The stimulation of superoxide anion production in guinea-pig peritoneal macrophages and neutrophils by phorbol myristate acetate, opsonized zymosan and IgG2-containing soluble immune complexes

The kinetics of superoxide anion production in guinea-pig peritoneal macrophages and neutrophils were determined following in vitro stimulation with phorbol myristate acetate (PMA), opsonized zymosan (OZ) and soluble immune complexes of guinea-pig IgG2 (SIC). Superoxide production was recorded as chemiluminescence (CL) arising from the reductive cleavage of lucigenin. With PMA, both macrophages and neutrophils displayed a two-phase response consisting of a rapid initial burst of CL, which preceded ligand ingestion, followed by a plateau in the CL response which persisted for more than 30 min. By contrast, OZ induced a slow progressive increase in CL in both phagocytes which was consistent with the development of an oxidative burst concomitant with ingestion. The phagocytes differed in their responses to SIC, the macrophages displaying CL kinetics similar to those observed with PMA, whereas the neutrophils responded in the manner observed with OZ. The relationship between disparity in the patterns of macrophage and neutrophil CL responses to SIC and differences in their expression of Fc receptors for IgG2 (Coupland & Leslie, 1983) is discussed.     

Two distinct cytolytic mechanisms of macrophages and monocytes activated by phorbol myristate acetate

Pulmonary alveolar macrophages (PAM) and peripheral blood monocytes (PBMO) of the miniature swine can be converted to cytolytically active effector cells by treating with phorbol myristate acetate (PMA) as determined by enhancement of cytotoxicity to various target cells. Kinetics of the PMA-activated PAM and PBMO in cytotoxicity show that the effective PMA concentration ranges from 10 to 1,000 ng/ml. Induction of cytotoxic macrophages and monocytes occurred as early as 30 min and to their maximum cytotoxicity after 1 hr exposure to PMA and the enhanced cytotoxic activity persisted up to 24 to 40 hr when PMA was removed by washing after 1 hr exposure, but prolonged exposure to PMA for more than 6 hr resulted in a drastic decrease of cytolytic activities suggesting the prolonged exposure to PMA causes macrophages and monocytes to become refractory to PMA stimulation. Target cells displayed varying degrees of cytotoxic sensitivity to the PMA-activated PAM and PBMO; PRBC, SRBC, and K562 were sensitive, WEHI-164 and U937 were relatively sensitive, and SB was very resistant to these activated effector cells. The mechanisms of PMA-induced cytotoxicity could largely be divided into two categories. One was the H2O2 mediated killing as shown by complete reduction of cytotoxicity after adding catalase in the assay. The other was the proteases mediated cytolysis, which could be blocked by protease inhibitors, Phenyl methyl sulfonyl fluoride (PMSF), and N- alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK). H2O2 was the only mediator produced in large enough quantities from PBMO to kill target cells, whereas PAM could produce both mediators (H2O2 and proteases). PRBC, SRBC, and K562 appeared to be killed by H2O2 produced by PAM and PBMO. In contrast, U937 and WEHI-164 appeared to be killed by proteases in PAM mediated cytolysis but by H2O2 in PBMO-mediated cytolysis. These results suggest that the observed cytolytic mechanisms can be differed by type of target cells as well as the source of mononuclear phagocytes within the individual animal.     

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.     

Polymorphonuclear leukocyte chemiluminescence induced by formylmethionyl-leucyl-phenylalanine and phorbol myristate acetate: Effects of catalase and superoxide dismutase

The effects of pristane (2,6,10,14-tetramethylpentadecane) on the cellular DNA of lymphoid cells from Copenhagen rats were examined by flow cytometry. Significant reductions in the mean relative fluorescent intensities of propidium iodide (PI) stained lymphocytes from peripheral blood, spleen, thymus and lymph nodes were observed after a single intraperitoneal injection of pristane. The altered PI staining characteristics were observed as early as 4 days and reached a maximum decrease between 1–4 weeks (depending upon the lymphoid cells examined) post pristane treatment. The pristane-induced effects on peripheral blood lymphocytes were observed to be dose dependent, transient and reinducible by a subsequent exposure to pristane. Further analyses, using gas-liquid chromatography to detect pristane in the blood and lymphoid tissues of treated rats, indicated, significant increases over normal amounts of pristane. Furthermore, correlations existed between the times of maximum decrease in the fluorescence of PI stained cells and the amounts of pristane detected within the respective lymphoid tissues. By contrast no changes in the PI staining characteristics of kidney cells were observed, even though appreciable amounts of pristane, were detected in this organ. Diphenylamine analyses indicated no differences in the amounts of DNA in lymphoid cells from pristane treated and untreated rats. Furthermore, lymphocytes from pristane-treated rats did not exhibit decreased fluorescence when fixed at pH 10 rather than pH 7.4 prior to PI staining. Collectively these results suggest that pristane, may preferentially induce qualitative rather than quantitative changes in the DNA of lymphocytes.This investigation was supported by PHS grant numbers CA33111 and AI22607.     

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.     

Effect of phorbol myristate acetate on cellular metabolism and lysozyme release from alveolar macrophages and polymorphonuclear leukocytes

Phorbol myristate acetate stimulated oxidative metabolism in alveolar macrophages and blood neutrophils. This compound also stimulated lysozyme release from neutrophils but not from alveolar macrophages. These findings suggest that the regulation of lysozyme release from alveolar macrophages is different than for polymorphonuclear leukocytes.     

Differential effects of nylon fibre adherence on the production of superoxide anion by human polymorphonuclear neutrophilic granulocytes stimulated with chemoattractants, ionophore A23187 and phorbol myristate acetate.

Human polymorphonuclear neutrophilic granulocytes were made adherent by passing them over protein-coated nylon fibre columns and compared with suspended cells for their production of superoxide anion as measured by cytochrome C reduction. The cells were stimulated with chemotactic factors, the ionophore A 23187, and the tumour promoter phorbol myristate acetate. There was no increased O2-. production by adherent cells in the absence of a stimulus. Adherent cells produced considerably higher amounts of superoxide than suspended cells when stimulated with formyl-methionyl-leucyl-phenylalanine, ionophore A 23187, C5a, C5adesArg, and the platelet activating factor 1-o-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine. In contrast, stimulation with phorbol myristate acetate did not result in higher superoxide release from adherent than from suspended cells, and leukotriene B4 and a mononuclear cell-derived chemotaxin did not stimulate either cell to release significant amounts of superoxide. It is suggested that the augmented production of oxygen radicals with certain stimuli contributes to inflammatory symptoms in situations involving adherent granulocytes.     

Tumor necrosis factor-alpha and interleukin-1 alpha synergistically enhance phorbol myristate acetate-induced superoxide production by rat bone marrow-derived macrophages.

Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) are secreted by macrophages in response to endotoxin challenge. In addition, macrophages express receptors for both of these cytokines. Macrophage function can therefore be modulated by regulation of both cytokine production and receptor levels. We have initiated studies to investigate the effects of TNF-alpha and IL-1 alpha on macrophage function. Macrophages were obtained by in vitro differentiation of rat bone marrow cells. The biologic response to TNF-alpha and IL-1 alpha was assessed by measurement of superoxide production quantitated by the reduction of cytochrome c in response to phorbol myristate acetate. Macrophages were treated with endotoxin (LPS), TNF-alpha, and IL-1 alpha, alone and in combination. None of these agents was a primary stimulus for superoxide production. However, after treatment with endotoxin or TNF-alpha for 24 h, macrophages were primed for enhanced production of superoxide. The priming effect of LPS was due, at least in part, to endogenously produced TNF-alpha, since anti-murine TNF-alpha antibodies blocked the LPS-mediated priming by approximately 30%. IL-1 alpha did not prime macrophages, but treatment with IL-1 alpha followed by TNF-alpha or LPS resulted in enhanced superoxide production. IL-1 alpha treatment of macrophages resulted in an increase in TNF-alpha receptors, which might explain the synergistic priming of TNF-alpha and IL-1 alpha.     

Phorbol myristate acetate modulates calcium ion-dependent superoxide anion generation induced by a monoclonal antibody raised against polymorphonuclear leukocytes.

We used a monoclonal antibody, YI 51, raised against human polymorphonuclear leukocytes (PMN) to induce superoxide anion (O2-) generation in cells. Although YI 51 alone played only a small part in inducing O2- generation in PMN, the amount of O2- generation induced in 5 X 10(5) PMN was 3.7 to 5.5 nmol/min when F(ab')2 fragments of rabbit anti-mouse immunoglobulin antibody were added as a cross-linking agent. This O2- -inducing activity was high compared with that of wheat germ agglutinin (WGA), insoluble immunoglobulin G immune complexes (IC), or phorbol myristate acetate (PMA). The binding of YI 51 and soluble immunoglobulin G IC to PMN was not reciprocally inhibitory, indicating that YI 51 does not interfere with ligand binding to the Fc receptor-binding site. In the absence of calcium ion (Ca2+), O2- generation induced by YI 51 decreased to 10 to 20% of that in the presence of Ca2+. In contrast, O2- generation in response to WGA, IC, or PMA under Ca2+-free conditions was not affected. When PMN were pretreated with low concentrations of PMA (10(-10) to 10(-9) M), the amount of O2- generation by the cells in response to YI 51 in Ca2+-free buffer was enhanced in a concentration-dependent manner. It also equaled the O2- generated by the cells in buffer containing Ca2+. In cells pretreated with PMA, the amount of O2- induced by WGA was enhanced two- to threefold over that in untreated cells. In contrast, there was no augmentation over untreated cells with stimulation by IC. These results suggest that YI 51, IC, and WGA induce O2- generation in human PMN in different manners.     

The effect of phorbol myristate acetate on human lymphocytes. An ultrastructural study.

Phorbol myristate acetate (PMA), a tumour promoter, acts as an agglutinin and as a stimulator of human lymphocytes. Agglutination begins within 1 hr and is macroscopically evident within 24 hr. The ultrastructural findings in lymphocytes stimulated with PMA after 48 and 72 hr show blast transformation of the nucleus, increase in cytoplasm and in ribosomes, and occasional “annulate lamellae.” Apart from the finding of “annulate lamellae” the changes are generally similar to those found in PHA stimulated lymphocytes. The function and significance of the “annulate lamellae” are not known but as they have been most often seen in malignant and embryonal cells they may be representative of a more fundamental dedifferentiation of the cells with PMA as opposed to PHA.     

IFN-beta-induced reduction of superoxide anion generation by macrophages.

Resident mouse peritoneal macrophages (M phi) produced significant amounts of superoxide anion (O2-) in response to phagocytic stimuli. When M phi were exposed in vitro for 20 hr to fibroblast interferon (IFN-beta), their capacity to release O2- was significantly reduced, such reduction being more evident with increasing IFN-beta concentrations. In contrast, O2- production by M phi exposed for 20 hr to the lymphokine macrophage activating factor (MAF) or treated with either MAF or IFN-beta for 4 hr was not significantly different from that of control cells. This pattern of activity closely followed that of M phi-mediated suppression of lymphocyte proliferation, which was dramatically reduced by 20 hr exposure of M phi to IFN-beta, but unchanged by treatment with MAF. No correlation was however found between superoxide anion generation and enhancement of tumoricidal capacity in IFN-beta-treated M phi. We thus concluded that O2- does not play a relevant role in IFN-beta-induced M phi cytolysis, whereas the reduction of O2- production could be of major importance in the decrease of M phi suppression induced by IFN-beta.     

Sensitivity of receptors for IgA on T560, a murine B lymphoma,to phorbol myristate acetate and to phosphatidylinositol-specific phospholipase C

A GALT-derived B lymphoma, T560, that bears IgAR is described. T560 is IgG2a kappa +, Ia+, B220+, J11d+, Thy-1-, CD3-, CD4-, CD5-, Mac 1-, Mac 2-, nonspecific esterase negative and binds bromelain-treated mouse RBC but not SRBC or ORBC. It presents antigen, secretes IL-1, IL-4 and IL-6 but not IL-2, IL-5 or TGF beta and appears to be related to the Lyt 1+(CD5) lineage of B cells though it lacks Lyt 1. T560 bears IgAR that, on the cell surface, are completely cross-inhibited by low concentrations of IgM and by high concentrations of IgG2a and IgG2b. They do not appear to represent a cell-surface form of galactosyl transferase. They are inducible by high concentrations of IgA, sensitive to trypsin and insensitive to neuraminidase. They are down-regulated by activation of PKC with PMA, but their recovery is not inhibited by cycloheximide, indicating that they are not degraded or shed. They may either lose their affinity for IgA or be internalized without degradation. Seventy percent of IgA receptor activity is lost when T560 is treated with PI-PLC; part of this loss of activity is due to activation of PKC and is inhibited by staurosporine, but approximately 30% of it is not protected by staurosporine indicating that some, or all, of the IgA receptor of T560 is connected to the cell membrane via a GPI linker. The T560 IgA receptor could be related to the poly-Ig or M cell receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-1 antichymotrypsin is increased in human alveolar macrophages by phorbol myristate acetate or lipopolysaccharide and released from these activated macrophages by glucocorticoid.

Alveolar macrophages were obtained from 23 patients and the effects of phorbol myristate acetate (PMA), lipopolysaccharide (LPS), and dexamethasone (DEX) on the proportion of cells with intracellular alpha-1 antichymotrypsin (ACT), and concentrations of ACT in the culture medium were studied in vitro. The alveolar macrophages were obtained by bronchoalveolar lavage at autopsy or from resected lungs at operation and were cultured in suspension for 3 days in medium containing PMA, LPS, DEX, PMA+DEX, or LPS+DEX. Both PMA and LPS significantly increased the percentage of macrophages with intracellular ACT. Dexamethasone did not increase the number of ACT-positive cells and significantly suppressed the increase induced by PMA or LPS, releasing ACT into the culture medium. The release of ACT from macrophages may contribute to the anti-inflammatory effects of corticoids.     

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).     

Heat shock treatment of macrophages causes increased release of superoxide anion.

Heat shock treatment of murine macrophages and the J774 cell line resulted in an enhanced capacity to release superoxide anion (O2-) upon stimulation. There was no concomitant increase in hydrogen peroxide production, and the macrophage microbicidal activity against Mycobacterium tuberculosis, Mycobacterium avium complex, and Staphylococcus aureus was not altered.     

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.     

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.     

Impaired release of colony stimulating activity by monocytes from Hodgkin's disease in response to phorbol myristate acetate activation

We assessed the humoral effect of resting and phorbol esters preincubated monocytes from Hodgkin's disease patients (HDMo) and healthy subjects (nMo), on granulocyte progenitors (CFU-dG) growth using a double diffusion chamber technique. The release of colony stimulating activity and indomethacin-dependent inhibitors by resting HDMo and nMo was found to be cell-concentration dependent. However, phorbol myristate acetate preincubated HDMo (PMA-HDMo) in contrast to nMo at low concentrations (2.5 x 10(4] were unable to increase the CFU-dG growth stimulation. On the other hand, at a higher cell number (5 x 10(4], phorbol treated HDMo stimulated the myeloid colony formation, whereas nMo suppressed the CFU-dG proliferation. Further enhancement of HDMo and nMo concentrations induced a pronounced inhibition of CFU-dG-derived colony formation, caused by an increased PGE2 production. After incubation with the cyclooxygenase inhibitor-indomethacin, PMA-HDMo showed considerably more granulocyte colony formation than nMo. Our results suggest that the observed abnormalities in the function of HDMo could be associated with an excessive production of PGE2 and a general dysfunction of these cells in Hodgkin's disease.