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.     

Asparagine-linked oligosaccharides on formyl peptide chemotactic receptors of human phagocytic cells

Formyl peptide chemotactic receptors affinity-labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (where Nle represents norleucine) and ethylene glycol bis(succinimidyl succinate) consist of two isoelectric forms with cell type differences in both apparent size and charge (neutrophils: 55-70 kDa, pI 5.8, and 6.2.; monocytes: 60-75 kDa, pI 5.6 and 6.0; differentiated HL-60 cells: 62-85 kDa, pI 5.6 and 6.0). Endo-beta-N-acetylglucosaminidase F (endo F) cleavage of N-linked oligosaccharides from formyl peptide receptor generates 40-50- and 33-kDa products that can be affinity-labeled. Whereas both pI forms of this receptor from neutrophils are cleaved by endo F to 33-kDa final products, this cleavage does not eliminate pI differences. Tunicamycin decreases expression of formyl peptide receptor on differentiating HL-60 and causes a dose-dependent decrease in size of the major product seen after affinity labeling (0.5 micrograms/ml: 38-48 kDa; 2 micrograms/ml: 32 kDa). Thus, the formyl peptide receptor polypeptide backbone from all three cell types contains at least two N-linked oligosaccharide side chains which contribute to the cell type differences in Mr and are not required for ligand binding. Papain treatment of intact cells generates a membrane-bound formyl peptide receptor fragment that can be affinity-labeled and is of similar size (29-31 kDa) in all three cell types. Endo F treatment of the affinity-labeled papain fragment of formyl peptide receptor does not alter its size, suggesting that this fragment does not contain the N-linked oligosaccharide cleaved by endo F from intact receptor. The results indicate that at least two N-linked oligosaccharide chains are located on the distal 1-3-kDa portion of the receptor polypeptide backbone.     

Phosphorylation of proteins in human neutrophils activated with phorbol myristate acetate or with chemotactic factor

In human neutrophils stimulated with phorbol myristate acetate (PMA) or with the chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLF) a number of proteins are phosphorylated, including proteins recovered in the membrane fraction corresponding to molecular masses of 130, 78, 46, 40, and 34 kDa and proteins recovered in the cytosol fraction corresponding to molecular masses of 65, 55, 48, 38, 36, 30, and 22 kDa. Phosphorylation of the membrane proteins was fourfold greater in cells stimulated with PMA, as compared to cells stimulated with fMLF, whereas both activators induced similar phosphorylation of proteins recovered in the cytosol fraction. Phosphorylation of membrane proteins appeared to be mediated by native protein kinase C (PKC) translocated from the cytosol to the plasma membrane. Thus phosphate incorporation was inhibited by retinal and a similar pattern of incorporation was reproduced in a reconstituted system composed of isolated cell membranes and purified PKC. Phosphorylation of cytosol proteins, on the other hand, appeared to be mediated by the proteolytically modified form of PKC. In this case, phosphate incorporation was inhibited by leupeptin, which prevents the conversion of native PKC to the proteolytically modified form, The phosphorylation pattern was reproduced when isolated cytosol fractions were incubated with the proteolytically modified form of the enzyme but not with the native PKC. These results demonstrate that responses to stimuli such as PMA or fMLF are mediated by different forms of PKC and that the proteolytically modified form is responsible for the major responses elicited by fMLF.     

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.     

Inhibition of thymine dimer excision by the phorbol ester, phorbol myristate acetate

The effect of the promoting agent, phorbol myristate acetate, on repair of UV-induced damage in HeLa cells was studied. The agent decreased survival and subsequent colony-forming ability of irradiated cells and inhibited removal of UV-induced thymine-containing dimers from DNA of irradiated cells.     

The effects of phorbol myristate acetate and chemotactic peptide on transmembrane potentials and cytosolic free calcium in mature granulocytes evolve sequentially as the cells differentiate

We isolated myeloid precursors from human marrow and studied the effects of phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) upon transmembrane potentials and cytosolic calcium ([Ca2+]i) as the cells matured. Using a panel of fluorescent probes, we found that membrane depolarization induced by PMA and fMLP in granulocytes, and elevation in [Ca2+]i stimulated by fMLP, were absent in myeloblasts. When we induced differentiation with granulocyte-macrophage colony-stimulating factors, we found that both ionic responses appeared at approximately the promyelocyte stage. By using di-O-C5(3), we detected an initial phase of fMLP-induced hyperpolarization which appeared ontogenetically earlier than depolarization and which could be evoked in mature granulocytes with lower concentrations of the ligand. Hyperpolarization was partially dependent on extracellular Na+, was abrogated by increasing the external K+ concentration, and was accompanied by mild acidification of the cytoplasm. Bordetella pertussis toxin abolished both hyperpolarization and depolarization. Our findings indicate that shifts in [Ca2+]i and membrane potential changes in response to PMA and fMLP evolve as granulocytes mature. In addition, transmembrane ionic fluxes induced by fMLP appear to be more complex than previously considered, involving at least two separable phases of membrane potential change.     

Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate

Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of 125I- thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of 125I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gamma-thrombin (0.5 microgram/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of 125I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation.     

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.     

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.     

Enhancement of the expression of a rat neutrophil-specific cell surface antigen by activation with phorbol myristate acetate and concanavalin A

A monoclonal antibody against rat neutrophils, RP-1, was produced by hybridizing a mouse myeloma (P3-X63-Ag8.653) with BALB/c mouse spleen cells sensitized with peritoneal neutrophils from Wistar-King-Aptakeman/Hok rats. RP-1 specifically reacted to neutrophils and bone marrow cells from rats of various strains. The expression of an antigen reactive to RP-1 on rat peritoneal neutrophils was enhanced by stimulation with phorbol myristate acetate and concanavalin A. The experimental result that the expression of an antigen reactive with another anti-neutrophil monoclonal antibody was not enhanced by stimulation with phorbol myristate acetate indicates that the antibody-binding capacity of stimulated neutrophils was not nonspecifically enhanced. The enhancement of antigen expression was temperature dependent. A glycolytic inhibitor, 2-deoxy-D-glucose, and an inhibitor of intracellular calcium mobilization, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester hydrochloride, which inhibited hydrogen peroxide release from stimulated neutrophils, did not inhibit enhancement of the expression of an antigen reactive with RP-1.     

Effect of catalase on the proliferation of human lymphocytes to phorbol myristate acetate

Phorbol esters have been documented to stimulate the proliferation of human blood mononuclear cell cultures. In addition, these agents are also known to stimulate the production and release of reactive oxygen species by monocytes. We demonstrated previously that H2O2, one of these oxygen metabolites, impairs the proliferative capacity of human blood lymphocytes. Therefore, in these experiments, we determined whether or not the H2O2 released by monocytes after activation by PMA modifies the proliferation of lymphocytes to this agent. Human blood mononuclear cells (80% lymphocytes and 20% monocytes) were incubated with PMA, and lymphoblastic transformation (LBT) was quantitated at 3 and 5 days by pulsing the cultures with thymidine. Initial experiments established that the concentration of PMA required for optimal LBT was 50 ng/ml. We then demonstrated that this concentration of PMA also induces a burst in hexose monophosphate shunt activity and H2O2 production of mononuclear cells as indicated by the enhanced oxidation of 14C-glucose and 14C-formate, respectively. The amount of H2O2 released into the medium was substantial. Our measurements indicate that the concentration of H2O2 could reach values as high as 0.008 mM during the first 2 hr of the cultures. The addition of catalase to PMA-treated cultures in concentrations sufficient to scavenge the H2O2 released by the monocytes was associated with an enhanced thymidine uptake (mean 79%). These results indicate that the hydrogen peroxide released by the monocytes modifies the response of lymphocytes to the PMA. Paradoxically, mononuclear cell cultures depleted of monocytes also had a lower proliferation to PMA than mononuclear cell cultures. This observation indicates that monocytes also produce factors required for lymphocyte proliferation to PMA such as an interleukin. In contrast, to PMA cultures, catalase did not alter the proliferation of mononuclear cell cultures stimulated by PHA. We previously documented that PHA does not stimulate an immediate burst in the oxidative metabolism of mononuclear cultures. Therefore, the effect of catalase in these two culture systems appears to correlate with the capacity of the mitogen to stimulate the oxidative metabolism of mononuclear cells. These observations suggest that the release of reactive oxygen species by monocytes may modify the response of lymphocytes to antigens both in vitro and in vivo.     

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.     

Detergent solubilization of the formyl peptide chemotactic receptor. Strategy based on covalent affinity labeling

The formyl peptide chemotactic receptor has been solubilized by digitonin treatment of purified human neutrophil membranes. Of several potential assay methods tested for their ability to separate receptor-bound from free ligand, only gel filtration through an acrylamide cross-linked agarose matrix yielded satisfactory results. Approximately 70% of the receptor initially present in the membrane was recovered in the digitonin extract. Binding of 125I-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys to the soluble receptor was rapid (t 1/2 at 22 degrees C less than 5 min), of high affinity (Kd = 2.2 nM) and saturable. The relative potencies of a small series of peptides as inhibitors of binding to the soluble receptor paralleled their potencies as inhibitors of the membrane-bound receptor. N-Formylation of the peptides was required for high affinity binding. Binding was maximal at pH 6.5 and was sulfhydryl-dependent; 20 microM p-chloromercuriphenylsulfonic acid decreased binding by 50%. 125I-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys was specifically cross-linked to the soluble receptor with ethylene glycol bis(succinimidyl succinate) and an apparent molecular weight of 55,000 to 70,000 and determined for the soluble receptor by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A strategy for obtaining an active, detergent-soluble receptor preparation based on covalent affinity labeling is presented.     

Changes in the expression of B cell surface markers on complement receptor-positive and complement receptor-negative B cells induced by phorbol myristate acetate

The ability of phorbol myristate acetate (PMA) to induce changes in the expression of B cell surface markers on CR- and CR+ B cells from normal mice in an in vitro culture system was examined. The markers studied were CR, sIgM, sIgD, and sIa. CR- B cells acquired the CR after overnight incubation with PMA. A twofold increase in sIa expression on CR- and CR+ B cells was also noted, whereas the staining intensity of sIgM and sIgD decreased on both B cell populations. These changes in the expression of surface markers took place without detectable increases in cell proliferation, cell size, or RNA content. Furthermore, the same effects were observed when CR- and CR+ B cells were prepared from a small B cell population purified by elutriation. It therefore appears that PMA can exert its effect directly on small, resting B cells.     

Differential activation of polymorphisms of the formyl peptide receptor by formyl peptides

We have investigated the role of two polymorphic sites (R190W and N192K) on the binding and activation of the formyl peptide receptor (FPR) by viral and formyl peptides. WEDWVGWI, a peptide with antiviral activity derived from the membrane proximal region of feline immunodeficiency virus, binds with high affinity to FPR. The three tryptophans in the peptide are all essential for FPR binding, just as they were essential for antiviral activity [S. Giannecchini, A. Di Fenza, A.M. D'Ursi, D. Matteucci, P. Rovero, M. Bendinelli, Antiviral activity and conformational features of an octapeptide derived from the membrane-proximal ectodomain of the feline immunodeficiency virus transmembrane glycoprotein, J. Virol. 77 (2003) 3724]. Formyl-NleWEDWVGWI behaved as a weak partial agonist with FPR W190/N192 but a stronger partial agonist with FPR R190/K192 and FPR R190/N192. Formyl-NleNleWEDWVGWI behaved as a full agonist toward all three FPRs but exhibited a much higher EC(50) with W190/N192 FPR (300+/-45 nM) than for R190/K192 FPR (40+/-3 nM) or R190/N192 (60+/-8 nM). Formyl-MYKWPWYVWL preferentially activated R190/K192 and R190/N192 FPRs by>5 fold compared to W190/N192 FPR. Formyl-MFEDAVAWF, a peptide derived from a protein in Mycobacterium avium subsp. paratuberculosis and formyl-MFTFEPFPTN, a peptide derived from the N-terminus of chemotaxis inhibitory protein of Staphylococcus aureus with an added N-terminal formyl-methionine exhibited the greatest selectivity for R190/K192 and R190/N192 FPRs with approximately 10 fold lower EC(50)s than that observed with FPR W190/N192. Thus, individuals with the W190 polymorphism may display a reduced ability to detect certain formyl peptides.     

Phenylarsine oxide and ethanol prevent cell death of porcine polymorphonuclear leucocytes induced by phorbol myristate acetate

In this study, we report that phenylarsine oxide and ethanol, both of which suppress a number of polymorphonuclear leucocyte functions including superoxide production, prevented the phorbol myristate acetate-induced cell death in a dose-dependent manner. These reagents had an inhibitory effect even after polymorphonuclear leucocytes were stimulated to produce superoxide by treatment with phorbol myristate acetate. The results indicate that activation of protein kinase C and subsequent superoxide release do not directly cause phorbol myristate acetate-induced cell death. Phenylarsine oxide or ethanol prevents cell death by affecting pathways downstream from those involved in the superoxide production.     

An urokinase receptor antagonist that inhibits cell migration by blocking the formyl peptide receptor

Urokinase receptor (uPAR) plays a key role in physiological and pathological processes sustained by an altered cell migration. We have developed peptides carrying amino acid substitutions along the Ser(88)-Arg-Ser-Arg-Tyr(92) (SRSRY) uPAR chemotactic sequence. The peptide pyro glutamic acid (pGlu)-Arg-Glu-Arg-Tyr-NH2 (pERERY-NH(2)) shares the same binding site with SRSRY and competes with N-formyl-Met-Leu-Phe (fMLF) for binding to the G-protein-coupled N-formyl-peptide receptor (FPR). pERERY-NH(2) is a dose-dependent inhibitor of both SRSRY- and fMLF-directed cell migration, and prevents agonist-induced FPR internalization and fMLF-dependent ERK1/2 phosphorylation. pERERY-NH(2) is a new and potent uPAR inhibitor which may suggest the generation of new pharmacological treatments for pathological conditions involving increased cell migration.     

Shear-induced resistance to neutrophil activation via the formyl peptide receptor

The application of fluid shear stress on leukocytes is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. The formyl peptide receptor (FPR) on neutrophils, which binds to formyl-methionyl-leucyl-phenylalanine (fMLP) and plays a role in neutrophil chemotaxis, has been implicated as a fluid shear stress sensor that controls pseudopod formation. The role of shear forces on earlier indicators of neutrophil activation, such as L-selectin shedding and α(M)β(2) integrin activation, remains unclear. Here, human neutrophils exposed to uniform shear stress (0.1-4.0 dyn/cm(2)) in a cone-and-plate viscometer for 1-120 min showed a significant reduction in both α(M)β(2) integrin activation and L-selectin shedding after stimulation with 0.5 nM of fMLP. Neutrophil resistance to activation was directly linked to fluid shear stress, as the response increased in a shear stress force- and time-dependent manner. Significant shear-induced loss of FPR surface expression on neutrophils was observed, and high-resolution confocal microscopy revealed FPR internalized within neutrophils. These results suggest that physiological shear forces alter neutrophil activation via FPR by reducing L-selectin shedding and α(M)β(2) integrin activation in the presence of soluble ligand.     

Detergent solubilisation of the rabbit neutrophil receptor for chemotactic formyl peptides

Digitonin was found to be the only detergent (out of 24 tested) capable of solubilising the chemotactic formyl peptide receptor from rabbit neutrophil membranes in a form which retained its [3H]fMet-Leu-Phe binding activity. The solubilised material retained many of the characteristics of the membrane-bound receptor. [3H]fMet-Leu-Phe binding to the digitonin extract was measured at 4 degrees C using an equilibrium dialysis assay. Binding was saturable and of high affinity (Kd = 3.5 +/- 0.7 nM). The potencies of a series of synthetic peptides as inhibitors of [3H]fMet-Leu-Phe binding to the soluble receptor showed the same rank order as for inhibition of the membrane-bound receptor. In addition, binding to both preparations was sulphydryl dependent showing a parallel inhibition by p-chloromercuribenzene sulphonate which could be partially reversed by subsequent incubation with dithiothreitol.     

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.