The effect of phorbol myristate acetate on the metabolism and ultrastructure of human alveolar macrophages.

In the present investigation we examined the influence of the surface-active agent phorbol myristate acetate (PMA) and opsonized heat-killed bacteria (HKB) on oxygen consumption, superoxide release, and glucose oxidation of human alveolar macrophages (AM). Both PMA and HKB produced a surge in oxygen consumption, superoxide release, and oxidation of 1-14C-glucose and 6-14C-glucose by human AM. Examination of AM by electron microscopy following stimulation by these two agents demonstrated membrane ruffling, loss of microvilli, and increased vacuolization in PMA-treated cells and phagocytic vacuoles containing bacteria in HKB-treated cells. The vacuolization produced by PMA-treated AM was much less striking than the vacuolization produced in PMA-treated leukocytes. The similarity in the metabolic and some of the physical responses of AM stimulated by PMA and HKB suggest that PMA may be a useful agent for evaluating cell-membrane-related events of phagocytosis in AM.     

Induction of human autorosette forming cells by phorbol myristate acetate.

Human peripheral blood lymphocytes were examined for rosette formation with autologous erythrocytes. When normal human lymphocytes were stimulated with phorbol myristate acetate (PMA) in the presence of autologous serum, the levels of autorosette forming cells (ARFC) were strongly enhanced. Pre-culture was necessary for the generation of ARFC by PMA and the maximal level of ARFC was observed at 72 hr of culture. ARFC appear to belong to a T cell subset and the induction of ARFC by PMA was noted in monocyte depleted lymphocyte fractions, indicating monocyte independency.     

Induction of human T-lymphocyte colonies by phorbol myristate acetate

Colony growth of human lymphocytes by phorbol myristate acetate (PMA) was studied. PMA was able to induce lymphocyte colony growth in methylcellulose semi-solid cultures in the absence of lectin mitogens, phytohaemagglutinin or concanavalin A. PMA-induced colonies were found in cultures of mononuclear cells, monocyte-depleted mononuclear cells and the T-enriched cell fraction, whereas no colonies were obtained from the non-T cell fraction. At least one million mononuclear cells were required to form colonies by PMA. The colony cells were mainly T cells as judged from sheep red blood cell rosette formation. Surface immunoglobulin positive cells and peroxidase positive cells were not detected in colony cells. Non-specific esterase positive cells were only found to be less than 1% of colony cells. T cells formed more colonies than did mononuclear cells, presumably because of a concentration of colony forming cells and/or co-operating cells. Colony formation by PMA was induced from monocyte-depleted mononuclear cells and monocyte-depleted T cells, suggesting independence of monocytes. When mononuclear cells were precultured at 37 degrees for 5 min or 30 min with PMA and then cultured in the semi-solid medium without PMA, only a small number of colonies grew. Further studies of PMA-induced T-cell colonies will provide information the identification and characterization of immunological states in various immunological diseases.     

Dual effect of phorbol myristate acetate (PMA) on murine thymocyte cultures

4 beta-phorbol 12-myristate 13-acetate (PMA), a potent tumor promoter, was found to have a dual effect on interleukin 1-(IL 1) stimulated murine thymocyte cultures. In the absence of 2-mercaptoethanol (2-ME) or other sulfhydryl containing compounds, PMA inhibited 3H-thymidine incorporation, while at the same concentrations but in the presence of sulfhydryl agents, PMA exerted a potent stimulatory effect. The other sulfhydryl compounds giving effects resembling 2-ME were dithiothreitol and cysteine, while glutathione and various oxygen radical scavengers (catalase, superoxide dismutase, histidine, alpha-tocopherol or mannitol) had no such effect. 2-ME enhanced the response of thymocytes incubated with PMA even when added 24 h after initiation of the cultures, but not at the 48 h interval. Harvesting the thymocyte cultures at various intervals showed that the inhibitory effect of PMA was already fully pronounced 48 h after initiation of the cultures. On the other hand, the stimulatory effect of PMA on cultures activated with IL 1 in the presence of 2-ME took place mainly during the last 24 h of the 72-h culture period. The modulation of the effect of PMA on thymocyte activation by 2-ME or similar agents may account for the apparent discrepancies in the literature concerning the action of PMA in lymphocyte cultures.     

The differential mobilization of human neutrophil granules. Effects of phorbol myristate acetate and ionophore A23187.

Phorbol myristate acetate (PMA, 2 to 100 ng/ml) and ionophore A23187 (10(-7) to 10(-6) M) cause human neutrophils to release up to 50% of the granule-associated enzyme lysozyme extracellularly without release of beta-glucuronidase or the cytoplasmic enzyme LDH. When azurophil and specific granules are separated from neutrophil lysates by sucrose density centrifugation, it is found that lysozyme release from neutrophils exposed to PMA or to A23187 reflects a selective disappearance of the small, peroxidase-negative (specific) granules from the cells. These studies demonstrate that neutrophils can mobilize the specific and azurophil granules independently. These studies also demonstrate that under certain conditions the specific granules of human neutrophils behave like the storage granules of secretory cells. Finally, these studies show that techniques of separating neutrophil granules according to their sedimentation characteristics successfully divide these granules into populations that are distinct not only by cytochemical and morphologic criteria but also according to their availability for mobilization and extracellular release. (APM J Pathol 87:273-284, 1977).     

Characterization of a direct effect of phorbol myristate acetate on human neutrophil cell membrane using 31D8 monoclonal antibody.

We found that 4-beta-phorbol 12-myristate 13-acetate (PMA) caused decreased expression of the polymorphonuclear neutrophil (PMN) surface antigen 31D8. In contrast to the rapid initiation of the oxidative burst caused by PMA, the effect was slow to start but increased during incubation periods up to 50 min. To study this apparent protein kinase C-independent late effect of PMA, we measured 31D8 expression in PMNs after incubation with various concentrations of PMA. The maximum PMA-induced inhibition was 76 +/- 2%, with an ID50 of 3.9 +/- 0.4 ng/ml. Oxidants and prooxidants (hydrogen peroxide, hypochlorite, taurine-chloramine, and ferrous iron, with or without H2O2) had no direct effect on 31D8 antigen expression. The following substances were not protective against the inhibitory affect of PMA: (1) antioxidants (superoxide dismutase, catalase, azide, dimethyl sulfoxide, Desferal, and ascorbate, with the exception of alpha-tocopherol), (2) inhibitors of protein kinase C (H7 and W7), (3) inhibitors of 5-lipoxygenase (A-63162, MK886, and high-dose indomethacin) and (4) inhibitors of cyclooxygenase (low-dose indomethacin). Myeloperoxidase-deficient PMNs had normal 31D8 antigen expression and a decrease of 31D8 antigen expression by PMA, as did normal PMNs. The inactive analog of PMA, 4-alpha-phorbol didecanoate, had no effect on 31D8 antigen expression. alpha-Tocopherol (50 micrograms/ml) and betamethasone (150 micrograms/ml) protected against the PMA effect by 30.5 +/- 7.3 (P less than .0005) and 52 +/- 15 (P less than 0.004) channels, respectively. These results indicate that PMA has a protein kinase C-independent late effect on human neutrophils, which can be prevented by pretreatment with alpha-tocopherol or the steroid betamethasone. These compounds probably exert their protective effect by membrane stabilization.     

Fine structural alterations induced in erythocytes by phorbol myristate acetate.

Prevous studies have demonstrated that PMA is a potent membrane-active agent causing cell-wall derived vacuole formation in neutrophils and granule labilization in platelets. The present investigation demonstrates that PMA also has marked effects on red blood cells. Erythrocytes exposed to PMA were converted into stomatocytes and stomatospherocytes. The effects of PMA on red cells were concentration-dependent, required removal of plasma, and occurred maximally at 37 C. Although the response of the red cell to PMA was not identical to that of other blood cells tested previously; the similarities suggest that the capacity of the agent to produce membrane invagination may be fundamental to its mode of action.     

Phorbol myristate acetate inhibits immunoglobulin synthesis by human peripheral blood lymphocytes

Phorbol myristate acetate (PMA), a known lymphocyte mitogen, has been shown to inhibit immunoglobulin synthesis by human peripheral blood lymphocytes. This effect occurs with low concentrations of the agent and following only 12 h contact with the lymphocytes. PMA was also capable of preventing pokeweed mitogen stimulation of lymphocytes. These experiments suggest that PMA prevents the complete differentiation of B-cells into plasma cells capable of secreting immunoglobulin.     

Acute and progressive lung injury after contact with phorbol myristate acetate.

Because of its potent ability to activate leukocytes and macrophages, resulting in the generation of large amounts of oxygen products (O-2, H2O2), phorbol myristate acetate (PMA) has been instilled into the air-ways of rats. The resulting acute lung injury is dose-dependent on the amount of PMA employed, is chiefly confined anatomically to the alveolar and interstitial compartments, is neutrophil-independent, and can be inhibited by catalase but not by superoxide dismutase. These data suggest that the generation of H2O2 is major mechanism involved in this method of acute lung injury. It has also been demonstrated that a progressive pattern of lung injury develops after exposure to PMA, with the onset of an interstitial fibrotic reaction by the sixth day. These data reinforce our recent studies, in which both acute and progressive injury occurs in lungs of rats when H2O2 is generated.     

Lymphocyte stimulation with phorbol myristate acetate in atopic and non-atopic individuals.

Phorbol myristate acetate (PMA) was found to be a potent stimulator of DNA synthesis in human whole blood cell cultures. Stimulation with pokeweed mitogen was strongly enhanced by addition of PMA-activated cells. PMA responsiveness varied with age, being low or absent in newborns and very pronounced in adults. In atopic children, PMA responsiveness was normal or increased. The ratio of phytohemagglutinin to PMA responsiveness was significantly reduced in cultures from such children. This finding would be compatible with the hypothesis of a relative suppressor cell deficiency in atopic disease. The results of tests designed to detect suppressor and helper cell activity, added further support to this hypothesis.     

Effects of tumour promoter phorbol myristate acetate on mouse lymphocytes: selective inhibition of B cell activation by mitogens and antigens.

The effect of the tumour promoter phorbol myristate acetate (PMA) on the responses of mouse lymphocytes to various stimuli was studied in vitro. Nanomolar concentrations of PMA inhibited B cell proliferation induced by lipopolysaccharide and by anti-Ig antibodies, and the polyclonal antibody response to lipopolysaccharide. Specific antibody responses to both T-dependent and T-independent antigens were similarly affected. In marked contrast, T cell proliferation elicited by various mitogens was 10- to 1000-fold more resistant to inhibition. Cell fractionation experiments suggest that the effects on B cell responses are the results of a direct anti-proliferative effect of PMA on responding B lymphocytes.     

Bactericidal capacity of phorbol myristate acetate-treated human polymorphonuclear leukocytes.

Thus far, the functional capacity of phorbol myristate acetate- (PMA)-treated human polymorphonuclear leukocytes has been undefined. PMA induced exocytosis of lactoferrin, the specific granule marker, but not of myeloperoxidase, the azurophil granule marker. This phenomenon was demonstrated both biochemically and with fluorescent antibody conjugates. PMA-treated neutrophils contained virtually no specific granules when viewed by electron microscopy. Separation of the granule classes by linear sucrose density gradient centrifugation revealed the loss, from PMA-treated neutrophils, of lactoferrin and the specific granule (D20(20) = 1.89) band usually resolved from normal neutrophils. Cells treated with PMA appeared to retain those functions normally associated with intraleukocytic microbicidal action. The hexose monophosphate shunt activated by phagocytic challenge was present in PMA-treated neutrophils. As demonstrated by electron microscopy, the azurophil granules of these cells appeared intact, and they retained the capacity for degranulation with translocation of myeloperoxidase to the site of phagocytized Escherichia coli. The PMA-treated neutrophils also remained capable of degrading the ingested microorganisms. PMA-treated neutrophils exhibited a decrease in phagocytic ability at all levels of bacterial challenge. In the presence of a high multiplicity of bacteria they demonstrated an impairment in killing. These same cells were able to kill low multiplicities of E. coli as well as control cells. It thus appeared that the loss of the specific granules, plus other undefined PMA-induced alterations, impaired neither the viability of these neutrophils nor their killing ability in the presence of a modest phagocytic challenge.     

Modulation of interleukin-2-induced proliferation and nonspecific cytotoxicity of rat lymphocytes by phorbol myristate acetate and staurosporine

It is shown that a 19-h pretreatment of rat splenocytes with 1 μM phorbol 12-myristate 13-acetate followed by a 42-h incubation with human recombinant interleukin-2 inhibits nonspecific cytotoxicity of these cells toward the target YAC-1 cells. By contrast, proliferation of splenocytes and thymocytes incubated under the same conditons increases considerably. The inhibitor of protein kinase C staurosporine (0.1 μM) significantly decreases nonspecific cytotoxicity of splenocytes after a 20-min incubation, while in a dose of 0.01 μM it stimulates lytic activity of splenocytes and thymocytes following a 3-day incubation with interleukin-2 in the presence of the inhibitor. Cell proliferation under these conditions is markedly decreased. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 10, pp. 394–398, October, 1996     

Identification and purification of human T-lymphocyte colony-enhancing factor, TLCEF: increased production by phorbol myristate acetate.

T-lymphocyte colony-enhancing factor (TLCEF), a growth factor for a minute subpopulation of T lymphocytes, is produced, along with other factors, by conditioned media (CM) of mononuclear cells following stimulation with T mitogens, such as phytohaemagglutinin (PHA). A combination of PHA and a co-mitogen, phorbol 12-myristate 13-acetate (PMA), has been shown to have a synergistic effect on the production of TLCEF, yielding levels of activity eight to fifteen times higher than those obtained with either PHA or PMA alone. TLCEF was purified by ammonium sulphate, fractionation hydrophobic interaction chromatography on phenyl-Sepharose and gel filtration. The last step of this purification procedure yielded two peaks of activity purified 13,000- and 29,000-fold, respectively. The first peak eluted from the column with an average molecular weight of 125,000, whereas the second peak was retained on the gel, probably due to non-specific interactions with it. The purified fractions contained none of the following activities: T-cell growth factor (TCGF), colony-stimulating factor-1 (CSF-1), interleukin-3 (IL-3) and interferon. TCGF activity, which is known to be unstable at high degrees of purification, was already lost after the ammonium sulphate fractionation step, most probably because of the low protein and albumin concentrations (0.33 and 0.11 mg/ml, respectively). TLCEF is thus a more stable immunoregulatory factor than TCGF and has a much higher apparent molecular weight.     

Comparison of the calcium ionophore and phorbol myristate acetate on the initiation of the respiratory burst in human neutrophils.

We investigated the ability of the calcium ionophore A23187 and phorbol myristate acetate to elicit a respiratory burst in human neutrophils. In general, the ionophore was considerably more potent than phorbol myristate acetate in generating a chemiluminescent response and slightly less active in generating superoxide anion. In contrast, the ionophore caused a much smaller stimulation of glucose oxidation via the hexose monophosphate shunt. This relative inability of the ionophore to stimulate the shunt could not be ascribed to an effect on glucose transport or to a direct inhibition of any of the enzymes involved in glucose metabolism. These data suggest that different stimuli may have markedly different effects on various activities associated with the respiratory burst and emphasize the necessity for measurement of more than one parameter to assess the oxidative metabolism of the neutrophil.     

An ultrastructural analysis of tumor-promoting phorbol diester-induced degranulation of human basophils.

Release reactions stimulated in human basophils by a variety of secretagogues show biochemical and morphologic differences as well as similarities. Biochemical differences include those of rate, amount, and order of mediator release, as well as mediator type released or generated. Morphologic diversity of release reactions includes prototypic anaphylactic degranulation (AND), or piecemeal degranulation (PMD), and a continuum of anatomic release comprised of PMD followed by AND that is seen when human basophils are stimulated by the bacterial peptide, formyl methionyl leucyl phenylalanine (FMLP). AND is characterized by extrusion of membrane-free granules through multiple plasma membrane pores; PMD is characterized by partially to completely empty, nonfused granule containers in the cytoplasm of basophils. AND is further characterized by diminished-to-absent granules and reduced cytoplasmic vesicles at peak histamine release intervals; PMD does not show decreases in numbers of granules, and cytoplasmic vesicles are plentiful. Smooth membrane-bound vesicles with granule particles and vesicles that appear empty comprise this organelle population. PMD is the single most evident activation change present in basophils that traffic into tissues in multiple diseases in vivo. In this study, we examined the ultrastructural kinetic morphology associated with stimulation of human basophils with tetradecanoyl phorbol acetate (TPA)--a tumor-promoting phorbol diester known to elicit histamine (but not LTC4) release. Partially purified human basophils were prepared for electron microscopy and examined either after control incubations in buffer alone or at 0 time, 1, 2, 5, 10, 30, and 45 minutes after TPA stimulation. Standard morphology and ultrastructural quantitation of vesicles and granules and contents of vesicles or alteration of granules was done and compared with previous ultrastructural kinetic analyses of human basophil release reactions stimulated by different triggers. Like biochemical studies that have determined that TPA is a unique secretogogue for human basophils, the morphology stimulated by TPA and associated with histamine release was also unique. For example, very minor images of AND were evident. Far greater amounts of PMD were imaged. PMD was associated with approximately 50% alteration of cytoplasmic granules by 45 minutes after TPA stimulation. This evidence of empty granules was associated with, and preceded by, a rapid, extensive, and sustained increase in particle-containing cytoplasmic vesicles, as compared with buffer controls (P < 0.001 for each TPA stimulation time compared with unstimulated basophils). In addition, previously undescribed interactions of releasing granules and their overlying plasma membranes characterized TPA-stimulated cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhancement of human polymorphonuclear leukocyte adherence to plastic and endothelium by phorbol myristate acetate. Comparison with human C5a.

The adherence of circulating neutrophils to vascular endothelium represents a necessary step in the chemotactic emigration of neutrophils to extravascular inflammatory sites. Studies of neutrophil adherence induced by phorbol myristate acetate (PMA) were undertaken to determine the ability of a nonchemotactic neutrophil stimulus to provoke increased adherence. The authors found that the adherence of human neutrophils to plastic surfaces or confluent monolayers of endothelial cells is enhanced in a concentration-dependent fashion by exposure of neutrophils to PMA. The effect of PMA concentration (0.1-5.0 ng/ml) on increased neutrophil adherence parallels that observed for superoxide anion generation and release of lysosomal enzymes from specific granules. Whereas complement C5a-treated neutrophils exhibited a fourfold to fivefold increase in adherence to endothelial cells, PMA-treated neutrophils showed a 10-fold to 20-fold increase. The ability of PMA to cause increased neutrophil adherence to endothelium appeared to be directed primarily at the neutrophil. Pretreatment of neutrophils with PMA was as effective as coincubation in causing increased adherence to plastic surfaces or confluent cultured endothelial cells, but pretreatment of endothelial cells with PMA failed to promote neutrophil adherence. Alteration of neutrophil cytoskeletal structures by cytochalasin B treatment did not prevent subsequent PMA-stimulated neutrophil adherence. These results demonstrate that increased neutrophil adherence to surfaces can be induced by a nonchemotactic stimulus and that neutrophil adherence is independent of organized microfilaments.     

Pretreatment with phorbol myristate acetate inhibits macrophage activity against intracellular protozoa

To further document the role of toxic oxygen intermediates in mononuclear phagocyte antiprotozoal activity, microbicidal macrophages were depleted of the capacity to generate superoxide anion (O-2) and hydrogen peroxide (H2O2) by pretreatment with phorbol myristate acetate (PMA), a soluble agent which triggers the macrophage respiratory burst. Treating cells for 90 min with 200 ng/ml of PMA inhibited the extracellular release of both O-2 and H2O2 by 90% upon subsequent restimulation with either PMA or opsonized zymosan. This effect persisted for 48 h, and could not be reversed by the addition of lymphokine. Intracellular nitro-blue tetrazolium reduction by PMA-treated cells was also inhibited by 66-95% upon rechallenge with either PMA or inert or viable particulate agents. In parallel, PMA pretreatment abolished or markedly impaired the ability of normal, lymphokine-stimulated, and in vivo activated macrophages to kill three diverse protozoa--Toxoplasma gondii, Leishmania donovani, and Trypanosoma cruzi. These studies illustrate an additional technique for investigating the antiprotozoal effects of macrophage-derived O-2 and H2O2 and reemphasize the importance of an intact respiratory burst mechanism in killing of intracellular parasites.     

Differentiation of ovine immature B cells upon exposure to phorbol myristate acetate

Immature B cells obtained from the ileal Peyer's patches ( IPPs ) of sheep, upon exposure to phorbol myristate acetate (PMA), expressed 20-30 times more sIgM per cell than nonstimulated IPP cells, and the sIgM level was the same as that of peripheral B cells. The exposure of IPP cells to PMA also induced IgM secretion. Macrophages were not required for the terminal differentiation of IPP cells in vitro.     

Comparison of phorbol myristate acetate and phytohaemagglutinin as stimulators of in vitro T lymphocyte colony formation of human peripheral blood lymphocytes. I. Surface markers of colony cells.

Human T lymphocyte colonies were grown in methylcellulose semi-solid cultures in the presence of phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA). Surface marker analysis showed lower percentages of OKT3- and OKT4-positive cells in PMA-induced colonies than those in PHA-induced colonies. The percentage of OKIa1-positive cells in PMA-induced colonies was approximately twice that in PHA-induced colonies. The percentage of OKT9-positive cells in PMA-induced colonies was significantly lower than that in PHA-induced colonies. These data suggest that the subsets of PMA-induced colony cells express a more immature phenotype than that of PHA-induced colony cells and that, among PMA-induced colony cells, there are fewer T cells in the proliferative status at the time tested. When 3 X 10(5)/ml monocyte-depleted T cells, at which concentration of seeded cells neither PHA nor PMA could induce colony growth, were cultured in the presence of both PHA and PMA, T cell colony growth was observed. In T cell colonies induced by a combination of PHA and PMA, the percentages of OKT3-, OKT4- and OKT8-positive cells were different from those in colonies induced by either PHA or PMA alone. These results suggest that PMA acts not only as a substitute for monocytes and/or interleukin-1, but may directly affect lymphocyte proliferation induced by a combination of PHA and PMA.