PMA-induced Chemiluminescence Research Articles

The effect of Intravenous Immunoglobulin (IVIG) on \textit{ex vivo} activation of human leukocytes.

Intravenous immunoglobulin (IVIG) has been widely used to treat various conditions, including inflammatory and autoimmune diseases. IVIG has been shown to have a direct influence on neutrophils, eosinophils and lymphocytes. However, many aspects IVIG's effect on neutrophils activation still remain unknown. To evaluate the effect of IVIG on PMA-induced activation of neutrophils, with and without priming with TNF-α, in a series of in vitro experiments performed on whole blood. Our data coincided with well-known literature indicating that the effect of phorbol 12-myristate 13-acetate (PMA) on human leukocytes includes activation of neutrophils, monocytes and eosinophils, increase of chemiluminescence (CL) and induction of netosis, resulting in assembly of traps. In presence of IVIG (10 mg/mL), CL was reduced by 25% in response to PMA compared to PMA-induced leukocyte activation without IVIG. Decreasing IVIG concentration to 1 mg/mL and below did not inhibit PMA-induced activation of CL.PMA-induced activation after TNF-α priming resulted in approximately 50% increase of amplitude of CL response to PMA. Moreover, maximum CL was reached by minute 5, which was more rapid than in the absence of TNF-α-priming (in this case maximum CL was reached by minute 15).The IVIG concentrations did not affect morphological changes of leukocytes after sequential addition of TNF-α and PMA. IVIG had no effect on leukocyte content and on PMA-induced CL of primed leukocytes.Addition of IVIG under TNF-α priming significantly increased the number of traps in the smears in response to PMA activation. Of note, such an increase in the number of traps was depended on the IVIG concentration in plasma. In conclusion, we suggest that IVIG is able to reduce the degradation of traps under priming with TNF-α. Moreover, IVIG might switch the activation of primed leukocytes to netosis.

Induction of inducible nitric oxide synthase increases the production of reactive oxygen species in RAW264.7 macrophages

Macrophages produce a large volume of ROS (reactive oxygen species) through respiratory burst. However, the influence of iNOS [inducible NOS (nitric oxide synthase)] activation on ROS production remains unclear. In the present study, the kinetic generation of ROS in RAW264.7 murine macrophages was monitored by chemiluminescence. PMA induces a robust chemiluminescence in RAW264.7 cells, suggesting PKC (protein kinase C)-related assembly and activation of NOX (NADPH oxidase). The effects of iNOS induction on ROS production were examined. Induction of iNOS expression in RAW264.7 cells with LPS (lipopolysaccharide; 1 microg/ml) causes a significant increase in PMA-induced chemiluminescence, which could be enhanced by the NOS substrate, L-arginine, and could be abolished by the NOS inhibitor, L-NNA (NG-nitro-L-arginine). Further experiments reveal that induction of iNOS expression enhances the PMA-stimulated phosphorylation of the p47phox subunit of NOX, and promotes the relocalization of cytosolic p47phox and p67phox subunits to the membrane. Inhibition of PKCzeta by its myristoylated pseudosubstrate significantly decreased the PMA-stimulated phosphorylation of the p47phox in LPS-pretreated cells, suggesting that PKCzeta is involved in the iNOS-dependent assembly and activation of NOX. Taken together, the present study suggests that the induction of iNOS upregulates the PMA-induced assembly of NOX and leads to the enhanced production of ROS via a PKCzeta-dependent mechanism.

AOPP-induced activation of human neutrophil and monocyte oxidative metabolism: A potential target for N-acetylcysteine treatment in dialysis patients

AOPP-induced activation of human neutrophil and monocyte oxidative metabolism: A potential target forN-acetylcysteine treatment in dialysis patients. Oxidative stress largely contributes to hemodialysis-associated lethal complications, thus explaining the urgent need of antioxidant-based therapeutic strategies in hemodialysis patients. We previously identified advanced oxidation protein products (AOPP) in the uremic plasma as exquisite markers of oxidative stress and potent mediators of monocyte activation. The present study was aimed at searching whether (1) AOPP can also trigger activation of polymorphonuclear neutrophils (PMN), and (2) whether AOPP-induced activation could be inhibited by N-acetylcysteine (NAC), a widely used compound which has been shown to prevent oxidative injury to kidney. Both human serum albumin (HAS) AOPP (i.e., HOCl-modified HSA in vitro preparations and AOPP extracted from plasma of hemodialysis patients) were tested for their capacity to trigger phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase (MPO)-dependent activities as measured by lucigenin- and luminol-amplified chemiluminescence (CL), respectively, as compared to receptor-dependent [opsonized zymosan or receptor-independent phorbol myristate acetate (PMA)]. The effect of PMN priming by platelet-activating factor (PAF), and the effect of NAC on normal monocyte and on normal or hemodialysis patient's (N = 16) PMN oxidative responses were compared. HSA-AOPP triggered in a HOCl dose-dependent manner both NADPH-oxidase- and MPO-dependent CL of PMN. This latter was further enhanced by PAF priming. Plasma-derived AOPP obtained from hemodialysis patients also triggered PMN respiratory burst. NAC significantly reduced HSA-AOPP-mediated responses of normal monocyte and of normal and uremic PMN but had no significant effect on opsonized zymosan- or PMA-induced CL responses. This dual potential of NAC to inhibit phagocyte oxidative responses induced by HSA-AOPP without affecting those mediated by compounds mimicking pathogens supports the proposal of a therapeutic trial with NAC aimed at reducing oxidative stress-related inflammation in hemodialysis patients.

HEMOGLOBIN POTENTIATES THE PRODUCTION OF REACTIVE OXYGEN SPECIES BY ALVEOLAR MACROPHAGES

The objectives of this investigation were (1) to determine the effects of hemoglobin on the production of reactive oxygen species by activated rat alveolar macrophages, (2) to determine a possible mechanism for these effects, and (3) to determine which part of the hemoglobin molecule is responsible for these effects. Production of reactive oxygen species by phorbol myristate acetate (PMA)-stimulated cells was assessed by measuring luminol-enhanced chemiluminescence (CL). Hemoglobin enhances PMA-stimulated CL in a dose-dependent manner. The effect is maximal at 0.5 1.0mu M hemoglobin where PMA-induced CL is increased by approximately 20-fold. Superoxide anion release from PMA stimulated cells is not affected by hemoglobin. However, the hemoglobin-induced enhancement of PMA stimulated CL is inhibited by superoxide dismutase, catalase, dimethylthiourea, or deferoxamine. These results suggest that hydroxyl radical may be formed from hydrogen peroxide which is derived from superoxide anion. Measurements of electron spin resonance spectra following spin trapping of radicals verify that hydroxyl radicals are produced by the cells in the presence of PMA and hemoglobin. The hemoglobin effects appear to require iron in a protoporphyrin complex, because hemin stimulates PMA induced CL, whereas neither ferrous nor ferric iron has any effect. These findings taken together suggest that hemoglobin can act as a biological Fenton reagent to enhance the production of reactive oxygen species from alveolar macrophages and potentially contribute to lung damage during leakage of blood into the alveolar spaces.

Selective inhibition of protein kinase C, mitogen-activated protein kinase, and neutrophil activation in response to calcium pyrophosphate dihydrate crystals, formyl-methionyl-leucyl-phenylalanine, and phorbol ester by O-(chloroacetyl-carbamoyl) fumagillol (AGM-1470; TNP-470)

The effect of O-(chloroacetyl-carbamoyl) fumagillol (AGM-1470; TNP-470) was investigated on protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation in neutrophils stimulated by plasma-opsonized crystals of calcium pyrophosphate dihydrate (triclinic) [CPPD(T)], formyl-Met-Leu-Phe (fMLP), and phorbol 12-myristate 13-acetate (PMA). Neutrophil respiratory burst responses also were determined in AGM-1470-pretreated cells stimulated with the same agonists, using chemiluminescence and superoxide anion generation assays. AGM-1470 (5 μM) effectively inhibited PKC activation in cells treated with CPPD(T) crystals (50 mg/mL, 2 min) and fMLP (1 μM, 1 min), but had no effect on PMA-treated cells (0.5 μM, 5 min). AGM-1470 blocked MAPK activity completely and reduced neutrophil activation induced by fMLP and PMA but not by CPPD(T). The degree of inhibition of the respiratory burst plateaued at approximately 46 ± 9 and 54 ± 3% in fMLP- and PMA-treated cells, respectively. These data indicate that activation of neutrophil respiratory burst activity may be mediated through the MAPK pathway. AGM-1470 pretreatment did not inhibit CPPD(T) crystal- or fMLP-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity. These findings, coupled with further observations that the PI 3-kinase inhibitor wortmannin (10 nM) inhibited fMLP- and CPPD(T) crystal-induced but not PMA-induced chemiluminescence, indicate that at least two distinct signaling pathways (mediated by PI 3-kinase or MAPK) lead to neutrophil respiratory burst responses. PKC may also be required in the MAPK-stimulated pathway. We propose that the inhibitory effect of AGM-1470 on the neutrophil respiratory burst may be due to its ability to inhibit PKC and MAPK activation.

Effect of decaglycerol mono-oleate on chemiluminescence of human neutrophils.

Decaglycerol mono-oleate (DGMO), a type of food additive (a polyglycerol ester of fatty acids), was evaluated for its in vitro effect on reactive oxygen species (ROS) generation by human neutrophils using a luminol-dependent chemiluminescence assay. Neutrophils were isolated by the Histopaque-1077/1119 double density gradient technique. Opsonized zymosan (OZ) and phorbol 12-myristate 13-acetate (PMA) were employed as artificial stimuli. DGMO inhibited the chemiluminescence response of OZ-stimulated neutrophils by 58% (p < 0.01) at a concentration of 0.5 mg/mL. DGMO inhibited the PMA-induced chemiluminescence by 40% (p < 0.01) at a concentration of 0.01 mg/mL. The inhibition phenomena could not be attributed to light absorption by DGMO alone, because the percentage chemiluminescence in the presence of DGMO is lower than the percentage transmittance of DGMO at 425 nm. These results suggest that DGMO downregulates ROS generation by human neutrophils.

Influence of nitric oxide on luminol-enhanced chemiluminescence measured from porcine-stimulated leukocytes.

The influence of endogenous nitric oxide (NO) and NO-releasing compounds on free radical release from porcine leukocytes was investigated by luminol-enhanced chemiluminescence (CL). The direct free radical-scavenging activity of the compounds was determined by a cell-free system using xanthine plus xanthine oxidase (X + XO). The NO donor, N-(2-hydroxyethyl)nicotinumide nitrate (nicorandil), markedly inhibited CL generated by phorbol myristate acetate (PMA)-stimulated leukocytes. In addition, nicorandil and S-nitrozo-N-acetylpenicillamine (SNAP) both decreased CL generated by X + XO. Conversely, C87 3754, a NO-releasing sydnonimine, decreased free radical release from leukocytes only when preincubated with the cells and had no effects on the X + XO system. None of the NO donors inhibited peroxynitrite-generated CL. L-, but not D-, arginine inhibited PMA-activated free radical generation without affecting X + XO-induced CL. L-Canavanine, N omega-nitro-L-arginine (L-NNA), and L-nitro-arginine methyl ester (L-NAME), inhibitors of the NO pathway, augmented PMA-induced CL. However, L-canavanine, but not L-NNA and L-NAME, produced a significant inhibition of X + XO-induced CL. It is concluded that endogenous NO may play an important role in the measurement of free radicals released from porcine leukocytes, assessed by luminol-enhanced CL, and that compounds with NO-releasing properties decrease CL, possibly by interfering with free radical generation.

Role of Pseudomonas aeruginosa lipase in inflammatory mediator release from human inflammatory effector cells (platelets, granulocytes, and monocytes.

Previously, we have shown that Pseudomonas aeruginosa lipase and phospholipase C (PLC), two extracellular lipolytic enzymes, interact with each other during 12-hydroxyeicosatetraenoic acid (HETE) generation from human platelets. In this regard. the addition of purified P. aeruginosa lipase to PLC-containing crude P. aeruginosa culture supernatants enhances the generation of the chemotactically active 12-HETE from human platelets. Therefore, we analyzed the interaction of purified P. aeruginosa lipase and purified hemolytic P. aeruginosa PLC with regard to inflammatory mediator release from human platelets, neutrophilic and basophilic granulocytes, and monocytes. Purified P. aeruginosa PLC, but not purified lipase by itself, induced 12-HETE generation from human platelets, the generation of leukotriene B4 (LTB4) and oxygen metabolites, enzyme release from human neutrophils, and histamine release from basophils but diminished interleukin-8 (IL-8) release from human monocytes in a dose-dependent manner. The addition of purified lipase enhanced PLC-induced 12-HETE and LTB4 generation, did not influence enzyme, histamine, or IL-8 release, but diminished the PLC-induced chemiluminescent response. Similar results were obtained when the hemolytic PLC from Clostridium perfringens was used instead of P. aeruginosa PLC. For further comparison, we used the well-defined calcium ionophore A23187 and phorbol-12-myristate-13-acetate (PMA) as stimuli. Lipase enhanced calcium ionophore-induced LTB4 generation and beta-glucuronidase release but reduced calcium ionophore-induced and PMA-induced chemiluminescence. In parallel, we analyzed the role of lipase in a crude P. aeruginosa culture supernatant containing PLC and lipase. Lipase activity in the P. aeruginosa culture supernatant was inhibited by treatment with the lipase-specific inhibitor hexadecylsulfonyl fluoride, leaving the activity of PLC unaffected. The capacity of "lipase-inactivated culture supernatant" to induce 12-HETE and LTB4 generation was diminished by 50 to 100%. Our results suggest that the simultaneous secretion of lipase and PLC by P. aeruginosa residing in an infected host may result in severe pathological effects which cannot be explained by the sole action of the individual virulence factor on inflammatory effector cells.

The Effect of C-Reactive Protein (CRP) on Respiratory Burst of Human Peripheral Blood Phagocytes

The effect of C-reactive protein (CRP) on the oxidative response of human peripheral blood monocytes and granulocytes was investigated. The respiratory burst of phagocytes induced by phorbol-myristate-13-acetate (PMA), phytohaemagglutinin (PHA) or opsonized zymosan (OZ) was measured in luminol-enhanced chemiluminescence.The effect of CRP on PMA-induced monocyte chemiluminescence (CL) depended both on CRP concentration and incubation time. A short incubation of cells with CRP (15–30 min.) enhanced the oxidative burst. Preincubation of cells for 1h (or longer) with low doses of CRP (about 2 μg/ml) increased, while with higher (&gt;10 μg/ml) inhibited PMA-stimulated chemiluminescence. CRP reduced also PHA or OZ-induced monocyte respiratory response. CRP diminished PMA, PHA, and OZ-induced granulocyte chemiluminescence, except the response to PHA in the presence of low doses CRP (about 5 μg/ml).The action of CRP on phagocytes probably involves activation of some intracellular mechanisms. During immune response, CRP could protect tissues against damage by excess of free oxygen and its biological active derivatives.

Ibudilast, an anti-allergic and cerebral vasodilator, modulates superoxide production in human neutrophils.

We evaluated the effect of ibudilast on superoxide generation in human neutrophils by chemiluminescence development using luciferine analog, FCLA. By incubating neutrophils with ibudilast (2-200 microM) for more than 10 minutes, fMLP- or PMA-induced chemiluminescence was enhanced. However, the fMLP-induced chemiluminescence was suppressed by incubation for less than 10 minutes. This suppressed effect was missing with PMA-induced chemiluminescence. On the both fMLP- and PMA-induced chemiluminescence, the priming effect of ibudilast was further enhanced by the treatment with H-7, a protein kinase C inhibitor. In contrast, the priming effect of ibudilast on the fMLP-induced chemiluminescence was abolished by the treatment with ST-638, a selective inhibitor of tyrosine kinase. Ibudilast showed a transient stimulatory effect on cyclic AMP accumulation which continued for only a few minutes. Ibudilast showed no significant effect on phospholipase D dependent chemiluminescence, 1,4,5 trisphosphate level, or protein kinase C activity. Ibudilast inhibited extracellular calcium influx. These results suggest that ibudilast acts on or through tyrosine kinase to achieved its priming effect on the fMLP-induced chemiluminescence. The early and transient increase in cyclic AMP level may explain the inhibitory effect of ibudilast on the fMLP-induced chemiluminescence after short time of incubation.

Contrasting influence of peplomycin and azelastine hydrochloride (Azeptin) on reactive oxygen generation in polymorphonuclear leukocytes, cytokine generation in lymphocytes, and collagen synthesis in fibroblasts.

The influence of peplomycin (PLM) and azelastine hydrochloride (Azeptin) on reactive oxygen (RO) and cytokine generation was examined in human peripheral blood mononuclear leukocytes, polymorphonuclear leukocytes (PMN), and rabbit alveolar macrophages (RAM). In addition, the influence of these drugs on DNA and collagen synthesis was investigated in human gingival and rabbit pulmonary fibroblasts. In vitro, PLM increased the FMLP- and PMA-induced chemiluminescence and superoxide (O2-) generation in human PMN and RAM in a dose-dependent manner. In contrast to PLM, Azeptin dose-dependently suppressed RO generation. Such contrasting actions of PLM and Azeptin were also observed in RAM and PMN obtained from rabbits treated with PLM or Azeptin. Even when human PMN were preincubated with 10-100 micrograms/ml of PLM, the increase in RO generation was negligible in the presence of 10(-5) M Azeptin in the culture medium. No increases in RO generation were observed in RAM or PMN obtained from rabbits that had received PLM (0.1 mg/kg per day) and Azeptin (0.04 mg/kg per day) concomitantly. PLM suppressed superoxide dismutase activity in RAM and human PMN, while Azeptin did not affect this activity. In vitro, PLM up-regulated the release of interleukin-1 beta, interleukin-6, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor both from human cells and from RAM and pulmonary fibroblasts. In the generation of these cytokines, Azeptin abrogated the up-regulatory action of PLM. PLM and Azeptin also had contrasting actions in [3H]thymidine and [3H]proline incorporation in human and rabbit fibroblasts. Furthermore, protein tyrosine phosphorylation, in particular that of a 115-kDa protein in human PMN, was suppressed by Azeptin and enhanced by PLM. These results seem to indicate that up-regulated RO and collagen generation are the causative factors of PLM-induced pulmonary fibrosis and that Azeptin may suppress the adverse effect.

Ibudilast, an anti-allergic and cerebral vasodilator, modulates superoxide production in human neutrophils

We evaluated the effect of ibudilast on superoxide generation in human neutrophils by chemiluminescence development using luciferine analog, FCLA. By incubating neutrophils with ibudilast (2–200 μM) for more than 10 minutes, fMLP- or PMA-induced chemiluminescence was enhanced. However, the fMLP-induced chemiluminescence was suppressed by incubation for less than 10 minutes. This suppressed effect was missing with PMA-induced chemiluminescence. On the both fMLP- and PMA-induced chemiluminescence, the priming effect of ibudilast was further enhanced by the treatment with H-7, a protein kinase C inhibitor. In contrast, the priming effect of ibudilast on the fMLP-induced chemiluminescence was abolished by the treatment with ST-638, a selective inhibitor of tyrosine kinase. Ibudilast showed a transient stimulatory effect on cyclic AMP accumulation which continued for only a few minutes. Ibudilast showed no significant effect on phospholipase D dependent chemiluminescence, 1,4,5 triphosphate level, or protein kinase C activity. Ibudilast inhibited extracellular calcium influx. These results suggest that ibudilast acts on or through tyrosine kinase to achieved its priming effect on the fMLP-induced chemiluminescence. The early and transient increase in cyclic AMP level may explain the inhibitory effect of ibudilast on the fMLP-induced chemiluminescence after short time of incubation.

Increased PMA-induced chemiluminescence from whole blood of patients with bronchial hyperreactivity.

Blood phagocytes from patients with asthma have an increased capacity to produce reactive oxygen metabolites. We studied whether whole blood luminol-dependent chemiluminescence could detect this phenomenon in patients with a normal spirometry but bronchial hyperreactivity as determined with a methacholine bronchial challenge test. Whole blood chemiluminescence, serum eosinophilic cationic protein (ECP), and serum myeloperoxidase (MPO) were determined from 50 patients referred for a methacholine challenge due to prolonged cough and/or dyspnoea. The chemiluminescence results were compared to those from 15 healthy persons. The hyperreactive patients (n = 18) had significantly higher phorbol 12-myristate 13-acetate (PMA)-induced whole blood chemiluminescence values (mean 18.8 mV.min-1; 95% confidence limits (C.L.) 16.3-21.3 mV.min-1) than the normoreactive patients (mean 14.2 mV.min-1; 95% C.L. 13.0-15.5 mV.min-1;) and the healthy controls (mean 12.8 mV.min-1; 95% C.L. 11.7-13.9 mV.min-1). There was no significant difference in PMA-induced chemiluminescence between the normoreactive patients and the controls. The hyperreactive patients had higher serum ECP values than the normoreactive patients, but there was no correlation between whole blood chemiluminescence and serum ECP levels or total eosinophil count. There was no significant difference in monocyte reactive oxygen metabolite production or serum MPO values between the normoreactive and the hyperreactive patients. We suggest that the increased PMA-induced whole blood chemiluminescence in bronchial hyperreactivity is due mainly to an activation of neutrophils, and that the assay might be useful as a systemic inflammatory marker in patients with pulmonary inflammatory processes resulting in bronchial hyperreactivity.

Glucagon modulates superoxide generation in human polymorphonuclear leucocytes

It has been found that leucocytes posses receptor sites for glucagon and glucagon was shown to increase during bacterial infection. To verify the interconnection between glucagon, leucocytes and bacterial infection we studied the effect of glucagon on superoxide generation and second messenger transduction in PMNs. We found that glucagon could not stimulate chemiluminescence by itself but it could enhance FMLP- but not PMA-induced chemiluminescence in a concentration (50–800 pg/ml) dependent manner. However, after incubation of PMNs with 10 μM of ST-638 (a tyrosine kinase inhibitor) the enhancement effect converted into inhibitory effect. We also found that glucagon treatment of PMNs increased both IP 3 and cyclic AMP levels as second messengers. ST-638 greatly attenuated the IP 3 increment in the glucagon-treated FMLP-stimulated PMNs. From these results we can conclude that glucagon could enhance superoxide generation from FMLP-stimulated PMNs by elevating IP 3. Inhibition of IP 3 increment by tyrosine kinase blockade uncover the inhibitory effect of the increasing cyclic AMP on superoxide production.

Nitric oxide: A modulator of human neutrophil oxigenating activity

The effect of O-(chloroacetyl-carbamoyl) fumagillol (AGM-1470; TNP-470) was investigated on protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation in neutrophils stimulated by plasma-opsonized crystals of calcium pyrophosphate dihydrate (triclinic) [CPPD(T)], formyl-Met-Leu-Phe (fMLP), and phorbol 12-myristate 13-acetate (PMA). Neutrophil respiratory burst responses also were determined in AGM-1470-pretreated cells stimulated with the same agonists, using chemiluminescence and superoxide anion generation assays. AGM-1470 (5 μM) effectively inhibited PKC activation in cells treated with CPPD(T) crystals (50 mg/mL, 2 min) and fMLP (1 μM, 1 min), but had no effect on PMA-treated cells (0.5 μM, 5 min). AGM-1470 blocked MAPK activity completely and reduced neutrophil activation induced by fMLP and PMA but not by CPPD(T). The degree of inhibition of the respiratory burst plateaued at approximately 46 ± 9 and 54 ± 3% in fMLP- and PMA-treated cells, respectively. These data indicate that activation of neutrophil respiratory burst activity may be mediated through the MAPK pathway. AGM-1470 pretreatment did not inhibit CPPD(T) crystal- or fMLP-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity. These findings, coupled with further observations that the PI 3-kinase inhibitor wortmannin (10 nM) inhibited fMLP- and CPPD(T) crystal-induced but not PMA-induced chemiluminescence, indicate that at least two distinct signaling pathways (mediated by PI 3-kinase or MAPK) lead to neutrophil respiratory burst responses. PKC may also be required in the MAPK-stimulated pathway. We propose that the inhibitory effect of AGM-1470 on the neutrophil respiratory burst may be due to its ability to inhibit PKC and MAPK activation.

Polyethylene Glycol-conjugated Superoxide Dismutase Attenuates Septic Lung Injury in Guinea Pigs

Reactive oxygen species (ROS), including superoxide anions, play an important role in mediating acute lung injury. We examined whether polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) attenuates lung injury in Escherichia coli-treated guinea pigs. Twenty-four guinea pigs were divided into four groups: (1) control group; (2) septic group, in which live E. coli (2 x 10(9)/kg) were injected intravenously; (3) pretreatment group, in which PEG-SOD (2,000 IU/kg) was injected intravenously 15 min before E. coli; and (4) posttreatment group, in which PEG-SOD (2,000 IU/kg) was injected intravenously 30 min after E. coli. Lung injury was assessed by the concentration ratio of 125I-labeled albumin in lung tissue and bronchoalveolar lavage (BAL) fluid relative to plasma (L/P and BAL/P), lung wet-to-dry weight ratio, and the number of neutrophils in BAL fluid. Plasma half-life of PEG-SOD in normal guinea pigs was 13.5 h. L/P, lung wet-to-dry weight ratio, and the number of neutrophils in BAL fluid decreased in both pretreatment and posttreatment groups compared with the septic group. BAL/P decreased in the pretreatment group but not in the posttreatment group compared with the septic group. After the animal model studies, we investigated the effect of PEG-SOD on the human neutrophil extracellular generation of ROS stimulated by phorbol myristate acetate (PMA) in lucigenin-dependent chemiluminescence (CL). PEG-SOD at concentrations greater than or equal to 0.1 U/ml inhibited PMA-induced CL in a dose-dependent manner. We also examined the effect of PEG-SOD on the neutrophil intracellular generation of ROS using flow cytometry to assess intracellular hydroethidine oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive intestinal peptide enhances phorbol myristate acetate-induced chemiluminescence in human lymphocytes

Phorbol-myristate-acetate (PMA) induced in lymphocytes the production or reactive oxygen intermediates in a process which was stimulated by the presence of vasoactive intestinal peptide (VIP) in a dose-dependent response at VIP concentrations in the range 10 −11–10 −8 M. The dissociation constant for the high-affinity receptors of VIP agreed with the ID 50 of the activation of adenylate cyclase, and the ID 50 for the stimulation by VIP of PMA-induced chemiluminescence, which were close to 0.2 nM VIP. Forskolin produced in lymphocytes an effect quite similar to VIP. A comparison of the response to VIP and forskolin of lymphocytes and monocytes showed that, in contrast to forskolin, VIP failed to induce the above describe effect in monocytes. A possible mechanism involving protein kinase C, which is activated by PMA, and an intracellular signal linked to VIP receptors is pointed out. This study further supports a role for VIP as a mediator in the neuroimmune system.

The Effects of Saikosaponin on Macrophage Functions and Lymphocyte Proliferation

The effects of saikosaponin-d (ssd), isolated from Bupleurum radix, on phagocytic functions of mouse peritoneal macrophages were investigated after treatment in vitro. The macrophages treated with ssd showed a significant increase in PMA-induced chemiluminescence. An increase in phagocytosis was detected after treatment with saikosaponin-b2 (0.1 microM) for 24 h in vitro, while a suppression of phagocytosis was observed following treatment with saikosaponins (0.5 microM). Treatment with ssd markedly increased the random migration of resident peritoneal macrophages, but did not affect the migration towards FMLP. We further investigated the effect of ssd on proliferative responses of spleen cells and found that ssd, which itself has no mitogenic activity, decreased spleen cell proliferative response to T-cell mitogen, but increased the response to B-cell mitogen.

Inhibition of chemiluminescence in granulocytes and alveolar macrophages by azelastine

The effect of azelastine, an orally effective antiasthmatic antiallergic drug on the generation of oxygen-derived free radicals in phagocytes was investigated using different chemiluminescence-assays. The chemiluminescence (CL) of both human polymorphonuclear granulocytes (PMNL) and guinea-pig alveolar macrophages (AM) was induced either by phorbol myristate acetate (PMA) or zymosan and amplified either by lucigenin or DMNH (7-dimethylamino-naphthalene-1,2-dicarbonic-acidhydrazide). The inhibitory effect of azelastine was dependent on the inducer employed and the condition and type of cells used. Azelastine reduced PMA-induced CL concentration-dependently in both PMNL (IC30 = 3.9 microM) and AM (IC30 = 9.8 microM). In AM zymosan-induced CL was inhibited 21.7% by 10 microM azelastine, whereas in PMNL it remained unchanged up to 10 microM azelastine. Azelastine has a significantly stronger inhibitory effect (IC30 = 4.2 microM) on oxygen free radical generation in AM primed by fetal calf serum than in unprimed AM. Based on present results it is likely that azelastine inhibits oxygen-derived free radical generation by interaction with protein kinase C.

Phagocyte oxidative metabolism in cyclosporine-or placebo-treated patients with insulin-dependent (Type I) diabetes mellitus of recent onset

Several lines of evidence suggest that phagocyte-mediated oxidative processes are involved in damage to pancreatic islet cells of Type I insulin-dependent diabetes mellitus (IDDM). This hypothesis, however, has not yet been explored at the clinical onset of IDDM. Similarly, the possibility that cyclosporine A (Cy-A) might exert a selective influence on these phagocyte-mediated oxidative reactions has also not yet been investigated as compared to a placebo. The present study tested both hypotheses in 32 patients with recently diagnosed IDDM who were part of the recent French multicenter randomized therapeutic trial of Cy-A. The production of reactive oxygen intermediates (ROI) by circulating polymorphonuclear (PMN) and mononuclear (MN) phagocytes was determined by luminol-dependent chemiluminescence (CL), both directly within microamounts of whole blood and in purified PMN or MN phagocyte suspensions. Lastly, CL production was measured in the absence (resting CL) and the presence of a panel of particular and soluble phagocyte membrane-stimulating agents. We found that on entry into the trial, i.e. within less than 2 months of the clinical onset of IDDM, patients had normal whole blood CL production in the absence of a stimulating agent and upon phagocytic challenge with latex or opsonised zymosan particles. By contrast, whole blood CL responses to soluble stimuli such as phorbol myristate acetate (PMA), concanavalin A (Con-A) and F Met-Leu-Phe (FMLP) were significantly higher than in the control group of 52 normal subjects ( P<0.01). In purified PMN and MN phagocyte suspensions, both resting and stimulated CL productions were normal, regardless of the type of stimulating agent. After 3 months of treatment, whole blood CL responses to Con-A and FMLP returned to almost normal levels in patients treated with Cy-A (15 cases) but not in those receiving the placebo (17 cases); PMA-induced CL responses were also decreased, but this was found in both groups of patients. In purified phagocyte suspensions we detected no effect of Cy-A on PMN, whereas MN phagocytes from Cy-A-treated patients showed reduced CL responses to FMLP but not to other stimuli. Altogether, these results demonstrate for the first time that the capacity of circulating PMN and MN phagocytes to generate ROI is normal at the clinical onset of IDDM and suggest that circulating substances increase oxidative responses to soluble, but not particulate, stimuli. With regard to the in vivo effect of Cy-A, it appears that Cy-A does not influence PMN oxidative response-potential, whereas it selectively alters MN oxidative responses to chemotactic agents such as FMLP. This latter finding suggests that, as already described for T lymphocytes, Cy-A may interfere with calcium-dependent activation pathways of monocytes.