Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes.

The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN.     

Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes

The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by Superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from Superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN.     

ESR studies on oxygen consumption during the respiratory burst of human polymorphonuclear leukocytes

Oxygen consumption during the respiratory burst of human polymorphonuclear leukocytes (PMN) stimulated with phorbol myristate acetate (PMA) was studied with spin probe oxymetry and using the transition metal ion CrOX (potassium trioxalatochromate) as a widening agent. The experimental results demonstrated that during the respiratory burst of PMN stimulated with PMA, oxygen consumption was found mainly in the intercellular medium but no change of oxygen concentration was found in the intracellular medium.     

Spin trap determination of free radical burst kinetics in stimulated neutrophils

Electron spin resonance spin-trapping methods were used to investigate the free radical production kinetics of neutrophils stimulated with phorbol myristate acetate (PMA) and opsonized zymosan (OPZ). Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, the principle spin adduct observed is DMPO-OH (trapped hydroxyl radical). The DMPO-OH ESR signal amplitude was observed to decay exponentially. In such cases a simple method may be used to analyze the raw kinetics amplitude data to yield true production rate and net production data. The method, pitfalls, and self-consistency criteria are illustrated with PMA and OPZ-stimulated neutrophils at 25 and 37 degrees C under varying oxygen tensions, and with noise-free simulated data. The simulations demonstrate that rate results are relatively insensitive to the precise choice of decay time constant, tc, while net production results are very sensitive to the choice of tc used to analyze the raw data. OPZ (0.6-2.4 mg/ml) yields a strong, sharp neutrophil burst which peaks in 2 min or less while PMA yields a slower burst which peaks in 3.4-14 min for PMA concentrations of 500-50 ng/ml, respectively. Increased oxygen tension during the PMA experiments increased the spin adduct lifetime. The methods presented are applicable to other cell systems or spin adducts which exhibit first order decay.     

Production and interaction of oxygen and nitric oxide free radicals in PMA stimulated macrophages during the respiratory burst

The activity of nitric oxide synthase (NOS) during the respiratory burst in phorbol-1,2-myristate-1,3-acetate (PMA) stimulated macrophages has been the topic of much debate in the literature. To help clarify the role of NOS, we have examined the chemiluminescence arising from peroxynitrite production, nitrite/nitrate and nitric oxide production, and oxygen consumption during the respiratory burst in PMA-stimulated macrophages. The Griess reaction was used to measure nitrite/nitrate, spin trapping with N-methyl D-glucamine dithiocarbamate (MGD)2-Fe2+ was used to quantify nitric oxide, and the spin probe 2,2,6,6-tetramethylpiperidine-N-oxyl-4-ol (TEMPOL) was used to measure oxygen consumption. Oxygen free radical production (hydroxyl and superoxide free radicals) was also investigated using the spin trap 5,5-dimethyl-1-pyroline-1-oxide (DMPO). The chemiluminescence emitted by the PMA-stimulated macrophages and nitrite/nitrate in the culture system were both found to increase. However, the rate of nitric oxide release remained constant, indicating that the activity of NOS is not enhanced during the respiratory burst in PMA stimulated macrophages.     

Roles of phospholipase D in phorbol myristate acetate‐stimulated neutrophil respiratory burst

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10‐fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA‐stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA‐stimulated respiratory burst. Using 1‐butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA‐stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA‐stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA‐stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA‐stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2‐dioctanoyl‐sn‐glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA‐stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA‐stimulated respiratory burst.     

Phorbol myristate acetate mediates redistribution of protein kinase C in human neutrophils: potential role in the activation of the respiratory burst enzyme

Protein kinase C may be important in leukocyte function, because it is activated by phorbol myristate acetate (PMA), a potent stimulus of the respiratory burst in neutrophils. The localization of protein kinase C was compared in unstimulated and PMA-stimulated human neutrophils. Protein kinase C was primarily cytosolic in unstimulated cells but became associated with the particulate fraction after treatment of cells with PMA. The particulate-associated kinase activity did not require added calcium and lipids, but when extracted by Triton X-100 (greater than or equal to 0.2%), calcium and phospholipid dependence could be demonstrated. The EC50 of PMA for stimulating kinase redistribution and activation of NADPH oxidase, the respiratory burst enzyme, were similar (30 to 40 nM). Redistribution of protein kinase C occurred rapidly (no lag) and preceded NADPH oxidase activation (30 sec lag). These results suggest that redistribution of protein kinase C is linked to activation of the respiratory burst in human neutrophils.     

Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals

With the use of the spin trapping methods, the scavenging effects of the extracts of green tea and other natural foods are studied. In stimulated polymorphonuclear leukocytes (PMN) system, water extract fraction 6 (F6) from green tea and green tea polyphenols (GTP) have the strongest scavenging effect on the active oxygen radicals, much stronger than vitamin C (Vc) and vitamin E (VE). Rosemary antioxidants (RA) and Curcumin (Cur) have weaker scavenging effects than Vc, but stronger than VE. In Fenton Reaction, Cur has the strongest scavenging effect (69%) on hydroxyl radicals. In irradiation, riboflavin system F6(74%) and GTP(72%) have very strong scavenging effects that are weaker than Vc, but much stronger than VE (23%). With the use of spin probe oxymetry, the oxygen consumption in respiratory burst of stimulated PMN were measured when the antioxidants existed in these systems. The results demonstrated that these antioxidants did not affect the respiratory burst of human polymorphonuclear leukocytes stimulated with PMA.     

Sesquiterpene lactone from Wunderlichia crulsiana inhibits the respiratory burst of leukocytes triggered by distinct biochemical pathways

The sesquiterpene lactone tubiferin was chemically purified from the brazilian native plant Wunderlichia crulsiana and identified by NMR and GC/MS data. Its ability to inhibit the respiratory burst of peritoneal inflammatory polymorphonuclear leukocytes (PMN) stimulated upon addition of phorbol miristate acetate (PMA), opsonized zymosan (OZ), and N-formyl-methionyl-leucyl-phenylalanine (fMLP) was evaluated. The tubiferin inhibition was more pronounced when PMN were stimulated through the protein kinase C pathway (PMA) compared to the alternative complement pathway (OZ). The inhibition when PMN were triggered by a chemoattractant stimulus (fMLP) was similar to that achieved with OZ-stimulated phagocytes. Tubiferin showed dose-dependent effects on the PMN respiratory burst triggered by the three different substances, and also decreased substantially the carrageenan-induced mice paw edema.     

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.     

Intracellular free calcium regulates the onset of the respiratory burst of human neutrophils activated by phorbol myristate acetate.

Thapsigargin was used to study the regulation of different static calcium level ([Ca2+]i) on the respiratory hurst of human neutrophils stimulated with phorbol myristate acetate (PMA). The result showed that the onset time of the respiratory hurst was obviously reduced by elevation of static [Ca2+]i but is still much longer than that stimulated with N-formylmethionylleucylphenylalanine (fMLP). To find the reason, the onset times of the respiratory burst stimulated with fMLP, 1,2-dioctanoyl-sn-glycerol (DiC8), and PMA were determined at different static [Ca2+]i. It turns out that although DiC8 was unable to induce the respiratory burst at low [Ca2+], the onset time of DiC8-stimulated response at high [Ca2+]i was almost the same as that stimulated with fMLP. The study revealed that the fast onset of the fMLP-stimulated respiratory burst in comparison with PMA-stimulated response is not only due to the transient rise of [Ca2+]i, but is also due to the higher efficiency of diacylglycerol (DAG) in activating protein kinase c (PKC). The determining step in governing the onset of a respiratory burst is the activation of PKC.     

Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals

With the use of the spin trapping methods, the scavenging effects of the extracts of green tea and other natural foods are studied. In stimulated polymorphonuclear leukocytes (PMN) system, water extract fraction 6 (F6) from green tea and green tea polyphenols (GTP) have the strongest scavenging effect on the active oxygen radicals, much stronger than vitamin C (Vc) and, vitamin E (Ve). Rosemary antioxidants (RA) and Curcumin (Cur) have weaker scavenging effects than Vc, but stronger thanVe. In Fenton Reaction, Cur has the strongest scavenging effect (69%) on hydroxyl radicals. In irradiation, riboflavin system F6(74%) and GTP(72%) have very strong scavenging effects that are weaker than Vc, but much stronger thanVe (23%). With the use of spin probe oxymetry, the oxygen consumption in respiratory burst of stimulated PMN were measured when the antioxidants existed in these systems The results demonstrated that these antioxidants did not affect the respiratory burst of human polymorphonuclear leukocytes stimulated with PMA.     

Scavenging effect of schizandrins on active oxygen radicals

The reactive oxygen radicals produced from human polymorphonuclear leukocytes (PMN) stimulated with PMA (phorbol myristate acetate), hydroxyl radicals generated by a Fenton reaction, and superoxide anion radicals produced by irradiating solutions of riboflavin in the presence of EDTA have been taken as the models for production of oxygen radicals. With the use of the electron spin resonance spin trapping method, the scavenging effects of schizandrol A (solA) (5 x 10(-4) M) and schizandrin B (sinB) (5 x 10(-4) M) have been studied and compared with the effects of vitamin E (5 x 10(-4) M) and vitamin C (5 x 10(-4) M). It has been found that in cell system the scavenging effects of sinB and solA, as judged by ESR spin trappings, on hydrpxyl radicals (.OH) are greater than vitamin E and vitamin C and the scavenging effects on superoxide anion (O2) are greater than vitamin E but lower than vitamin C. With respect to the Fenton reaction, sinB has the strogest scavenging effect on .OH (77%) and solA has strong scavenging effect on .OH (63%), both of them larger than that of vitamin E (35%) and vitamin C (56%). In the riboflavin/EDTA system, the scavenging effect of sinB (46%) is smaller than that of vitamin C (96%) but larger than that of vitamin E (23%); the scavenging effect of solA is not obvious (14%). With the use of spin probe oximetry, the oxygen consumption during the respiratory burst of stimulated PMN has been measured when exposed to schizandrins. The experiment results demonstrated that they do not affect the activity of production of active oxygen radicals in the respiratory burst of PMN stimulated with PMA.     

Comparison of the roles of calmodulin and protein kinase C in activation of the human neutrophil respiratory burst

The roles of calmodulin and protein kinase C in the activation of the human neutrophil respiratory burst were characterized pharmacologically. The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) did not inhibit superoxide anion generation by neutrophils stimulated for 30 minutes with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). However, H-7 did depress superoxide production during the first 5 minutes following stimulation. In contrast, the specific calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and the dual calmodulin antagonist/protein kinase C inhibitor trifluoperazine (TFP) were potent inhibitors of the response throughout the 30 minute incubation. Stimulation of neutrophils with submaximal doses of FMLP or PMA failed to promote inhibition of the respiratory burst by H-7 or H-9, but did stimulate a respiratory burst response which was not inhibited by TFP or W-7. These results suggest that while protein kinase C may play a role in the initiation of the respiratory burst response, propagation of the response is dependent on calmodulin-dependent processes. The inability of TFP and W-7 to inhibit superoxide anion generation in response to submaximal stimulatory doses of FMLP or PMA suggests that calmodulin-independent processes may also be involved in activation of the respiratory burst.     

Effects of liposomes of different phospholipid composition on the induction of respiratory burst in human blood monocytes and alveolar macrophages.

Liposomes made from phosphatidylcholine (PC) or PC-fatty acid ester mixtures have been shown to induce an activation of the respiratory burst in human blood monocytes and alveolar macrophages (AM). Incorporation of 1,2-diacylglycerol or arachidonic acid into PC liposomes significantly enhanced the effect. In the case of AM, the effect of PC liposomes was similar to those of phorbol 12-myristate 13-acetate (PMA) and the ionophore A 23187, while in monocytes, PMA and A 23187 induced a stronger respiratory burst than PC liposomes. In the presence of PMA, higher liposomal concentrations were required to produce the maximum activation of the respiratory burst in both types of cells.     

The efficacy of a commercial beta-glucan preparation, EcoActiva, on stimulating respiratory burst activity of head-kidney macrophages from pink snapper (Pagrus auratus), Sparidae.

This study investigated the in vitro effects of a commercial beta-glucan preparation, EcoActiva, on the respiratory burst activity of head-kidney macrophages isolated from pink snapper (Pagrus auratus), a marine fish cultured in Australia. Macrophages incubated with EcoActiva displayed morphological characteristics of activation, and were stimulated to produce superoxide. Pre-incubation with low levels of EcoActiva significantly increased the response to phorbol myristate acetate (PMA) and lipopolysaccharide (LPS), indicating that EcoActiva could prime these macrophages. Co-culturing macrophages with both LPS and PMA, or EcoActiva and PMA, increased burst activity compared with the response to PMA alone, however, this increase was additive and not synergistic. These results suggest that EcoActiva is able to stimulate non-specific immunity in snapper through increased respiratory burst activity of macrophages, an important component of the host defence network.     

Effect of isorhapontigenin on respiratory burst of rat neutrophils

The effect of Isorhapontigenin (Iso) isolated from Belamcanda chinensis on respiratory burst of rat neutrophils was investigated. Iso (1, 10, 100 mmol/l) showed an inhibitory effect on superoxide anion and hydrogen peroxide production in phorbol myristate acetate (PMA) activated rat neutrophils in a concentration-dependent manner. Scanning electron microscopy detected that Iso (100 mmol/l) protected against surface changes in rat neutrophils stimulated with PMA. Also, 100 mmol/l Iso inhibited the release of beta-glucuronidase from the activated neutrophils. Electron-spin resonance (ESR) detected that Iso scavenged oxygen free radicals generated in the PMA activated Neutrophils. These results suggest that Iso inhibits respiratory burst of PMA-activated rat neutrophils by scavenging oxygen free radicals.     

Deactivation of the respiratory burst in activated macrophages: evidence for alteration of signal transduction

Enhanced function of the respiratory burst, measured as stimulated release of superoxide anion (O2-) or hydrogen peroxide, characterizes activated macrophages. Activated macrophages undergo a decline in their capacity to release O2- (a deactivation) when placed in culture for 3 days. To better understand the molecular basis for the enhanced respiratory burst of activated macrophages, we explored the mechanisms underlying deactivation of activated mouse peritoneal macrophages. Deactivation was observed when the assay was performed in a physiologic Na+ buffer, and by day 3 of culture, release of O2- from activated macrophages stimulated with phorbol myristate acetate (PMA) was almost identical to that in resident (nonactivated) macrophages. In contrast, when the assay was performed in a buffer in which Na+ was replaced by K+, release of O2- from activated macrophages on day 3 was equal to or greater than that on day 0, suggesting that the enzyme responsible for the respiratory burst was not altered during culture. The number and affinity of PMA receptors were not changed during culture and were not affected by high external K+. Continuous assay of O2- release by coverslip-adherent macrophages in a cuvette indicated that the lag time between addition of stimulus and release of O2- was reduced, and the initial rate of O2- release was enhanced in K+ buffer. The potency of monovalent cations to support O2- release was K+ greater than Rb+ greater than choline+ greater than Cs+ = Na+ greater than Li+, suggesting that characteristics such as ionic radius or molecular size influence this effect, and the effect is not due simply to absence of Na+. Extracellular Ca2+ or Mg2+ was required for the maximal effect of high external K+, and enhancement by high K+ and divalent cations increased progressively during culture. These findings suggest that deactivation is caused primarily by changes in signal transduction from PMA receptors to the respiratory burst enzyme, rather than by changes in these receptors or the enzyme itself, and that signal transduction can differ in different macrophage populations.     

Acute in vitro hypoxia and high-altitude (4,559 m) exposure decreases leukocyte oxygen consumption

Hypoxia impairs metabolic functions by decreasing activity and expression of ATP-consuming processes. To separate hypoxia from systemic effects, we tested whether hypoxia at high altitude affects basal and PMA-stimulated leukocyte metabolism and how this compares to acute (15 min) and 24 h of in vitro hypoxia. Leukocytes were prepared at low altitude and ～24 h after arrival at 4559 m. Mitochondrial oxygen consumption (JO?) was measured by respirometry, oxygen radicals by electron spin resonance spectroscopy, both at a Po? = 100 mmHg (JO?,???) and 20 mmHg (JO?,??). Acute hypoxia of leukocytes decreased JO? at low altitude. Exposure to high altitude decreased JO?,???, whereas JO?,?? was not affected. Acute hypoxia of low-altitude samples decreased the activity of complexes I, II, and III. At high altitude, activity of complexes I and III were decreased when measured in normoxia. Stimulation of leukocytes with PMA increased JO?,??? at low (twofold) and high altitude (five-fold). At both locations, PMA-stimulated JO? was decreased by acute hypoxia. Basal and PMA-stimulated reactive oxygen species (ROS) production were unchanged at high altitude. Separate in vitro experiments performed at low altitude show that ～75% of PMA-induced increase in JO? was due to increased extra-mitochondrial JO? (JO?(,res); in the presence of rotenone and antimycin A). JO?(,res) was doubled by PMA. Acute hypoxia decreased basal JO?(,res) by ～70% and PMA-stimulated JO?(,res) by about 50% in cells cultured in normoxia and hypoxia (1.5% O?; 24 h). Conversely, 24 h in vitro hypoxia decreased mitochondrial JO?,??? and JO?,??, extra-mitochondrial, basal, and PMA-stimulated JO? were not affected. These results show that 24 h of high altitude but not 24 h in vitro hypoxia decreased basal leukocyte metabolism, whereas PMA-induced JO? and ROS formation were not affected, indicating that prolonged high-altitude hypoxia impairs mitochondrial metabolism but does not impair respiratory burst. In contrast, acute hypoxia impairs respiratory burst at either altitude.     

The efficacy of a commercial β-glucan preparation, EcoActiva™, on stimulating respiratory burst activity of head-kidney macrophages from pink snapper (Pagrus auratus), Sparidae

This study investigated the in vitro effects of a commercial β-glucan preparation, EcoActiva™, on the respiratory burst activity of head-kidney macrophages isolated from pink snapper (Pagrus auratus), a marine fish cultured in Australia. Macrophages incubated with EcoActiva™ displayed morphological characteristics of activation, and were stimulated to produce superoxide. Pre-incubation with low levels of EcoActiva™ significantly increased the response to phorbol myristate acetate (PMA) and lipopolysaccharide (LPS), indicating that EcoActiva™ could prime these macrophages. Co-culturing macrophages with both LPS and PMA, or EcoActiva™ and PMA, increased burst activity compared with the response to PMA alone, however, this increase was additive and not synergistic. These results suggest that EcoActiva™ is able to stimulate non-specific immunity in snapper through increased respiratory burst activity of macrophages, an important component of the host defence network.