Inhibitory effect of local anaesthetics on reactive oxygen species production by human neutrophils

BACKGROUND: Reactive oxygen species (ROS) generated from neutrophils accumulated in various major organs are thought to play a pivotal role in the pathogenesis of host auto-injury. Lidocaine has been shown to reduce the injury. We investigated the effect of local anaesthetics (lidocaine, mepivacaine and bupivacaine) on ROS production by neutrophils using an in vitro system. METHODS: We measured the production of superoxide (ferricytochrome c method), hydrogen peroxide (H2O2: scopoletin fluorescence technique), and hydroxyl radical (OH.: ethylene gas method) by neutrophils isolated from human adult volunteers in the absence and presence of lidocaine (2-200 micrograms/mL), mepivacaine (3-300 micrograms/mL), and bupivacaine (3-300 micrograms/mL). We also measured the ROS generation in a cell-free (xanthine-xanthine oxidase) system. RESULTS: Lidocaine and mepivacaine at higher levels significantly decreased the production of ROS by neutrophils. However, these local anaesthetics at clinically relevant blood concentrations had no effect on the levels of ROS. Furthermore, lidocaine and mepivacaine failed to reduce ROS generated by the cell-free system. Bupivacaine did not decrease ROS generation by either generating system. CONCLUSION: In conclusion, in the present in vitro system, only concentrations of lidocaine and mepivacaine 100-fold higher than clinically feasible ones reduced ROS production by human neutrophils. However, the local anaesthetics at clinically relevant blood concentrations had no suppressive effect. Further studies using in vivo systems are required to elucidate the inhibitory effects of local anaesthetics on ROS generation in clinical settings.     

The local role of tumor necrosis factor alpha in the modulation of neutrophil function at sites of inflammation

OBJECTIVE: To examine the hypothesis that tumor necrosis factor alpha (TNF-alpha) is an important local modulator of neutrophil function in the inflammatory microenvironment. DESIGN: In vitro studies of host defense. PATIENTS: A volunteer sample of healthy subjects. INTERVENTION: Exudative neutrophils were collected from skin-blister chambers and functionally compared with blood neutrophils. METHODS: Tumor necrosis factor alpha levels at sites of inflammation and neutrophil exudation were determined and compared with serum concentrations. Flow cytometry was used to evaluate neutrophil microbicidal activity and N-formyl-methionyl-leucyl-phenylalanine-induced changes in intracellular calcium and superoxide production. In vitro TNF-alpha was used to evaluate the nature and dose response of TNF-alpha-induced changes in neutrophil function. RESULTS: Exudative neutrophils have an increased responsiveness to subsequent N-formyl-methionyl-leucyl-phenylalanine stimulation, as determined by changes in intracellular calcium. Microbicidal activity and superoxide production are also up-regulated compared with circulating neutrophils. The exudative microenvironment contains TNF-alpha at local levels that are capable of significantly enhancing neutrophil host defense. CONCLUSIONS: Tumor necrosis factor alpha may serve to enhance neutrophil function at sites of inflammation. Neutrophils become more cytotoxic and have an enhanced ability to respond to weak environmental signals.     

Protection against UVA damage and effects on neutrophil-derived reactive oxygen species by beta-carotene

OBJECTIVE: Phagocyte-derived reactive oxygen species (ROS) are involved in microbicidal activities as well as in tissue damage at sites of inflammation. Carotenoids play an important function in protecting cells from oxidant damage. We investigated the in vitro and in vivo effect of 13-cis and 9-cis-beta-carotene on human neutrophils. METHODS: Neutrophils from healthy donors in the presence of 0.25 mumol/L-1 mumol/l beta-carotene and from subjects under beta-carotene supplementation and UVA or UVA/B exposure were stimulated by opsonized zymosan and the generation of ROS was measured by electron spin resonance spectroscopy. RESULTS: Our in vitro results show different effects of the two isomers on stimulated neutrophils. 9-cis-beta-carotene did not produce any change, whereas 13-cis-beta-carotene significantly and concentration-dependent inhibited the ROS generation by stimulated neutrophils. Further, in a controlled study, we were able to demonstrate an in vivo protective effect of beta-carotene on neutrophils against UVA damage by beta-carotene supplemented subjects.     

Association between neutrophil functions and periparturient disorders in cows

Neutrophil functions were examined in healthy periparturient dairy cows (n = 46) and in cows with retained placenta and metritis complex (n = 20); metritis (n = 18); or mastitis (n = 13). Blood samples (50 ml) were collected from each cow via jugular vein twice weekly from 1.5 weeks before to 4 weeks after parturition. Neutrophil function was evaluated, using 6 tests: random migration, chemotaxis, ingestion, myeloperoxidase activity (iodination), superoxide production (cytochrome C reduction), and antibody-dependent cell-mediated cytotoxicity. Ability to ingest bacteria and random migration activity of neutrophils from clinically normal cows were high around parturition and increased immediately after parturition, whereas myeloperoxidase activity and antibody-dependent cell-mediated cytotoxicity ability of neutrophils from these cows decreased after parturition. Measurement of neutrophil function in 4 ovariectomized cows revealed significant (P < 0.0005) seasonal changes in results of all 6 functional assays. We observed various defects of neutrophil function in all cows with abnormal conditions after parturition. Before parturition, superoxide production activity by neutrophils from cows with metritis and chemotaxis by neutrophils from cows with mastitis were significantly (P < 0.001 and P < 0.05, respectively) lower, indicating that a defect of neutrophil function may be a predisposing factor in the development of these disorders. In conclusion, the host defense role of neutrophils in periparturient cows was impaired, principally because of a defect in killing capacity, which may increase susceptibility to infections. We also investigated the in vitro effects of arachidonic acid metabolites and recombinant human colony-stimulating factors (rhCSF) on functions of neutrophils from clinically normal and postparturient cows with abnormalities, including retained placenta, metritis, or mastitis (n = 5/group). Each abnormal cow was matched for postpartum period with a clinically normal cow. Neutrophils from individual cows were preincubated with arachidonic acid metabolites (prostaglandin F2 alpha, 10(-7) M; prostaglandin E2, 10(-6) M; leukotriene B4, 10(-8) M; and lipoxin B, 10(-8) M) and rhCSF (rh-granulocyte-CSF, 1,000 or 6,000 U/ml; rh-granulocyte-macrophage-CSF, 5 or 15 ng/ml) in a 37 C water bath for 30 minutes before submitting them to function assays. There was no response by neutrophils from either clinically normal or abnormal postparturient cows to treatment with either arachidonic acid metabolites or rhCSF in any of the 6 functional assays. However, preincubation of neutrophils alone in a 37 C water bath for 30 minutes resulted in some alteration of neutrophil function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Superoxide anion production during reperfusion is reduced by an antineutrophil antibody after prolonged cerebral ischemia

Neutrophils may be involved in the pathophysiology of reperfusion injury following cerebral ischemia. One potential mechanism of reperfusion injury by neutrophils is through production of the superoxide anion. We hypothesized that, due to progressive endothelial damage during ischemia, neutrophil activation would be more prominent after longer periods of ischemia prior to reperfusion. Thus, neutrophils would contribute more to pathological processes such as superoxide anion formation after longer than after shorter periods of ischemia. A reversible middle cerebral artery occlusion model in rats was employed and superoxide anion concentration was measured with a cytochrome c coated electrode placed on the cortical penumbral region. Occlusion times were varied from 60 min to 2 h, and neutrophils were inhibited with an antiCD18 antibody administered prior to occlusion. Neutrophil accumulation and reduction with antibody treatment was confirmed immunohistochemically. Superoxide anion (O2*-) concentration was detected during the hours following 60 min of occlusion, and increased further with 2 h of occlusion. Treatment with the antiCD18 antibody had no effect on O2*- concentration during reperfusion in the 60-90 min occlusion groups, but O2*- concentration was significantly lower in the antiCD18 antibody treated group than in the control group during reperfusion after 120 min of ischemia. The antibody also reduced cortical neutrophil accumulation in the 120 min ischemia group. These results indicate for the first time that superoxide production by neutrophils becomes more important with longer periods of ischemia, and other quantitatively less important sources of superoxide predominate with shorter periods of ischemia. This phenomenon may explain some of the variation seen between different models of ischemia with different durations of ischemia when targeting reactive oxygen species, and supports an approach to combination therapy to extend the therapeutic window and reduce the deleterious effects of reperfusion.     

Aortoiliac stent placement in patients treated for intermittent claudication

Inhaled nitric oxide (NO) is an important new therapeutic agent used to treat pulmonary arterial hypertension in a variety of disease states. However, the effects of NO on cells in the lung are uncertain. Previously, we have shown that NO gas depresses neutrophil oxidative cell function and increases neutrophil cell death. The purpose of this in vitro study was to determine the mechanism of neutrophil death. We hypothesized that NO hastened cell death by inducing apoptosis. To mimic the clinical environment of patients with respiratory failure, we also studied the effects of hyperoxia on neutrophil cell viability and apoptosis. Isolated human neutrophils were exposed to 80% O2 (O2), NO at 20 ppm in room air (NO/RA), 20 ppm NO blended with 80% O2 (NO/O2), or RA alone (control) for 2 to 24 h. Experiments were repeated with NO concentrations of 5 and 50 ppm and with 20 ppm in the presence of superoxide dismutase (SOD). Neutrophils were also incubated in the absence or presence of neutrophil stimulant fMLP (10 nM). Neutrophil cell viability was measured by fluorescence viability/cytotoxicity assay. Neutrophil apoptosis was assessed by cell death detection ELISA for histone-associated DNA fragments, TdT transferase-mediated fluorescence-labeled dUTP nick end labeling (TUNEL) assay, and DNA fragmentation gel electrophoresis. NO/O2-exposed neutrophils showed decreased viability at 2 h (31.7 +/- 3.7%, mean % viability +/- SD) compared with control (94.7 +/- 4.7%), O2 (75.6 +/- 9.3%), and NO/RA (62.8 +/- 14.9%; P < 0.05 by ANOVA; n = 9). Although control neutrophils demonstrated marked apoptosis at 24 h, there was no significant apoptosis at 2, 4, or 6 h (P < 0.001 by Kruskal-Wallis, n = 20) as assessed by ELISA and TUNEL assays. When compared with RA controls at 2 h, neutrophils exposed to NO/O2 showed significantly more apoptosis (292% of control, range: 106 to 2,488%, P < 0.001 by ANOVA and Kruskal-Wallis) but not with exposure to NO/RA or O2 alone. These findings were confirmed by TUNEL assay (n = 4, P < 0.05). NO/ RA and NO/O2-exposed neutrophils demonstrated both evidence of necrosis and enhanced DNA fragmentation at 2 h by gel electrophoresis (n = 2). Fifty parts per million NO produced similar findings, but exposure to 5 ppm NO did not induce significant DNA fragmentation. Coincubation with SOD inhibited NO/ O2-associated apoptosis, suggesting peroxynitrite contributed to cell death. Stimulation with fMLP did not alter apoptosis induced in neutrophils exposed to NO/RA or NO/O2. We conclude that exogenous NO gas, at clinically relevant concentrations under hyperoxic conditions, induces cell death in neutrophils in part by enhancing DNA fragmentation.     

Differentiation induction in non-lymphocytic leukemia cells upon treatment with mizoribine

The effectiveness of an ideal antimicrobial agent depends on its ability to kill microbes with minimal toxicity to host cells. Depending on the treatment regimen, antimicrobial agents come into contact with host cells for various intervals of time. Sanguinarium (SANG), chlorhexidine (CHX) and tetracycline (TET) are 3 antimicrobial agents frequently used in the management of periodontal infections. However, their effects on host immune cells during different treatment regimens are not known. Due to their ability to serve as the first line of host defense against microbial infections, we have compared the effects of these antimicrobial agents on human neutrophil functions and viability. The results show that SANG is not lytic to neutrophils from peripheral blood or crevicular fluid, at all concentrations tested. However, exposures of neutrophils to very low concentrations of SANG (0.001%) inhibits neutrophil chemotaxis, oxidative metabolism and degranulation within 5 min. Increasing the exposure time results in a similar inhibition of neutrophil functions, albeit at 50-100 fold lower concentrations of SANG. CHX rapidly disrupts the cell membrane of both crevicular and peripheral blood neutrophils at concentrations above 0.005% within 5 min, and inhibition of all neutrophil functions is due to its lytic properties. While TET is least toxic to neutrophils, a dose dependent inhibition of neutrophil functions is dependent on the calcium concentrations of the cellular environment, and is observed only above 0.04% or higher concentrations in the absence of calcium. The data suggest that a critical cumulative concentration of these drugs is essential for their toxicity and inhibition of neutrophil functions. Therefore, both the length of exposure and the dose of the drug both are critical while considering the effectiveness of SANG, CHX or TET in the treatment of infections. Furthermore, due to differences in their mechanisms of action, the consequences of their effects on neutrophils may have significant bearing on tissue pathology as well as on their therapeutic efficacy.     

Parasite infection and Japanese cedar pollinosis in monkeys

We describe here a specific calcineurin activity in neutrophil lysates, which is dependent on Ca2+, inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both the A and B chains of calcineurin. Neutrophils treated with cyclosporin A or FK 506 showed a dose-dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H2O2), where catalase was inhibited with aminotriazole, exhibited a specific inhibition of calcineurin activity. However, the addition of reducing agents to neutrophil extracts partially reversed the inhibition caused by H2O2. A similar inhibitory effect of H2O2 on calcineurin activity was observed to occur in isolated lymphocytes. This is the first demonstration that redox agents modulate calcineurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide-induced activation of NF-kappaB in human neutrophils is inhibited by cell pretreatment with H2O2 in a dose-dependent manner. These data indicate that calcineurin activity regulates the functional activity of lipopolysaccharide-induced NF-kappaB/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of calcineurin activity and that the H2O2-dependent NF-kappaB inactivation in neutrophils occurs in concert with inhibition of calcineurin.     

Significant levels of oxidants are generated by isolated cardiomyocytes during ischemia prior to reperfusion

Oxidants such as reactive oxygen species (ROS) have been shown to participate in myocardial ischemia/reperfusion injury. While many studies report a burst of ROS at reperfusion, few reports have presented evidence of significant ROS generation during ischemia. Our previous studies of cultured cardiomyocytes indicated that antioxidants are most effective when given prior to reperfusion during ischemia. Therefore, we hypothesized that significant ROS generation may occur during ischemia prior to reperfusion. We tested this in a perfused isolated cardiomyocyte system (i.e. without neutrophils, endothelial cells, or xanthine/xanthine oxidase) during simulated ischemia/reperfusion while measuring oxidant generation using intracellular fluorescent probes. During ischemia, the ROS probes dihydroethidium and 2',7'-dichlorofluorescin were significantly oxidized, suggesting superoxide and H2O2 generation. At reperfusion following 1 h ischemia, these probes suggested a further burst of H2O2 and hydroxyl radicals. The antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline used during ischemia attenuated oxidant generation, increased cell viability, and improved return of contraction after ischemia. To further evaluate the relationship between residual O2 and ROS generation, we administered O2 scavengers during ischemia and measured corresponding changes in oxidant generation, cell viability and contraction during reperfusion. Enzymatic scavenging of residual O2 during ischemia (reducing PO2 from 3.5 to 2.5 tau) paradoxically improved subsequent viability and contraction. These results indicate that cultured cardiomyocytes generate significant ROS during ischemia. This ROS generation is related to residual O2 present during ischemia and contributes significantly to the cellular injury seen at reperfusion.     

Borrelia burgdorferi outer surface protein A (OspA) activates and primes human neutrophils

Lyme disease is caused by infection with the spirochete Borrelia burgdorferi and is characterized by bacterial persistence and inflammation of many host tissues. B. burgdorferi express outer surface lipoproteins, including OspA, with inflammatory properties that could contribute to the localized tissue inflammation. Neutrophils are the predominant infiltrate into the inflamed arthritic joints, and are crucial for controlling the spirochete infection. They may also contribute to the joint pathology associated with Lyme arthritis. This study examines the effect of OspA on the activities of the neutrophil. Picomolar concentrations of OspA induce surface markers associated with neutrophil activation: increased CD10 and CD11b expression; decreased CD62-L expression; and an increased adherence to extracellular matrix. These events were similar in kinetics and magnitude to those induced by the strong activators LPS and FMLP. Like LPS, OspA could prime neutrophils for FMLP-induced release of lysosomal granules and production of superoxide. Thus, models of Lyme arthritis should include the possible contribution of direct activation of neutrophils to both defense and disease.     

IMGT, the international ImMunoGeneTics database

Propofol emulsion supports bacterial growth. Extrinsic contamination of propofol has been implicated as an etiological event in postsurgical infections. When added to propofol, local anesthetics (e.g., lidocaine) alleviate the pain associated with injecting it. Because local anesthetics have antimicrobial activity, we determined whether lidocaine would inhibit microbial growth by comparing the growth of four microorganisms in propofol and in mixtures of propofol and lidocaine. Known quanta of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans were inoculated into solutions of 1% propofol, 0.2% lidocaine in propofol, 0.5% lidocaine in propofol, 0.5% lidocaine in isotonic sodium chloride solution, and 0.9% isotonic sodium chloride solution. All microorganisms were taken from stock cultures and incubated for 24 h. Growth of microorganisms in each solution was compared by counting the number of colony-forming units grown from a subculture of the solution at 0, 3, 6, 12 and 24 h. Propofol supported the growth of E. coli and C. albicans. Propofol maintained static levels of S. aureus and was bactericidal toward P. aeruginosa. The addition of 0.2% and 0.5% lidocaine to propofol failed to prevent the growth of the studied microorganisms. The effect of 0.5% lidocaine in isotonic sodium chloride solution did not differ from the effects of isotonic sodium chloride solution alone. We conclude that lidocaine, when added to propofol in clinically acceptable concentrations, does not exhibit antimicrobial properties. IMPLICATIONS: Local anesthetics such as lidocaine have antimicrobial activity. Propofol supports the growth of bacteria responsible for infection. Bacteria were added to propofol and propofol mixed with lidocaine. The addition of lidocaine to propofol in clinically relevant concentrations did not prevent the growth of bacteria. The addition of lidocaine to propofol cannot prevent infection from contaminated propofol.     

Treatment failure and methadone dose in a public methadone maintenance treatment programme in Geneva

PURPOSE: To determine the effect of an anaesthetic with antioxidant potential, propofol, on red blood cell (RBC) antioxidant enzyme activities and RBC susceptibility to peroxidative challenge. METHODS: Propofol was administered by intravenous bolus (2.5 mg.kg-1) and continuous infusion (36 and 72 ml.hr-1 in nine swine; 216 ml.hr-1 in two swine), to achieve serum concentrations between 5 and 30 micrograms.ml-1 for two hours at each rate. Arterial blood sampling was at 0, 10, 30, 60, and 120 min for each rate of infusion, for measurement of plasma propofol concentration, activities of plasma and RBC superoxide dismutase, glutathione peroxidase, glutathione reductase, RBC catalase, and RBC malondialdehyde (MDA) formation in response to ex vivo oxidative challenge with t-butyl hydrogen peroxide (tBHP; 1.5 mM). Antioxidant mechanisms were determined by in vitro study of MDA formation, GSH depletion, and oxidation of haemoglobin to methaemoglobin in human erythrocytes exposed to propofol 0-75 microM. The antioxidant potential of propofol was compared with that of alpha-tocopherol utilising the reaction with 2,4,6-tripyridyl-s-triazine (TPTZ). RESULTS: Propofol had no effect on plasma or RBC antioxidant enzyme activities. It inhibited RBC MDA production over the range of 0-20 micrograms.ml-1 (y = -18.683x + 85.431; R2 = 0.8174). Effective propofol concentrations for 25% and 50% reductions in MDA levels were 7-12 and 12-20 micrograms.ml-1, respectively. Propofol has a similar effect on human erythrocytes in vitro (R2 = 0.98). CONCLUSION: Propofol antagonises the effects of forced peroxidation of red cells at anaesthetic and sub-anaesthetic concentrations in swine. Its actions include scavenging of oxygen derived free radicals in a tocopherol-like manner.     

Oxidative stress in malaria; implications for prevention and therapy

Malaria affects world-wide more than 200 million people, of which 1-2 million die every year. New drugs and treatment strategies are needed to face the rapidly increasing problems of drug resistance. During a malaria infection, both host and parasite are under oxidative stress. Increased production levels of reactive oxygen species (ROS, e.g superoxide anion and the hydroxyl radical) are produced by activated neutrophils in the host and during degradation of haemoglobin in the parasite. The effects of ROS in malaria can be both beneficial and pathological, depending on the amount and place of production. Enhanced ROS production after the administration of pro-oxidants, which is directed against the intra-erythrocytic parasite, inhibits the infection both in vitro and in vivo. However, ROS are also involved in pathological changes in host tissue like damage of the vascular endothelial lining during a malaria infection (cerebral malaria). Pro-oxidants support the host defense against the parasite when working in or near the infected cell but potentially cause vascular damage when working on or near the vascular lining. Examples of pro-oxidants are found among xenobiotics and food components. Important new drugs belonging to the class of pro-oxidants are artemisinin and its derivatives. Anti-oxidants potentially counteract these agents. Treatment with anti-oxidants or chelators of metals to prevent their catalytic function in the generation of ROS may prevent vascular pathology. In addition, the iron chelator desferrioxamine, exhibits an antiparasitic activity, because iron is also essential for the proliferation of the parasite. Cytokines play an important role in ROS-related pathology of malaria, though their mechanism of action is not completely elucidated. This field might bring up new treatment concepts and drugs. Drugs which prevent host pathology, such as the cerebral complications might be life saving.     

Molecular cloning and sequence analysis of interleukin 16 from nonhuman primates and from the mouse

We analysed the occurrence of anti-neutrophil cytoplasmic antibodies (ANCA) in sera of 191 patients with glomerulonephritis (76 females and 115 males) by the standard indirect immunofluorescence method (IIF). The presence of ANCA was demonstrated in sera of 4.4% (8/181) patients with idiopathic glomerulonephritis (GN) and in 30% (3/10) of patients with rapidly progressive glomerulonephritis (RPGN), as a form of renal limited vasculitis. In the experimental part of our study we analysed the influence of GN ANCA-negative sera on the neutrophil function in vitro and compared with the effect of ANCA-positive sera (titre > or = 4:40) from systemic vasculitis (SV) patients with renal involvement. The activation of neutrophils was established by reactive oxygen species (ROS) production and the ability of superoxide anion to reduce ferrocytochrome c. Among 30 ANCA-negative GN sera 20% (6/30) revealed the ability to activate neutrophils isolated from healthy donor. Remaining ANCA-negative GN sera and all sera from normal healthy individuals (negative control group) did not affect the neutrophil function and did not induce the superoxide anion production. Their effect was similar to the second negative reference system without serum. Only 33% (3/9) of high titre ANCA-positive sera (> or = 1:40) from SV patients were able to activate neutrophils and to produce the superoxide anion with following ferrocytochrome c reduction, but the effect of activation was most powerfully expressed (three times greater than by GN ANCA-negative sera). The remaining ANCA-positive sera and all SV ANCA-negative sera did not affect the neutrophil function in vitro. These experimental data indicate that the presence of ANCA in GN sera is not necessary to induce neutrophil activation in vitro. On the other hand the influence of the SV ANCA-positive sera was most powerful expressed, although only 33% of sera were able to activate neutrophils in vitro. Our results indicate that not always ANCA presence in serum was connected with the ability to neutrophil activation in vitro. It is possible that in ANCA-negative sera other factors were able to activate neutrophils in vitro, but the effect of activation was markedly lower.     

Modulation of neutrophil apoptosis by psychological stress and glucocorticoid

Neutrophils play a pivotal role in host defense against bacterial infection, however, these cells may have a harmful effect on normal tissues under certain pathological conditions. We demonstrate here that apoptosis of these cells is modulated by psychophysical stress and its related hormones, suggesting that psychoneural systems may exert an effect on host defense through modulating neutrophil survival.     

The effect of midazolam and propofol on interleukin-8 from human polymorphonuclear leukocytes

Anesthetics and sedatives contribute to postoperative immunosuppression. Interleukin-8 (IL-8) is a chemotactic and activating factor that mediates neutrophil adhesion and margination and is essential for host defense. We investigated the effect of anesthetics on isolated human polymorphonuclear leukocyte production of IL-8. Healthy human polymorphonuclear leukocytes were isolated using a single-step density gradient and stimulated with lipopolysaccharide in the presence of varying concentrations of propofol or midazolam for up to 20 h. IL-8 was measured in both culture supernatants and cell lysates using enzyme immunoassay, and IL-8 mRNA in cells was measured using Northern blotting and phosphorimaging. Data were analyzed using Kruskal-Wallis analysis of variance or the Mann-Whitney U-test as appropriate. Lipopolysaccharide increased extracellular accumulation of interleukin-8, which was suppressed by both propofol (P = 0.025) and midazolam (P = 0.028). However, intracellular IL-8 increased with exposure to lipopolysaccharide (P = 0.028) and remained increased with both anesthetics. Northern blot analysis also revealed increased IL-8 mRNA levels in the presence of both midazolam and propofol, which was confirmed by molecular imaging. These data strongly suggest that the anesthetics modulate transport or secretion of IL-8 protein from the cell. Suppression of IL-8 by anesthetics and sedatives may predispose postoperative and intensive care patients to infection. Implications: Anesthesia causes immune suppression and alters neutrophil function. We investigated the effect of propofol and midazolam on interleukin-8, a neutrophil chemotactic agent in human neutrophils. Both anesthetics decreased extracellular interleukin-8 accumulation, but intracellular levels and mRNA remained high. This suggests that propofol and midazolam alter interleukin-8 secretion from cells.     

Endothelial barrier dysfunction and oxidative stress: roles for nitric oxide?

Endothelial dysfunction has an important role to play in the pathophysiology of human vascular disease. The maintenance of barrier function is critical to the role of vascular endothelium in cardiovascular haemostasis and this function can be compromised by inflammatory mediators, cytokines or oxidants. Under conditions of oxidative stress a variety of reactive oxygen species (ROS) may be generated, which increase the permeability of the endothelial monolayer to fluid, macromolecules and inflammatory cells. The endothelium-derived nitric oxide radical (NO), whose physiological actions include effects on vascular smooth muscle, is normally inactivated by the superoxide radical anion. While large amounts of NO have cytotoxic potential, it is now becoming clear that combinations of NO with ROS can produce either cytotoxic or cytoprotective effects, depending on the relative amounts of each which are present in the target cell or its environment at a particular time. The contribution of NO to oxidant-mediated endothelial barrier dysfunction can be assessed in vitro in endothelial monolayers grown on porous membrane supports. In this model, using hydrogen peroxide (H2O2) as the oxidant, H2O2-induced losses of barrier function can be enhanced or partially offset by NO donor drugs, depending on the concentration of NO donor used. Furthermore, the injurious or cytoprotective effects of these agents appear to be determined by the quantity of NO generated. Since NO is administered clinically by inhalation in conditions such as pulmonary hypertension or the adult respiratory distress syndrome, which are themselves associated with generation of ROS, it is likely that low concentrations of NO may protect the pulmonary vascular endothelium while high concentrations might be expected to combine with ROS to yield intermediates capable of causing further endothelial injury or loss of barrier function.     

Biochemistry and cell biology of phospholipase D in human neutrophils

Neutrophils play a major role host defense against invading microbes. Recent studies have emphasized the importance of the phospholipase D (PLD) in the signalling cascade leading to neutrophil activation. Phospholipase D catalyzes the hydrolysis of phospholipids to generate phosphatidic acid with secondarily generation of diradylglycerol; both of these products have been implicated as second messengers. Herein, we discuss the regulation and the biochemistry of the receptor-regulated PLD in human neutrophils. In vivo and in vitro studies suggest an activation mode in which initial receptor-linked activation of phospholipase C generates diacylglycerol and inositol trisphosphate. The resulting calcium flux along with the diacylglycerol activate a conventional isoform of protein kinase C (PKC), probably PKC beta 1. This PKC, in turn phosphorylates a plasma membrane component resulting in PLD activation and a second outpouring of diradylglycerol. The small GTP-binding proteins, RhoA and ARF, also participate in this process, and synergize with a 50 kDa cytosolic regulatory factor.     

Effects of thalidomide on neutrophil respiratory burst, chemotaxis, and transmigration of cytokine- and endotoxin-activated endothelium

Vascular endothelium activated by endotoxin and cytokines plays an important role in organ inflammation and blood leukocyte recruitment. Neutrophils, which are a homogeneous population of effector cells, are rapidly attracted in large numbers to sites of inflammation where they form an early response to infection or injury. Excessive production of various interleukins, TNF, arachidonic acid metabolites, and other substances by neutrophils and macrophages results in systemic endothelial cell injury, a fundamental problem. In the present study, we investigated in vitro the effects of thalidomide (THD) on activation of endothelial cells for enhanced transmigration of neutrophils by lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF), and interleukin-1 (IL-1). Modulation of endotoxin- and cytokine-induced neutrophil chemotaxis and respiratory burst by THD were also studied. Treatment of HUVEC with THD in combination with LPS, TNF, and IL-1, respectively, antagonized LPS-activated transmigration of neutrophils but stimulated the effects of TNF and IL-1. All of the agents used-THD, LPS, TNF, and IL-1-inhibited neutrophil chemotaxis. Addition of THD to the neutrophils had no effect on LPS-inhibited chemotaxis whereas the TNF- and IL-1-induced chemotaxis was modulated in a bimodal manner. However, THD failed to influence neutrophil respiratory burst activity. Results demonstrate that THD differentially affects mediator-induced activation of HUVEC and neutrophils.