Summary. The oxidative burst response of neutrophils to bacteria is crucial for effective host defence. It has been shown that inhalation anaesthetics interfere.
British Journal of Anaesthesia 1997; 78: 718–723
Effects of volatile anaesthetics on human neutrophil oxidative response to the bacterial peptide FMLP
D. FRÖHLICH, G. ROTHE, B. SCHWALL, P. SCHMID, G. SCHMITZ, K. TAEGER AND J. HOBBHAHN
Summary The oxidative burst response of neutrophils to bacteria is crucial for effective host defence. It has been shown that inhalation anaesthetics interfere with neutrophil function and the object of this study was to characterize the mechanisms of interaction of volatile anaesthetics with the oxidative response of neutrophils. H2O2 production by neutrophils after stimulation with the bacterial peptide, N-formyl- L -methionyl- L -leucyl-phenylalanine (FMLP) and phorbol-12-myristate-13-acetate (PMA) was measured by oxidation of the indicator dye dihydrorhodamine using flow cytometry. FMLP binds to a specific surface receptor on neutrophils and initiates via receptor specific signal transduction respiratory burst as an all-ornone event, whereas PMA is an artificial activator of protein kinase C, which bypasses receptormediated signal transduction. In the presence of halothane, enflurane and sevoflurane, there was an increase in activation threshold on FMLP stimulation. Overall, this correlated with reduced H2O2 production. Isoflurane had no effect. In the presence of desflurane, however, H2O2 production of neutrophils increased two-fold, followed by transient suppression of neutrophil function. PMAinduced H2O2 generation was unchanged in the presence of volatile anaesthetics. We conclude that volatile anaesthetics modulated FMLP receptordependent signal transduction upstream of protein kinase C activation, leading to a reduced response in the presence of halothane, enflurane and sevoflurane and to an increased response in the presence of desflurane. (Br. J. Anaesth. 1997; 78: 718–723). Key words Immune response. Blood, leucocytes. Blood, neutrophils. Anaesthetics volatile, desflurane. Anaesthetics volatile, enflurane. Anaesthetics volatile, halothane. Anaesthetics volatile, isoflurane. Anaesthetics volatile, sevoflurane.
Neutrophil granulocytes are a major component of the non-antigen-specific, cell-mediated immune system. Activated by chemotactic signals, neutrophils migrate towards infected tissue ingesting and killing bacteria. A crucial mechanism of bacterial
killing by neutrophils is generation of reactive oxygen derivatives. Because of the high oxygen uptake during generation of reactive oxygen species, this reaction has also been called respiratory burst.1 The defence mechanism of oxygen radical production may be impaired, for example after drug exposure or during disease. Numerous studies have shown suppression of neutrophil oxidative function with halothane2–6 and enflurane.5–7 Isoflurane appears to have no, or only a minimal effect on neutrophils.3–5 7 We have investigated the effects of several halogenated, volatile anaesthetics on the production of reactive oxygen derivatives in human neutrophils. Two different stimuli, N-formyl-methionyl-leucylphenylalanine and phorbol-13-myristate-12-acetate, were used to trigger respiratory burst in order to discriminate between receptor-dependent and independent mechanisms, respectively. The flow cytometric assay used in this study allows quantification of the heterogenous oxidative response of neutrophils at the single cell level with low degrees of stimulation.
Materials and methods The volatile anaesthetics used were obtained from the following companies: halothane from Hoechst, Germany; enflurane, sevoflurane and isoflurane from Abbott, Germany; desflurane from Pharmacia, Sweden. Carboxy - seminaphthorhodafluor -1 acetoxymethylester (SNARF1/AM) and dihydrorhodamine-123 (DHR) were obtained from Molecular Probes, Eugene, OR, USA. Dulbecco’s phosphatebuffered saline (PBS) with Ca2; and Mg2; was obtained from Gibco-Life Technologies, Germany. N-formyl- L -methionyl- L -leucyl-phenylalanine (FMLP) and phorbol-12-myristate-13-acetate (PMA) were obtained from Sigma Chemicals, DIETER FRÖHLICH*, MD, BARBARA SCHWALL, MD, KAI TAEGER, MD, JONNY HOBBHAHN, MD (Department of Anaesthesia); GREGOR ROTHE, MD, GERD SCHMITZ, MD (Institute of Clinical Chemistry and Laboratory Medicine); University of Regensburg, Germany. PETER SCHMID, PHD, Institute of Toxicology, Swiss Federal Institute of Technology, Schwerzenbach, Switzerland. Accepted for publication: February 11, 1997. *Address for correspondence: Department of Anaesthesia, University of Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany.
Neutrophil response in the presence of volatile anaesthetics Deisenhofen, Germany. Propidium iodide (PI) was purchased from Serva, Heidelberg, Germany. DHR, SNARF1/AM and FMLP were kept as stock solutions in dimethylformamide (DMF) and diluted in PBS yielding working concentrations of 100 mol litre91 for DHR, and 10 mol litre91 for SNARF1/AM, FMLP and PMA. Stock solutions were stored at 970⬚C. Standard dye-beads (Quantum 26) for calibration of the flow cytometer were obtained from Flow Cytometry Standards Europe based in Leiden, The Netherlands. ISOLATION OF LEUCOCYTES
This study was approved by the local Ethics Committee, and after receiving written informed consent, venous blood was obtained from healthy donors with no history of infection for the 2 weeks before the experiments. The donors (n:9) had a mean age of 32 (range 25-39) yr. They had a mean white blood cell count of 6200 (SD 1300)/l and haemoglobin concentration was 15.2 (1.8) g dl91. Mean differential leucocyte count was: neutrophils 53 (8) %, lymphocytes 32 (7) %, monocytes 8 (3) %, eosinophils 3 (2) % and basophils 1 (1) %. Blood counts were performed before each experiment with a Technicon H*3 counter (Bayer Diagnostics, Tarrytown, NY, USA). No specimens had to be excluded because of abnormal blood cell counts. Blood was collected in lithium heparin coated disposable blood sampling tubes (Sarstedt, Nürmbrecht, Germany). Leucocyte isolation was carried out by sedimenting erythrocytes on Ficoll. Heparinized (10 u. ml91) whole blood (3 ml) was layered on top of 3 ml of lymphocyte separation medium (density 1.077 g ml91) from Sigma Chemicals, Deisenhofen, Germany. Erythrocytes aggregated at the interface and settled at room temperature without centrifugation. After 40 min the upper 800 l of supernatant leucocyte-rich plasma were withdrawn, avoiding contact with the plasma fraction close to the interface with the separation medium. To avoid artificial activation of cells, the isolation process did not involve lysis, centrifugation or washing procedures. RESPIRATORY BURST ASSAY
Each volatile anaesthetic was tested separately in vitro with neutrophils from the nine donors at four different concentrations (0, 0.5, 1.0, 2.0 MAC). The MAC values used for this study were: 0.8% for halothane, 1.7% for enflurane, 2% for sevoflurane, 1.2% for isoflurane and 6% for desflurane.8 9 All anaesthetics were administered as volatile anaesthetic/air mixtures. The respiratory burst assay was performed in a 75 L airtight glovebox. Fresh gas was prepared using a gas mixing unit with calibrated vaporizers (Draeger, Lübeck, Germany). Fresh gas flow, after equilibration of the atmosphere inside the box with high flow rates, was 5 litre min91 during the experiments. Inside the glovebox, samples were kept at 37⬚C with a heated aluminium block. To maintain the amount of evaporated water from the samples low, tubes were sealed with gas-permeable
719 parafilm during all incubations. Volatile delivery under the hood was monitored continuously at the site of the sample tubes by a multi-gas analyser (Capnomac Ultima, Datex, Sweden). Before filling the polypropylene tubes (diameter 10 mm) and adding leucocytes, Dulbecco’s PBS was equilibrated with the anaesthetic inside the glovebox by bubbling fresh gas through for 30 min at 37⬚C. The leucocyte-rich plasma was suspended 1:50 (20 l of leucocytes and 980 l of PBS) in preequilibrated Dulbecco’s PBS and incubated for 20 min at 37⬚C. The leucocytes were loaded with the fluorogenic substrates DHR and SNARF1/AM for 10 min. Cells were pre-exposed to the volatile anaesthetics for 30 min. The final concentrations were 1 mol litre91 for DHR and 0.2 mol litre91 for SNARF1/AM. Thereafter, FMLP or PMA (10 l) at a final concentration of 100 nmol litre91 were added to initiate H2O2 generation. After 15 min of incubation at 37⬚C the reaction was stopped on ice. Dead cells were counterstained with PI (10 l) at a final concentration of 30 mol litre91. Samples were stored on ice in the dark and measured within 1 h. For evaluation of recovery of neutrophil function, the assay was repeated sequentially for both unexposed control and exposed samples: during exposure and 0.5 h, 1 h and 2 h after the end of exposure. For analysis we used a FACScan flow cytometer (Becton Dickinson, San José, CA, USA) with argonion laser excitation at 488 nm, measuring 104 cells of each stained sample. Data were acquired and processed using LYSIS-II software provided by the cytometer manufacturer. In order to obtain absolute calibration of the fluorescence measurements the arbitrary fluorescence units of the cytometer were transformed into molecule equivalent of soluble fluorochrome (MESF) units. Therefore, after each experiment five populations of standard beads (Quantum 26) with a known content of fluorochrome were measured and a linear regression line was calculated. The MESF units were used for absolute quantification of cellular fluorescence, allowing inter-assay and inter-laboratory comparison of data. Dead cells were assessed by their PI fluorescence (above 600 nm). Leucocyte esterase activity, characteristic for viable cells, was determined based on SNARF1-related orange fluorescence. SNARF1/ AM is cleaved in vital leucocytes by esterases to fluorescent SNARF1. Neutrophils were identified by their typical side scatter light (SSC) and forward scatter light (FSC) patterns. SSC depends on the granularity of cells, whereas FSC is related to the size of cells. GAS CHROMATOGRAPHY/MASS SPECTROMETRY
For assessment of the concentrations of volatile anaesthetics in the fluid phase of our experiments, we used gas chromatography/high resolution mass spectrometry (GC/HRMS) with a HRGC Mega 2 series (Fisons Instruments SpA, Rodano, Italy). Elution was performed on a 20 m0.27 mm glass capillary column coated with a 2.1-m film of PS 090 (Petrarch Systems Inc., Bristol, PA 19007,
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British Journal of Anaesthesia
USA). The carrier gas was hydrogen at a head pressure of 30 kPa (linear flow rate 0.3 m s91). Samples of 0.75 l were injected into the split injector (temperature 250⬚C, split ratio 1:20); oven temperature was kept at an ambient temperature of 24⬚C. The mass spectrometer MAT 95 (Finnigan MAT, Bremen, Germany) was run in electron impact mode at 70 eV electron energy (ion source temperature 180⬚C). The mass resolution m/⌬m was set to 7500; the multiplier voltage was 1.4 kV. Typical ions were recorded by single ion monitoring (SIM) using perfluorotributylamine and carbon dioxide, respectively, as references; cycle time was 0.2 s. Quantification was based on signal areas in the mass chromatograms.
Results Figure 1A shows flow cytometric analysis of resting, non-stimulated leucocytes, whereas figure 1B displays the heterogenous reaction of neutrophils on FMLP stimulation. In contrast, the PMA-induced reaction of all cells was homogenous (fig. 1C). The percentage of reacting and non-reacting neutrophils is given on the left of each dot plot. The percentage of dead cells was lower than 0.1% independent of drug exposure or stimulation. In the presence of halothane the reactive oxygen derivative generation of neutrophils and monocytes was significantly reduced. At 1 MAC mean fluorescence was 50% compared with control (fig. 1D). In the presence of enflurane (2 MAC), H2O2 generation reached only 60% of control (table 1). The decreased H2O2 production correlated with a decreased number of reacting neutrophils. Percentages of neutrophils reacting on FMLP stimulation are given in table 2. The effects observed in the presence of halothane and enflurane were also visible with sevoflurane, although the decrease in H2O2 production was not significant. Isoflurane did not interfere with neutrophil reaction on FMLP stimulation. In contrast with all other anaesthetics, the oxidative response after stimulation was enhanced in the presence of desflurane. Both mean fluorescence and percentage of neutrophils responding with a
STATISTICAL ANALYSIS
All data are presented as mean (SD) and range. Analysis of variance and the Student–Newman– Keuls test for multiple pairwise comparison at a significance level of P0.05 were used to compare controls and drug-exposed samples. MAC was designated as the independent variable for analysis of variance to evaluate the effects of different concentrations of volatile anaesthetics. To test the significance of differences in the recovery kinetic data, the Student’s t test was used. We compared mean fluorescence of unexposed controls processed in parallel with volatile anaesthetic-exposed aliquots.
Table 1 Influence of different concentrations of volatile anaesthetics on neutrophil oxidative response. Hydrogen peroxide production after stimulation with FMLP or PMA is given as 103 molecule equivalents of soluble fluorochrome (MESF) units (mean (SD)) of nine independent experiments (donors)). *P0.05, **P0.01 FMLP 100 nmol litre91
PMA 100 nmol litre91
Concentration
Control
0.5 MAC
1 MAC
2 MAC
Control
2 MAC
Halothane Enflurane Sevoflurane Isoflurane Desflurane
133 (47) 152 (51) 146 (45) 137 (16) 136 (35)
76 (38)* 111 (45) 111 (35) 108 (41) 125 (38)
63 (47)** 101 (38) 102 (41) 111 (47) 154 (54)
80 (51)* 92 (35)** 102 (19) 133 (45) 226 (47)**
6972 (891) 6556 (666) 6469 (1052) 6327 (1011) 6328 (751)
7443 (1666) 7189 (1225) 7296 (940) 7881 (1427) 6187 (617)
Table 2 Influence of different concentrations of volatile anaesthetics on the percentage of respiratory burst positive neutrophils FMLP stimulation. Results are expressed as the percentage of reacting neutrophils from all neutrophils (mean (SD) of nine independent experiments (donors)). *P0.05, **P0.01 FMLP 100 nmol litre91 Concentration
Control
0.5 MAC
1 MAC
2 MAC
Halothane Enflurane Sevoflurane Isoflurane Desflurane
69 (10) 54 (12) 69 (10) 66 (15) 55 (7)
57 (7) 43 (19) 61 (8) 54 (13) 51 (8)
47 (21)* 33 (9)* 61 (16) 53 (17) 51 (8)
55 (17) 28 (12)** 53 (13)** 58 (14) 67 (12)*
Table 3 Recovery of H2O2 production after FMLP stimulation following exposure to different volatile anaesthetics at 1 MAC for 30 min. Ratio of fluorescence between exposed and unexposed cells from five independent experiments (mean (SD)). *P0.05, **P0.01 Time after exposure
0h
0.5 h
1h
2h
Halothane Enflurane Sevoflurane Isoflurane Desflurane
0.46 (0.09)** 0.67 (0.13) 0.76 (0.12) 0.77 (0.05) 1.08 (0.22)
0.73 (0.11)* 0.80 (0.21) 1.03 (0.18) 0.77 (0.14) 0.73 (0.14)*
0.92 (0.12) 0.96 (0.13) 0.97 (0.16) 0.91 (0.17) 0.70 (0.10)*
0.93 (0.08) 0.90 (0.14) 0.83 (0.13) 0.78 (0.11) 0.78 (0.10)
Neutrophil response in the presence of volatile anaesthetics
721
Figure 1 Flow cytometric analysis of intracellular H2O2 of 10 000 leucocytes from a representative experiment. The X-axis indicates the side scatter (SSC) of leucocytes as a marker of cellular granularity, while the Y-axis represents H2O2 production (rhodamine-123 (123Rho) fluorescence) in arbitrary units (neutrophils (pmn), monocytes (mo), lymphocytes (ly)). A: Unstimulated cells; B: cells stimulated with FMLP 100 nmol litre91; C: cells stimulated with PMA 100 nmol litre91; D and E: cells stimulated with FMLP 100 nmol litre91 in the presence of halothane (1 MAC) and desflurane (2 MAC). B serves as a control for D and E. The ratio between reacting and non-reacting cells is given to the right of each dot plot.
respiratory burst reaction were increased compared with controls (fig. 1E, tables 1, 2). Recovery data are given as the ratio between fluorescence of the exposed and non-exposed samples (table 3). Significant differences between control and drug exposed samples were found for halothane up to 30 min after exposure. Desflurane exposed neutrophils showed transient depression of respiratory burst activity on FMLP stimulation. The impairment lasted less than 2 h. By GC/HRMS the following buffer/gas partition coefficients and concentrations of volatile anaesthetics for 37⬚C and 1 MAC were confirmed: halothane 0.663 and 0.24 (0.01) mmol litre91, enflurane 0.548 and 0.42 (0.02) mmol litre91, isoflurane 0.438 and
0.24 (0.01) mmol litre91, sevoflurane 0.370 and 0.33 (0.01) mmol litre91 and desflurane 0.234 and 0.63 (0.03) mmol litre91. Equilibration was complete after 10 min for all anaesthetics. Ten minutes after removal of the anaesthetic, concentrations in the fluid phase decreased to less than 2% of the initial concentration. The relationship between fluid- and gas-phase concentration was linear in the range relevant to this study.
Discussion In this in vitro study we have investigated the ability of human neutrophils to form reactive oxygen products after FMLP stimulation during and after exposure to volatile anaesthetics.
722 Unlike other investigators we used MAC-related concentrations in order to compare equipotent concentrations of anaesthetics. The measured buffer/gas partition coefficients were lower than those coefficients given for blood by other authors and met the coefficients published for saline.10–12 This may lead to underestimation of the effects presented in this report. In terms of molarity, the MAC-derived differences in concentration were smaller in the fluid phase than in the gas phase of the experiment. For example, 1 MAC of halothane (0.8 vol%) corresponded to 0.24 mmol litre91, whereas 1 MAC of desflurane (6.0 vol%) was only 0.63 mmol litre91. The small amounts of intracellular H2O2 generated after stimulation were assessed by quantifying the intracellular oxidation of the indicator dye dihydrorhodamine-123 to rhodamine-123.13 The former is a non-fluorescent and membranepermeable fluorogenic substrate whereas the latter oxidation product emits green light (510–530 nm) on excitation.14 This method has several advantages over those used previously for quantifying neutrophil oxidative function. First, purification of neutrophils, which often leads to artefactual activation of cells, is unnecessary. Second, flow cytometric results are not affected by variations in concentration of neutrophils in the assay, as each individual neutrophil is measured and not the average response of all those assayed. Thus we can show the heterogenous response of neutrophils in an all-or-none reaction after stimulation; only a subset of all neutrophils respond with the respiratory burst reaction on FMLP stimulation while the others remain resting (fig. 1B). In our study, halothane and enflurane caused a reversible decrease in the formation of H2O2, which is in accordance with the results of other investigators.2–7 This inhibition was characterized by a decrease in production of respiratory burst products per neutrophil, and reduction in the number of neutrophils recruited for respiratory burst. Desflurane enhanced the neutrophil response significantly, and a transient decrease in neutrophil oxidative burst activity ensued after removal of anaesthetic. The increased activity not only correlated with increased recruitment of neutrophils, but also enhanced intracellular H2O2 per cell. Enhanced neutrophil function in the presence of isoflurane has also been shown by Erskine and James4 and by Nakagawara and colleagues5 for a concentration corresponding to 2 MAC. The reported effects of isoflurane were not significant in our experiments. Using the receptor independent stimulus PMA, none of the anaesthetics at any concentration had an effect on the oxidative response. The finding of unchanged PMA-induced respiratory burst in the presence of all volatile anaesthetics excludes two potential causes of altered H2O2 production: scavenging of reactive oxygen derivatives by volatile anaesthetics and direct interference with H2O2 generating enzymes. Furthermore, Nakagawara and colleagues5 tested the ability of halothane to scavenge respiratory burst products in celldependent and cell-free systems. Neither halothane nor enflurane were able to reduce the amount of
British Journal of Anaesthesia generated reactive oxygen products. Thus the significant decrease in H2O2 production of monocytes in the presence of halothane (fig. 1D) was most likely caused by interference with regulation of the spontaneous oxidative burst of monocytes. This regulation involves signalling pathways different from those mediating neutrophil respiratory burst. Halothane, enflurane and sevoflurane increased and desflurane decreased the threshold for FMLPdependent activation such that the number of neutrophils displaying respiratory burst on stimulation declined or increased. PMA activates respiratory burst through the same signal transduction chain as FMLP but at a later activation step. Nakagawara and colleagues5 investigated the affinity of the FMLP receptor for its agonist in the presence of volatile anaesthetics by measuring the uptake of radioactive labelled FMLP. They found an unchanged affinity of the receptor in the presence of halothane and enflurane. Thus halothane, enflurane and sevoflurane appear to interfere with intracellular signal transduction downstream of the FMLP receptor and upstream of the activation site of PMA. The bacterial peptide FMLP is a physiological agonist for specific receptors on neutrophils. FMLP has been shown to be a major chemotaxin for neutrophils, and phagocytosis of Escherichia coli is mediated mainly by FMLP and its receptor on the neutrophil surface.15 The concentration of 100 nmol litre91 for FMLP was chosen to allow analysis of the heterogenous response of neutrophils. This concentration also allows comparison with results of other authors.5 FMLP triggers multiple responses, including chemotaxis, exocytosis of certain enzymes and superoxide generation by the NADPH oxidase complex, the so-called respiratory burst.1 16–18 Binding of FMLP to the G-protein-coupled receptor induces a temporary increase in phosphatidylinositol-1,4,5triphosphate, cytosolic calcium, 1,2 diacylglycerol (DAG) and other activators of protein kinase C (PKC).19 20 Human neutrophils express four PKC isoenzymes (␣, I, II, ) PKC and are the most abundant isoforms, while PKC␣ is hardly detectable. After stimulation with FMLP the PKC isoforms I, II and are redistributed from the cytosol to the plasma membrane. PMA is a synthetic analogue of DAG and therefore induces a similar redistribution and activation of PKC, but unlike FMLP the redistribution of PKC is irreversible.21 The membrane bound form of PKC activates, by intermediate steps, NADPH oxidase. By a single electron transfer, this oxidase transforms molecular oxygen into superoxide anion. Superoxide then dismutates to hydrogen peroxide (H2O2) inside the phagosomes or in the extracellular space.1 17 Activation of respiratory burst depends on calmodulin,22 23 as deduced from experiments using specific antagonists. Calmodulin, an ubiquitous lowmolecular-weight protein, regulates the calciumdependent activity of various enzymes. Périanin, Pedruzzi and Hakim22 demonstrated that the calmodulin antagonist W-7 dose-dependently enhances and depresses the respiratory burst reaction. Levin and Blanck24 showed that halothane and isoflurane interfered with the affinity of
Neutrophil response in the presence of volatile anaesthetics calmodulin for calcium. Low concentrations of halothane (0.57%) decreased the affinity, whereas higher concentrations (5.7%) significantly increased the ability to bind calcium. The latter effect was found to be irreversible.24 Isoflurane behaved in a similar way, but halothane was more potent when compared on the basis of MAC values. These findings may explain the ability of desflurane to enhance and of halothane, enflurane and sevoflurane to lower respiratory burst by interfering with calmodulin, in a manner similar to the known calmodulin antagonist W-7.22 Thus desflurane might interfere in the same manner as high-dose isoflurane in the experiments of Levin and Blanck,24 increase the ability of calmodulin to bind calcium and subsequently enhance respiratory burst. This interaction should be irreversible, so that temporary depression of neutrophil function after desflurane exposure might depend on protein synthesis leading to restoration of calmodulin with a normal affinity for calcium. In contrast, halothane or enflurane might decrease the ability of calmodulin to bind calcium and should therefore lead to an immediately reversible inhibition of respiratory burst. The clinical relevance of impairment of neutrophil function cannot be deduced as our experimental design was not suitable for quantifying clinical effects.
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723 7. Welch WD. Effect of enflurane, isoflurane, and nitrous oxide on the microbicidal activity of human polymorphonuclear leukocytes. Anesthesiology 1984; 61: 188–192. 8. Frink EJ, Brown BR. Sevoflurane. Anaesthetic Pharmacological Review 1994; 2: 61–67. 9. Jones RM, Nay PG. Desflurane. Anaesthetic Pharmacological Review 1994; 2: 51–60. 10. Strum DP, Eger EI. Partition coefficients for sevoflurane in human blood, saline, and olive oil. Anesthesia and Analgesia 1987; 66: 654–656. 11. Eger EI. Partition coefficients of I-653 in human blood, saline, and olive oil. Anesthesia and Analgesia 1987; 66: 971–973. 12. Steward A, Allott PR, Cowles AL, Mapleson WW. Solubility coefficients for inhaled anaesthetics for water, oil and biological media. British Journal of Anaesthesia 1973; 45: 282–293. 13. van Pelt LJ, van Zwieten R, Weening RS, Roos D, Verhoeven AJ, Bolscher BGJM. Limitations on the use of dihydrorhodamine 123 for flow cytometric analysis of the neutrophil respiratory burst. Journal of Immunological Methods 1996; 191: 187–196. 14. Rothe G, Valet G. Flow cytometric assays of oxidative burst activity in phagocytes. Methods in Enzymology 1994; 233: 539–548. 15. Marasco WA, Phan SH, Krutzsch H. Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli. Journal of Biological Chemistry 1984; 259: 5430–5439. 16. Murphy MM, Özcelik T, Kenney RT, Tiffany HL, McDermott D, Francke U. A structural homologue of the Nformyl peptide receptor. Journal of Biological Chemistry 1992; 267: 7637–7643. 17. Clark RA. The human neutrophil respiratory burst system. Journal of Infectious Disease 1990; 161: 1140–1147. 18. Fujishima S, Aikawa N. Neutrophil mediated tissue injury and its modulation. Intensive Care Medicine 1995; 21: 277–285. 19. Tsu RC, Lai HWL, Allen RA, Wong YH. Differential coupling of the formyl peptide receptor to adenylate cyclase and phospholipase C by pertussis toxin-insensitive Gz protein. Biochemical Journal 1995; 309: 331–339. 20. McPhail LC, Qualliotine-Mann D, Agwu DE, McCall CE. Phospholipases and activation of the NADPH oxidase. European Journal of Haematology 1993; 51: 294–300. 21. Dang PM, Hakim J, Périanin A. Immunochemical identification and translocation of protein kinase C zeta in human neutrophils. FEBS Letters 1994; 349: 338–342. 22. Périanin A, Pedruzzi E, Hakim J. W-7, a calmodulin antagonist, primes the stimulation of human neutrophil respiratory burst by formyl peptides and platelet-activating factor. FEBS Letters 1994; 342: 135–138. 23. Rao KMK, Padmanabhan J, Kilby DL, Cohen HJ, Currie MS, Weinberg JB. Flow cytometric analysis of nitric oxide production in human neutrophils using dichlorofluorescin diacetate in the presence of calmodulin inhibitor. Journal of Leukocyte Biology 1992; 51: 496–500. 24. Levin A, Blanck TJJ. Halothane and isoflurane alter the Ca2; binding properties of calmodulin. Anesthesiology 1995; 83: 120–126.
