behind this TPA action.     

Dexamethasone inhibition and phorbol myristate acetate stimulation of plasminogen activator in human embryonic lung cells

Treatment of human embryonic lung cells with dexamethasone resulted in a decrease in plasminogen activator activity measured in the fibrinolytic assay. The decrease in activity could at least partially be explained by the presence of an inhibitory substance(s) based on the following observations of lysates of dexamethasone-treated vs. control cells: a) an increase in specific activity following subcellular fractionation; b) an increase in fibrinolytic activity following separation by gel electrophoresis; c) an increase in fibrinolytic activity following mild acid-treatment; and d) a decrease in urokinase-directed fibrinolytic activity in mixing experiments. Phorbol myristate acetate increased plasminogen activator activity without affecting the level of inhibitory substance.     

Modulation of the activity of the platelet-derived growth factor receptor by phorbol myristate acetate

Phorbol myristate acetate (PMA) weakly activates Na+/H+ exchange in NR-6 cells. Simultaneously, PMA blocks the activation of Na+/H+ exchange by platelet-derived growth factor or by serum. Phorbol esters that do not activate protein kinase C do not show this metabolic response. We conclude that activation of Na+/H+ exchange by platelet-derived growth factor or serum does not require the intermediate activation of protein kinase C. We postulate from this and previous observations that a major role of protein kinase C is to act as an inhibitor of the activity of cell surface receptors, in particular mitogen receptors.     

Induction of cytotoxic effector activity in the HL-60 promyelocytic cell line by incubation with phorbol myristate acetate: a model system of human spontaneous monocyte-mediated cytotoxicity

In an attempt to develop a constant and reproducible in vitro system for a detailed analysis of cytotoxic effector mechanisms of nonimmune mononuclear phagocytes, the HL-60 promyelocytic cell line was studied for its cytotoxic action on chicken erythrocyte target cells. HL-60 cells cultured in complete medium were found to be noncytotoxic for chicken erythrocytes in an 18-hr 51Cr-release assay. These cells have been shown to acquire several characteristics of mature macrophages upon incubation with phorbol myristate acetate (PMA), and when PMA was included in the medium during the assay, the HL-60 cells became strongly cytotoxic to the target cells in the absence of exogenous antibody, lectin, or serum complement. Freshly isolated peripheral blood monocytes also became cytotoxic in the presence of PMA, whereas peripheral blood lymphocytes and the U937 histiocytic cell line did not. Detectable target lysis was observed between 4 and 8 hr after HL-60 stimulation with PMA, and HL-60 cells prestimulated with PMA for 24 hr retained their cytotoxic activity following washing and assay in PMA-free medium. Cytotoxic HL-60 cells developed after exposure to 10(-6) to 10(-9) M PMA, and significant target cell lysis occurred at effector:target cell ratios as low as 0.5:1. The PMA-induced HL-60-mediated cytotoxic response was markedly inhibited by blockers of protein synthesis, inhibition of microfilament function, and depletion of cellular superoxide and hydrogen peroxide. Interestingly, cytotoxicity of HL-60 cells for chicken erythrocyte targets was modulated by the direct addition of certain simple saccharides to the assay in a fashion similar to that observed with spontaneously cytotoxic mononuclear cells from several vertebrate and invertebrate species. Thus, the cytolytic effector function induced in HL-60 cells by incubation with PMA presents a useful model for the study of cellular cytotoxic mechanisms as well as the mechanisms utilized by nonimmune cells in the recognition of non-self.     

Mechanism of phorbol myristate acetate-induced lymphotoxin production by a human T cell hybridoma

Various hydroxyl radical scavengers markedly inhibited phorbol myristate acetate (PMA)-induced lymphotoxin (LT) production by a human T cell hybridoma, AC5-8. Among those we tested, tetramethylurea (TMU) was the most potent scavenger, and it was revealed that TMU must be added before 2 h have elapsed after PMA addition in order for LT production to be inhibited. In concordance with this fact, soluble NADPH dependent O2- forming enzyme(s) were activated several fold by PMA. PMA also induced DNA strand breaks, a process markedly inhibited by TMU. As expected, ADP-ribosyl transferase (ADPRT), which is well known to require DNA strand breaks for its enzymatic activity, was activated by PMA treatment. In addition, specific inhibitors for ADPRT, namely 3-amino-benzamide and nicotinamide, inhibited PMA-induced LT production. Taken together, these three successive events, activation of soluble NADPH dependent O2- forming enzyme(s), DNA strand breaks and activation of ADPRT, may be required for PMA-induced LT production by AC5-8.     

Effect of phorbol myristate acetate on processing of formyl peptide receptors by human neutrophils

We examined the effect of phorbol myristate acetate on the ability of human neutrophils to process formyl peptide receptors. The receptor was affinity-labeled and its extracellular localization assessed over time, by cleavage of extracellular labeled receptor with papain. Neutrophils were capable of internalizing (and/or recycling) affinity labeled formyl peptide receptor in the absence of extracellular calcium. This phenomenon was dependent upon stimulation with phorbol myristate acetate, suggesting a role for protein kinase C in this process.     

Human granulocyte-macrophage colony-stimulating factor plus phorbol myristate acetate stimulate a promyelocytic cell line to produce an IL-1 inhibitor

We recently described an IL-1 inhibitor found in urine of febrile patients. It is a 26-kDa glycoprotein that acts by blocking the binding of IL-1 to its receptor. In a search for a cell source for the urinary IL-1 inhibitor, we tested three promyelocytic cell lines, H-161, AML-193, and HL-60, for their ability to produce this protein. Under normal culture conditions none of these cell lines produce detectable IL-1 inhibitory activity. The H-161 cells were treated with differentiation-inducing agents, i.e., sodium butyrate, hemin, retinoic acid, DMSO, vitamin D3, and PMA alone or in combination with IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, TNF-alpha, IFN-gamma, granulocyte-CSF, macrophage-CSF, granulocyte/macrophage-CSF (GM-CSF), and Con A and tested for the production of IL-1 inhibitor. Production of IL-1 inhibitor was detected in cell supernatant, when H-161 cells were differentiated to adherent macrophage-like cells under the influence of PMA followed by a second signal provided by GM-CSF. Treatment of the other two cell lines, AML-193 and HL-60, with PMA plus GM-CSF also yielded similar IL-1 inhibitor protein. Partial purified H-161-derived IL-1 inhibitor showed specific binding to IL-1R-bearing cells and blocked the binding of IL-1 to its receptor and is thus similar to the urinary-derived molecule. We conclude the GM-CSF provides a signal to adherent macrophage-like cells to become "inhibitory macrophages" and to produce a competitive inhibitor of IL-1.     

Induction of multinucleated giant cell formation from human blood-derived monocytes by phorbol myristate acetate in in vitro culture

Multinucleated giant cells (MGC) of mononuclear phagocyte origin occur in different tissues in various inflammatory states and pathological conditions. Although MGC are believed to be derived from monocyte-derived macrophages by fusion, their mechanism of formation is not known. In this study, we investigated the role of PMA, a protein kinase C activator, in the induction and formation of MGC from blood monocyte-derived macrophages in in vitro culture. The addition of PMA (1 x 10(-9) to 8 x 10(-8) M) to 3-wk-old cultures of blood-derived monocytes induces cell fusion with a 30% to 80% fusion rate. Moreover, IFN-gamma-treated blood-derived monocyte cultures showed an additional enhancement of fusion rate with the addition of PMA. 1(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a protein kinase inhibitor, inhibited the formation of macrophage-derived giant cells when added before phorbol ester and IFN-gamma. These findings suggest that protein kinase C may play an important role in the formation of macrophage-derived MGC.     

IL-4 induces LFA-1 and LFA-3 expression on Burkitt's lymphoma cell lines. Requirement of additional activation by phorbol myristate acetate for induction of homotypic cell adhesions

LFA-1 and LFA-3 expression is absent or low on Burkitt's lymphoma cell lines and low on the EBV-transformed B cell line UD61. Incubation of cells of BL2 and of UD61 with various concentrations of IL-4 resulted in induction of LFA-1 and LFA-3 expression in a dose dependent fashion. This effect was already observed after 16 h of incubation whereas maximal expression was obtained after 72 h. Induction of LFA-1 and LFA-3 expression seemed to be specific for IL-4, because IL-1, IL-2, IL-3, IFN-alpha, IFN-gamma and a low m.w. B cell growth factor were ineffective. LFA-1 and LFA-3 induction by IL-4 was blocked specifically by an anti-IL-4 antiserum. Induction of LFA-1 expression by IL-4 was furthermore confirmed at the specific LFA-1 beta-chain mRNA level. IL-4 was unable to induce LFA-1 expression on EBV-transformed lymphoblastoid cell lines of two LFA-1-deficient patients. BL2 grows as single cells, but induction of LFA-1 and LFA-3 expression by IL-4 was insufficient to induce homotypic cell adhesions and required PMA as a second signal. PMA alone did not induce LFA-1 antigen expression and was unable to induce adhesions between BL2 cells in the absence of IL-4 in 22 h assays. Addition of PMA to BL2 cells that expressed LFA-1 Ag upon incubation with IL-4 resulted in aggregate formation within 30 min. Adhesions between BL2 cells induced by IL-4 in combination with PMA were blocked by anti-LFA-1 beta or anti-LFA-1 alpha-chains mAb. In addition, these mAbs dispersed preformed aggregates of BL2 cells. Our results indicate that IL-4 can induce the adhesion molecules LFA-1 and LFA-3 on B cell lines, but that an additional activation signal provided by PMA was required for the induction of homotypic cell adhesions.     

Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and beta-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate.

Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdown and intracellular Ca2+ ([Ca2+]i) mobilization by phorbol esters in platelets. In this study, we have examined the effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced granule secretion and correlated it with agonist-induced [Ca2+]i mobilization, arachidonate and thromboxane (Tx) release in human platelets. With increasing times of incubation with PMA (10 s-5 min), the rise in [Ca2+]i induced by thrombin and the TxA2 mimetic, U46619, was increasingly inhibited (90-100% with 5 min incubation) and, correlating with this, thrombin-induced [3H]arachidonate, TxB2 and beta-thromboglobulin (beta TG) release were also inhibited. In addition, the conversion of exogenously added arachidonate to TxB2 was inhibited (50-80%) by a 10 s-5 min pretreatment with PMA. However, secretion of 5-hydroxy[14C]tryptamine (5HT) induced by thrombin or U46619 was not inhibited by 10 s-2 min incubations with PMA and, on the contrary, with low agonist concentrations, was potentiated by PMA in the absence of a significant rise in [Ca2+]i or endogenous Tx formation, to levels significantly greater than or equal to the sum of that obtained when agonist and PMA were added separately. With longer times of incubation with PMA (5 min), these synergistic effects became less pronounced as inhibitory effects of PMA on agonist-induced [14C]5HT secretion became apparent. The results indicate that, while PMA may cause an inhibition of agonist-induced [Ca2+]i mobilization resulting in an inhibition of agonist-induced arachidonate, TxB2 and beta TG release, its effects on agonist-induced 5HT secretion may be complicated by [Ca2+]i-independent synergistic effects of agonist and PMA.     

Activation of the NADPH oxidase in a cell-free system from human neutrophils stimulated by phorbol myristate acetate

Kinetics of activation of the NADPH oxidase in a fully soluble cell-free system from phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system in which Mg2+ and sodium dodecyl sulfate, an anionic detergent required for the activation of NADPH oxidase are contained, cytosol prepared from PMA-stimulated neutrophils failed to activate PMA-stimulated neutrophil oxidase. However, cytosol prepared from resting (control) neutrophils was capable of activating PMA-stimulated neutrophil oxidase in a cell-free system in which its Km for NADPH was almost similar to that of control neutrophil oxidase. Cytosol from PMA-stimulated neutrophils could not activate control neutrophil oxidase, although it did not contain any inhibitors of NADPH oxidase activation. These results suggest that, in PMA-stimulated neutrophils, cytosolic activation factors may be consumed or exhausted, and that the affinity for NADPH of PMA-stimulated neutrophil oxidase may be the same as that of control neutrophil oxidase.     

Suppression of an IL-13 autocrine growth loop in a human Hodgkin/Reed-Sternberg tumor cell line by a novel IL-13 antagonist

IL-13 has been proposed to be an autocrine growth factor for Hodgkin/Reed-Sternberg tumor cells (H/RS cells). Since we have recently identified and produced a novel IL-13 antagonist (IL-13E13K) that can suppress the biological activity of IL-13, here we examined whether IL-13E13K can inhibit growth of Hodgkin lymphoma (HL)-derived cell lines. IL-13E13K not only inhibited the growth of an unstimulated H/RS cell line (L1236) but also cells that were stimulated by exogenous IL-13 in a dose-dependent manner. Several HL-derived cell lines expressed IL-13 message and protein and message for various chains of IL-13R. H/RS cell lines expressed mRNA for the IL-13R alpha 1, IL-4R alpha, and IL-2R gamma chains. However, none of these cell lines expressed the IL-13R alpha 2 chain. An H/RS cell line (L1236) internalized the ligand-receptor complex after binding to a fusion protein composed of IL-13 and a mutated form of Pseudomonas exotoxin A (IL-13-PE38QQR, or IL-13 cytotoxin), as IL-13 cytotoxin was specifically cytotoxic to H/RS cells in vitro. These results indicate that IL-13E13K and IL-13 cytotoxin can effectively suppress growth of a L1236 H/RS cell line. Therefore, additional studies should be performed to determine the expression of IL-13 and IL-13R in primary clinical samples of Hodgkin's lymphoma and both agents should be further tested in vitro and in vivo as possible therapeutic agents for HL.     

Coregulation of collagenase and collagenase inhibitor production by phorbol myristate acetate in human skin fibroblasts

Phorbol myristate acetate (PMA), a tumor promotor known to stimulate collagenase production in fibroblasts and endothelial cells, was examined with regard to its ability to regulate the expression of the collagenase inhibitor secreted by human skin fibroblasts. Confluent human skin fibroblasts were incubated with concentrations of PMA ranging from 10(-11) to 10(-7) M, and the conditioned medium was analyzed by enzyme-linked immunosorbent assay for both immunoreactive collagenase and collagenase inhibitor. PMA stimulated the production of both collagenase and collagenase inhibitor in several cell lines to maximal rates that were very similar, 300 to 350 vs 230 to 330 pmol 10 micrograms DNA-1 48 h-1, respectively. Due to differences in the basal levels of expression of these proteins, such rates reflected a two- to sevenfold stimulation in collagenase production, in comparison to a more uniform two- to threefold enhancement in inhibitor synthesis. Production of inhibitor was 50% of maximal at 7 X 10(-9) M and maximal at 10(-7) M phorbol. This concentration-dependent effect was very similar to that observed for collagenase expression. Total protein synthesis by the phorbol-conditioned cells, as studied by incorporation of [3H]leucine into newly synthesized protein, was not significantly increased, nor was cellular DNA content. The onset of the effect of PMA on inhibitor production occurred between 4 and 8 h, was maximal by 8 h, and continued undiminished for at least another 64 h. After the first 8 h, inhibitor production continued at a roughly constant rate of approximately 10 pmol 10 micrograms DNA-1 h-1. Interestingly, following the removal of phorbol from culture medium, such fibroblasts continued to produce increased quantities of inhibitor protein for at least 72 h. Metabolic labeling studies in which fibroblasts were exposed to [3H]leucine followed by immunoprecipitation using inhibitor-specific antibody suggested that stimulation of inhibitor production by PMA was mediated via an increased synthesis of new inhibitor protein. Therefore, in response to the tumor promoter, PMA collagenase and collagenase inhibitor expression by human skin fibroblasts appear to be coregulated.     

Enhancement of selective tumor cell binding by activated murine macrophages in response to phorbol myristate acetate

The fundamental biology of how stable cell-cell bonds develop between activated macrophages and tumor cells, although essential to lysis of the neoplastic targets, remains poorly understood. To investigate whether this phenomenon could be pharmacologically manipulated, we analyzed the effect of phorbol diesters on tumor cell binding by macrophages. Activated murine peritoneal macrophages, treated in vitro with as little as 1 ng/ml of phorbol myristate acetate (PMA), bound significantly more tumor cells than did untreated macrophages. The effect was induced rapidly by PMA (i.e., maximum enhancement was seen within 15 min) and resulted in an average approximately twofold increase in the number of targets bound. The interaction between PMA-treated activated macrophages and tumor cells was completed much more rapidly than by untreated macrophages. The enhanced binding was seen only in macrophages treated with biologically active phorbol esters. Only the selective interaction between activated macrophages and tumor cells was affected (i.e., PMA treatment had no effect on nonselective interactions between activated macrophages and non-neoplastic targets or between nonactivated macrophages and any type of target). Pretreatment of activated macrophages with PMA apparently altered the requirements for microfilaments and microtubules in establishing binding, because cytochalasin B and colchicine, which inhibited control binding, as well as phagocytosis, had no effect on PMA-enhanced binding. PMA treatment did not alter energy requirements for binding, however, because low temperature (4 degrees C) or inhibitors of glycolysis and oxidative phosphorylation blocked both control and PMA-enhanced binding. The enhancement of binding apparently was not due to large quantities of secreted oxygen metabolites but did correlate closely with increased spreading and surface area of the macrophages. PMA treatment resulted in enhanced expression of trypsin-sensitive tumor-cell binding sites on the macrophage surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of macrophage membrane proteins labeled with 125I by the lactoperoxidase method revealed at least four trypsin-sensitive cell surface proteins that were re-expressed after PMA treatment. The data suggest that rearrangement and/or induced expression of surface binding sites may be an important step in the binding of tumor cells and indicate that PMA is a useful pharmacologic probe in dissecting the establishment of such binding into discrete steps.