Changes in phosphatidylinositol and phosphatidic acid in stimulated human neutrophils. Relationship to calcium mobilization, aggregation and superoxide radical generation

Human neutrophils aggregate and release mediators of inflammation, such as active oxygen species and lysosomal enzymes, when exposed to the chemoattractant, fMet-Leu-Phe, or the tumor promotor, phorbol myristate acetate. In order to 'stage' events which may lead to such neutrophil responses, we determined the temporal relationship between stimulus-induced changes in the endogenous phospholipids phosphatidylinositol (PI) and phosphatidic acid, the mobilization of calcium, and the onset of aggregation and generation of superoxide anion during the initial 2 min of cell activation. Within 5 s after addition of fMet-Leu-Phe (10(-7) M) neutrophils accumulated phosphatidic acid and the levels of PI decreased, as determined by two-dimensional thin-layer chromatography and phosphorus determinations. By 5 s, phosphatidic acid levels rose approximately 3.5-fold and at 15 s the loss of PI exceeded the quantity of phosphatidic acid generated. In response to phorbol myristate acetate (1 microgram/ml), however, changes in PI or phosphatidic acid were not observed until after 60 s. Accumulation of phosphatidic acid in fMet-Leu-Phe-stimulated cells was not inhibited by chelation of extracellular calcium. Neutrophils exposed to either fMet-Leu-Phe or phorbol myristate acetate also showed rapid decrements in fluorescence of cell-associated chlorotetracycline (used as an indirect probe of mobilization of intracellular membrane-associated calcium) and took up 45Ca2+ from the extracellular medium (under 60 s). The results indicate that changes in calcium mobilization, together with the alterations in phospholipid metabolism (under 5 s) anteceded aggregation and the generation of O2-. (10-15 s) induced by fMet-Leu-Phe. In contrast, when neutrophils were exposed to phorbol myristate acetate, changes in PI and phosphatidic acid (over 60 s) were observed after the mobilization of calcium (under 5 s) and the onset of O2-. generation and aggregation (30-35 s).     

Phorbol myristate acetate potentiates superoxide release and membrane depolarization without affecting an increase in cytoplasmic free calcium in human granulocytes stimulated by the chemotactic peptide, lectins and the calcium ionophore

We investigated the inter-relationships of superoxide (O2-) release, membrane depolarization and an increase in cytoplasmic free Ca2+, [Ca2+]i, in human granulocytes stimulated by various agonists. When concanavalin A or the Ca2+ ionophore ionomycin was used as stimulus, an increase in [Ca2+]i clearly preceded the onset of membrane depolarization, which was followed by O2- release. On the other hand, when N-formylmethionylleucylphenylalanine or wheat-germ agglutinin was used as stimulus, no demonstrable lag was seen in any of the responses. O2- release and membrane depolarization stimulated by all these agonists were markedly potentiated in parallel by pretreatment of cells with a low concentration of phorbol myristate acetate (0.25 ng/ml), whereas an increase in [Ca2+]i was not affected or minimally potentiated. The lag time between addition of the stimulus (concanavalin A or ionomycin) and onset of membrane depolarization or O2- release was significantly reduced by pretreatment of cells with phorbol myristate acetate, whereas the lag time between addition of concanavalin A and onset of the increase in [Ca2+]i was not affected. The dose-response curves for triggering of O2- release and membrane depolarization by each of receptor-mediated agonists in phorbol myristate acetate-pretreated or control cells were identical. These findings suggest that; (a) an increase in [Ca2+]i stimulates membrane depolarization indirectly; (b) a low concentration of phorbol myristate acetate potentiates membrane depolarization and O2- release by acting primarily at the post-receptor level, in particular, at the level distal to an increase in [Ca2+]i, but not by augmenting an increase in [Ca2+]i; and (c) the system provoking membrane depolarization and the system activating NADPH oxidase share a common pathway, which may be susceptible to a low concentration of phorbol myristate acetate.     

Labelling of lipids and phospholipids with [3H]arachidonic acid and the biosynthesis of eicosanoids in U937 cells differentiated by phorbol ester

Phorbol esters induce morphologic and biochemical differentiation in U937 cells, a monocyte/macrophage-like line derived from a human histiocytic lymphoma. We are interested in the phorbol ester-stimulated release of arachidonic acid from cellular membranes and the subsequent synthesis of eicosanoids, as it may prove to correlate with the induced cellular differentiation. Undifferentiated log-phase U937 cells released little recently incorporated [3H]arachidonic acid, but phorbol 12-myristate 13-acetate increased its apparent rate of release to that of cells differentiated by exposure to phorbol myristate acetate for 3 days. Exposure of washed differentiated cells immediately prelabelled with [3H]arachidonic acid to additional phorbol myristate acetate did not augment the release of [3H]arachidonic acid. The basal release of nonradioactive fatty acids from differentiated cells was 5-10 times that of undifferentiated cells, and phorbol myristate acetate increased their release from both types of cell 2- to 3-fold. Differentiated cells immediately prelabelled with [3H]arachidonic acid exhibited greater incorporation into phosphatidylinositol and phosphatidylcholine, and contained more radioactive free arachidonic acid, compared with undifferentiated cells. Undifferentiated cells contained more radioactivity in phosphatidylserine, phosphatidylethanolamine and neutral lipids. Phorbol myristate acetate caused differentiated cells to release [3H]arachidonic acid from phosphatidylinositol, phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine, but release from neutral lipids was reduced, and the content of [3H]arachidonic acid increased. In undifferentiated cells incubated with phorbol myristate acetate, radioactivity associated with phosphatidylserine, phosphatidylethanolamine and neutral lipid was reduced and [3H]arachidonic acid was unchanged. Synthesis of cyclooxygenase products exceeded that of lipoxygenase products in both differentiated and undifferentiated cells. Phorbol myristate acetate increased the synthesis of both types of product, cyclooxygenase-dependent more than lipoxygenase-dependent, especially in differentiated cells. The biological significance of these changes in lipid metabolism that accompany phorbol myristate acetate-induced differentiation are yet to be established.     

Adhering lung macrophages produce superoxide demonstrated with desferal-Mn(IV)

It has been shown that H2O2, the dismutation product of O2., is produced at cell-surface interfaces. Nevertheless, the relationships between the degree of attachment itself, type of surface, and O2. production are not clear. Superoxide production can be measured by the O2.-dependent reduction of nitroblue tetrazolium to an insoluble formazan. Superoxide dismutase (SOD) may be unable to scavenge O2. produced between alveolar macrophages (AM) and a surface. Desferal-Mn(IV) (Des-Mn), a low molecular weight mimic of SOD, is protective against paraquat toxicity in vivo, presumably because of specificity for O2-. Using that assumption, Des-Mn was used to measure O2. production that occurred during adherence of AM. AM suspensions were placed on fibronectin-coated glass coverslips or uncoated glass coverslips or non-stick tissue culture plates. Adherence to the surfaces varied with fibronectin greater than glass greater than non-stick and the percent formazan positive cells was 60, 24, and 4, respectively. With SOD present, the percentage of formazan positive cells were 40, 17, and 2; however, in the presence of Des-Mn the percent stained cells was 4, 4, and 0. When phorbol myristate acetate (PMA) was added during adherence, the percent of formazan positive cells was 82, 57, and 44, respectively. With PMA, Des-Mn was able to inhibit 88-100% of formazan staining whereas SOD inhibition decreased more markedly with increasing adherence. These results indicated that the degree of attachment correlated with both the degree of NBT reduction and the relative effectiveness of Des-Mn versus SOD to scavenge O2..     

Cultured human monocytes require exposure to bacterial products to maintain an optimal oxygen radical response

Freshly isolated human blood monocytes displayed a vigorous oxygen radical response, measured as release of superoxide anion (O2-), after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. High O2- release was observed with cells isolated by using a variety of procedures. Monocytes cultured in endotoxin-free medium M199 with or without 5% heat-inactivated autologous serum gradually lost this ability to produce O2- in response to PMA over the course of 4 days. The decreased responsiveness to PMA was accompanied by decreased adherence and viability. The loss of function, adherence, and viability was prevented by supplementing the culture medium with either bacterial lipopolysaccharide (LPS) or muramyl dipeptide (MDP). The O2- response of monocytes cultured for several days without bacterial products could be partially restored by the addition of LPS on day 2 or 3 of culture. Partial restoration could be detected in monocytes after only 1 hr of exposure to LPS, although a maximal response required a 2-day exposure. The minimum effective concentration of MDP was 1 ng/ml; stereoisomers of MDP, which are inactive as adjuvants, had no effect at 1 micrograms/ml. The minimum effective concentration of LPS was 1 pg/ml, corresponding to fewer than 10 molecules of LPS per monocyte. These results suggest that exposure to LPS or other bacterial products, represented here by MDP, may be required to preserve the microbicidal potential of human monocyte-macrophages in vivo.     

Relationships between in vitro selenium supply, glutathione peroxidase activity, and phagocytic function in the HL-60 human myeloid cell line

Utilizing the HL-60 human promyelocytic leukemia cell line cultured in defined medium, we examined the quantitative and temporal relationships between Se supply and the activity of the selenoenzyme glutathione peroxidase, as well as the effects of selenium deficiency on phagocytic function. Glutathione peroxidase activity depended on the medium Se concentration up to 2.6 X 10(-8) M (sodium selenate, 5 ng/ml), above which a plateau occurred. HL-60 cells grown in medium without Se supplementation became GSH peroxidase deficient, with activity 1-3% that of Se-replete cells. Replenishment of the medium with sodium selenate returned enzyme activity to 23% that of replete cells by 24 h and to 85% by 7 days, a process blocked by cycloheximide. Se-deficient HL-60 cells induced to granulocytic differentiation by dimethylformamide showed decreased hexose monophosphate shunt activity in response to phorbol myristate acetate and to an exogenous enzymatic H2O2-generating system. However, Se-deficient and -replete cells showed equal responses to methylene blue, which stimulates the shunt independently from the glutathione cycle. Se-deficient mature HL-60 cells stimulated with phorbol myristate acetate released 2.3-fold more H2O2 than Se-replete cells and only slightly (not significantly) less O2. Se-deficient and -replete differentiated HL-60 cells did not differ significantly in their capacities for cell motility or for ingestion of serum-opsonized bacteria. Differences between the findings of the present study and previous in vivo rat studies may reflect both the defined in vitro environment of the cell line and the inverse ratios of catalase and glutathione peroxidase activities in human and rat granulocytes.     

Regulation of the growth and differentiation of a human monocytic cell line by lymphokines. I. Induction of superoxide anion production and chemiluminescence

The U937 human monocytic cell line was studied to determine its ability to generate a respiratory burst after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. U937 cells cultured in normal medium produced virtually no superoxide anion or chemiluminescence in response to either stimulus. In contrast, U937 cells cultured in medium containing soluble factors from activated lymphocytes produced significant O2- and chemiluminescence when stimulated with PMA or opsonized zymosan. The chemiluminescence in response to PMA was maximal in U937 cells precultured with these soluble factors for 3 days, whereas maximal responsiveness to opsonized zymosan was not observed until 5 to 6 days of lymphokine exposure. Although this ability to generate a respiratory burst persisted for a number of days in U937 cells that were subsequently recultured in normal medium, this responsiveness was gradually lost in the continued absence of these factors. The data indicate that the U937 monocytic cell line can be activated or induced to differentiate by soluble factors released by activated lymphocytes. In the process, these cells acquire the ability to generate a respiratory burst. The U937 cell line may serve as a useful model for the study of the ontogeny and regulation of the respiratory burst during human monocytic differentiation.     

Up-regulation of interleukin 4 receptor expression on immature (Lyt-2-/L3T4-) thymocytes

In this report, the effect of interleukin 4 (IL-4) on the growth and differentiation of Lyt-2-/L3T4-(2-4-) thymocytes was investigated. It was found that these thymocytes proliferated extensively when cultured in the presence of IL-4 + phorbol myristate acetate without apparent differentiation to Lyt-2+ or L3T4+ cells. We also demonstrated that 2-4- thymocytes constitutively express a high affinity (dissociation constant of 20 to 40 pM) receptor for IL-4. Freshly isolated 2-4- thymocytes expressed on average about 100 IL-4 receptors per cell, but the number of receptors increased approximately 8-fold within 3 days after activation by IL-4 + phorbol myristate acetate. These findings suggest that IL-4 may play an important role in T cell ontogeny by promoting self-renewal of stem cells within the thymus.     

Effect of an inhibitor of protein kinase C on human polymorphonuclear leukocyte degranulation

The protein kinase C inhibitor C-I reduced superoxide production by human neutrophils in response to phorbol myristate acetate by greater than 50%. In contrast to its effects in oxidative metabolism, 100 microM C-I caused minimal inhibition (5-18%) of lysozyme release in response to phorbol myristate acetate. Enzyme release produced by the formylated oligopeptide FMLP was enhanced by 23-54% in neutrophils pretreated with 100 microM C-I. These findings suggest that protein kinase C activation is not required for phorbol myristate acetate induced enzyme release. Enhancement of FMLP stimulated degranulation by C-I suggests that protein kinase C activation may have inhibitory effects on the release of granule enzymes by human neutrophils.     

Intracellular calcium does not appear to be essential for arachidonic acid release from stimulated macrophages as shown by studies with Quin-2

The present investigation was undertaken to study the potential role of intracellular calcium on the release of arachidonic acid from mouse peritoneal macrophages activated by inflammatory stimuli. The intracellular calcium concentration, as measured using fluorescent probe Quin-2, was 112 +/- 8.4 nM. The chelation of intracellular calcium with Quin-2 did not affect the release of arachidonic acid from macrophages upon stimulation with phorbol myristate acetate, opsonized zymosan or calcium ionophore A23187. However, the removal of calcium from the extracellular medium resulted in a 30-50% decrease in arachidonic acid release from phorbol myristate acetate- and zymosan-stimulated macrophages and also the stimulation of arachidonic acid release from calcium ionophore-stimulated cells were nullified. These studies indicated the existence of calcium-dependent and independent mechanisms modulating the release of arachidonic acid from macrophages subjected to inflammatory stimuli.     

Changes in phosphatidylinositol and phosphatidic acid in stimulated human neutrophils: Relationship to calcium mobilization,aggregation and superoxide radical generation

Human neutrophils aggregate and release mediators of inflammation, such as active oxygen species and lysosomal enzymes, when exposed to the chemoattractant, fMet-Leu-Phe, or the tumor promotor, phorbol myristate acetate. In order to ‘stage’ events which may lead to such neutrophil responses, we determined the temporal relationship between stimulus-induced changes in the endogenous phospholipids phosphatidylinositol (PI) and phosphatidic acid, the mobilization of calcium, and the onset of aggregation and generation of superoxide anion during the initial 2 min of cell activation. Within 5 s after addition of fMet-Leu-Phe (10^?7 M) neutrophils accumulated phosphatidic acid and the levels of PI decreased, as determined by two-dimensional thin-layer chromatography and phosphorus determinations. By 5 s, phosphatidic acid levels rose approximately 3.5-fold and at 15 s the loss of PI exceeded the quantity of phosphatidic acid generated. In response to phorbol myristate acetate (1 μg/ml), however, changes in PI or phosphatidic acid were not observed until after 60 s. Accumulation of phosphatidic acid in fMet-Leu-Phe-stimulated cells was not inhibited by chelation of extracellular calcium. Neutrophils exposed to either fMet-Leu-Phe or phorbol myristate acetate also showed rapid decrements in fluorescence of cell-associated chlorotetracycline (used as an indirect probe of mobilization of intracellular membrane-associated calcium) and took up ⁴⁵Ca²⁺ from the extracellular medium (under 60 s). The results indicate that changes in calcium mobilization, together with the alterations in phospholipid metabolism (under 5 s) anteceded aggregation and the generation of O^?₂ (10–15 s) induced by fMet-Leu-Phe. In contrast, when neutrophils were exposed to phorbol myristate acetate, changes in PI and phosphatidic acid (over 60 s) were observed after the mobilization of calcium (under 5 s) and the onset of O^?₂ generation and aggregation (30–35 s).     

Prostaglandins E1 and E2 enhance the stimulation of superoxide release by 1-oleoyl-2-acetylglycerol from human neutrophils

Superoxide release from human neutrophils was stimulated either by receptor activation (using fMet-Leu-Phe) or by activating, independently, each of the two pathways considered to be involved in signal transduction--calcium mobilization (using the ionophore, A23187) and protein kinase C activation (using phorbol myristate acetate or 1-oleoyl-2-acetylglycerol). Prostaglandin E1 (3 X 10(-5) M) decreased fMet-Leu-Phe-stimulated superoxide release, had no effect on superoxide release stimulated by A23187, or by phorbol myristate acetate, and markedly enhanced the superoxide release stimulated by 1-oleoyl-2-acetylglycerol. Similar enhancement was obtained with prostaglandin E2.     

Potentiation of arachidonic acid release by phorbol myristate acetate in platelets is not due to inhibition of arachidonic acid uptake or incorporation into phospholipids

Activators of protein kinase C, such as tumor-promoting phorbol esters (e.g., phorbol myristate acetate), mezerein, (-)-indolactam V and 1-oleoyl 2-acetoyl glycerol, potentiate arachidonic acid release caused by elevation of intracellular Ca2+ with ionophores. This action of protein kinase C-activators required protein phosphorylation, and was attributed to enhanced hydrolysis of phospholipids by phospholipase A2 (Halenda, et al. (1989) Biochemistry 28, 7356-7363). Recently Fuse et al. ((1989) J. Biol. Chem 264, 3890-3895) reported that the apparent enhanced release of arachidonate was actually due to inhibition of the processes of re-uptake and re-esterification of released arachidonic acid. They attributed this to loss of arachidonyl-CoA synthetase and arachidonyl-CoA lysophosphatide acyltransferase activities, which were measured in membranes obtained from phorbol myristate acetate-treated platelets. In this paper, we show that phorbol myristate acetate, at concentrations that strongly potentiate arachidonic acid release, does not inhibit either arachidonic acid uptake into platelets or its incorporation into specific phospholipids. Furthermore, the fatty acid 8,11,14-eicosatrienoic acid, a competitive substrate for arachidonyl-CoA synthetase, totally blocks arachidonic acid uptake into platelets, but, unlike phorbol myristate acetate, does not potentiate arachidonic acid release by Ca2+ ionophores. We conclude that the action of phorbol myristate acetate is to promote the process of arachidonic acid release by phospholipase A2.     

Reversible activation of the neutrophil superoxide generating system by hexachlorocyclohexane: correlation with effects on a subcellular superoxide-generating fraction

gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M gamma-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, gamma-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10(6) cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messengers for activation of the NADPH-dependent O-2-generating system, we compared neutrophil responses to gamma-hexachlorocyclohexane with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, gamma-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of gamma-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that in intact cells indicated that concurrent degranulation was not required for sustained O-2 generation in response to this agent. When neutrophils or neutrophil cytoplasts exposed to gamma-hexachlorocyclohexane were centrifuged and resuspended in stimulus-free medium, O-2 generation ceased entirely but could be reinitiated by addition of the same stimulus. This finding was in contrast to the continued O-2 production by phorbol myristate acetate-stimulated neutrophils similarly washed and resuspended in stimulus-free medium. Unlike subcellular fractions of phorbol myristate acetate-stimulated neutrophils, corresponding fractions prepared from gamma-hexachlorocyclohexane-stimulated neutrophils contained almost no detectable NADPH-dependent O-2-generating activity. Subcellular oxidase activity was not recovered when cells and membrane fractions were continuously exposed to gamma-hexachlorocyclohexane during disruption and fractionation after cell stimulation, nor could it be induced by the addition of the stimulus to the subcellular fractions. Thus, the stimulus dependence of continuous neutrophil superoxide release evoked by gamma-hexachlorocyclohexane does not merely reflect a physical interaction of the agonist with the enzyme system involved.(ABSTRACT TRUNCATED AT 400 WORDS)     

The human promyelocytic HL60 cell line: a model of myeloid cell differentiation using dimethylsulphoxide, phorbol ester and butyrate.

The release of the reactive oxygen species that accompanies the oxidative burst was studied in HL60 cells differentiated with either dimethylsulphoxide, butyrate or phorbol myristate acetate in order to establish the extent to which differentiated cells are phenotypically similar to human neutrophils, monocytes and macrophages. When phorbol myristate acetate was used as a stimulus, the rates of superoxide production by dimethylsulphoxide and butyrate differentiated HL60 cells was not significantly different from those observed in neutrophils and monocytes isolated from normal peripheral blood. Similar results were obtained when luminol-dependent chemiluminescence was measured in the presence of horseradish peroxidase using phorbol myristate acetate as the stimulus. However, in the absence of horseradish peroxidase, the luminol-dependent chemiluminescence in the dimethylsulphoxide and butyrate-differentiated HL60 cells was significantly lower than that of the control cells isolated from human blood, reflecting the absence of myeloperoxidase in the differentiated cells. In contrast, HL60 cells differentiated by phorbol myristate acetate failed to show any increased generation of superoxide or luminol-dependent chemiluminescence upon stimulation. Impaired release of lysosomal enzymes by the chemically differentiated cells suggests impairments in the extent of differentiation resulting in cells with defective azurophilic degranulation processes. It is concluded that HL60 cells differentiated by the above agents are somewhat controversial models of promyelocyte differentiation into typical neutrophilic, monocytic and macrophage-like cells.     

Effect of interferon-gamma and 1 alpha,25-dihydroxyvitamin D3 on superoxide anion, prostaglandins E2, and mononuclear cell factor production by U937 cells

Both interferon-gamma (IFN-gamma) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) induce changes in the human monocytic cell line U937 that may reflect cellular differentiation. The effects of recombinant IFN-gamma and 1 alpha,25(OH)2D3 on U937 cells with regard to the release of superoxide anion (O2-), prostaglandin E2 (PGE2), and mononuclear cell factor (MCF) after stimulation with phorbol myristate acetate (PMA) were examined. PMA did not induce O2- production in untreated cells. A 3-day preincubation with IFN-gamma or 1 alpha,25(OH)2D3 resulted in a 5- to 10-fold increase in PMA-stimulated production of O2- as compared to cells preincubated in medium alone. The response was related to IFN-gamma and 1 alpha,25(OH)2D3 concentrations. In contrast, the PMA-induced production of PGE2 and MCF does not require preincubation with either IFN-gamma or 1 alpha,25(OH)2D3. These results suggest that O2- production and cytokine production (i.e., PGE2 and MCF) are modulated by different signals related to maturation processes.     

In vitro modulation of leucocyte-function-associated antigen-1 expression on two leukemic cell lines

Summary Leukocyte-function-associated antigen-1 (LFA-1) expression on two widespread tumor cell lines: K562 (an erythroleukemia) and MOLT-4 (a T leukemia), was investigated using two monoclonal antibodies specific for the chain of this surface antigen, and flow cytometry analysis. When K562 cells are in the exponential phase of growth, they display very low levels of LFA-1. By contrast, cells from the plateau phase exhibit a strong labelling, which disappears rapidly when they are allowed to resume division by changing the culture medium. Using the same experimental conditions, we failed to detect any LFA-1 expression on MOLT-4 cells. However, after stimulation of these cells by phorbol myristate acetate, we observed a significant labelling, which occurred within 2 days of treatment. The LFA-1 expression disappears progressively after removal of the phorbol ester. From these results it may be concluded that (a) LFA-1 expression can vary considerably according to the culture conditions, (b) the expression of this antigen on the surface of non-expressing variants can be induced by phorbol ester, and (c) in both cases, the change in expression can be reversed completely by replacing the culture medium or by removing phorbol myristate acetate from it. Abbreviations used: LFA-1, leukocyte-function-associated antigen-1; PMA, phorbol myristate acetate; TNBS, trinitrobenzenesulfonic acid     

Superoxide anion release by human endothelial cells: synergism between a phorbol ester and a calcium ionophore

In order to study the signal transduction mechanism of human endothelial cells (EC), the regulation of superoxide anion (O2-)release in EC has been investigated using the calcium ionophore A23187 and phorbol myristate acetate (PMA), a potential activator of the Ca2+ activated, phospholipid-dependent protein kinase, designated "protein kinase C." PMA enhanced O2- release from EC, and this enhancement occurred regardless of the presence or absence of extracellular Ca2+. A similar increase was produced by A23187; omission of extracellular Ca2+ prevented this increase. Simultaneous stimulation with PMA and A23187 produced a large increase in O2- release at submaximal concentrations of these agents, which, when added separately, caused minimal effects. These findings indicate that the activation of protein kinase C and mobilization of Ca2+ evoked by PMA and A23187 respectively are synergistically effective for eliciting a full physiological response of EC in the generation and release of O2-.     

Phagocytosis and giant cell formation at 0° C by macrophage (MØ) of Notothenia coriiceps

Macrophages adhered to round glass coverslips that had been implanted into the abdominal cavity of Notothenia coriiceps , collected from Admiralty Bay, King George Island, and removed and incubated in vitro for 4 h at 0° C at various intervals. The macrophages phagocytosed yeast Saccharomyces cerevisiae cells suspended in saline. Bi-nuclear macrophages were first observed after 6 h. The formation of giant cells and their phagocytic activity was observed only in fish which had been injected with 1 ml of Bacillus of Calmette and Guerin (BCG)-suspension 24 h before coverslip implantation and the coverslips removed 15 days after the implantation. Phagocytosis and the formation of giant cells in an Antarctic fish at 0° C is described for the first time.     

Delineation of the catalytic components of the NADPH-dependent O2- generating oxidoreductase of human neutrophils

Four catalytic components of the NADPH-dependent O2- generating oxidoreductase of human neutrophils have been identified. DCIP reductase, cytochrome c reductase and a chromophore 450-455 reductase are present in phorbol myristate acetate stimulated neutrophils and absent in resting cells and phorbol myristate acetate stimulated chronic granulomatous disease cells. Quinol dehydrogenase activity has also been demonstrated in activated and resting cells. Furthermore, a chromophore absorbing in the reduced state at 450-455 nm participates in superoxide production. This chromophore is reduced by NADPH or duroquinol and is missing in cell lysates derived from a patient with chronic granulomatous disease.