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Effects of isoflurane and sevoflurane on the neutrophil myeloperoxidase system of horses Grégory Minguet a,∗ , Thierry Franck b , Jean Joris a , Justine Ceusters b , Ange Mouithys-Mickalad b , Didier Serteyn b,c , Charlotte Sandersen c a
Department of Anaesthesia and Intensive Care Medicine, CHU de Liège, Domaine Universitaire du Sart-Tilman, 4000 Liège, Belgium Center for Oxygen Research and Development, Institute of Chemistry B6a, University of Liège, Domaine Universitaire du Sart-Tilman, 4000 Liège, Belgium c Department of Clinical Sciences, Anaesthesiology and Equine Surgery, Faculty of Veterinary Medicine, University of Liège, Domaine Universitaire du Sart-Tilman, 4000 Liège, Belgium b
a r t i c l e
i n f o
Article history: Received 18 November 2014 Received in revised form 10 February 2015 Accepted 26 February 2015 Keywords: Volatile anaesthetics Isoflurane Sevoflurane Neutrophil Myeloperoxidase Oxidative stress
a b s t r a c t Volatile anaesthestics have shown to modulate the oxidative response of polymorphonuclear neutrophils (PMNs). We investigated the effects of isoflurane and sevoflurane on the degranulation of total and active myeloperoxidase (MPO) from horse PMNs and their direct interaction with MPO activity. Whole blood from horse was incubated in 1 and 2 minimal alveolar concentrations (MAC) of isoflurane or sevoflurane for 1 h and PMNs were stimulated with cytochalasin B (CB) plus N-formyl-méthionyl-leucyl-phenylalanine (fMLP). After stimulation, the plasma was collected to measure total and active MPO by enzymelinked immunosorbent assay (ELISA) and specific immunological extraction followed by enzymatic detection (SIEFED) respectively. The effects of 1 and 2 MAC of isoflurane and sevoflurane on the peroxidase and chlorination activity of pure MPO were assessed by fluorescence using Amplex red and 3 -(p-aminophenyl) fluorescein (APF) respectively and in parallel with a SIEFED assay to estimate the potential interaction of the anaesthetics with the enzyme. Although isoflurane and sevoflurane had inconsistent effects on total MPO release, both volatile agents reduced active MPO release and showed a direct inhibition on the peroxidase and the chlorination activity of the enzyme. A persistent interaction between MPO and anaesthetics was evidenced with isoflurane but not with sevoflurane. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Myeloperoxidase (MPO) is a pro-oxidant haemic enzyme from azurophilic granules of polymorphonuclear neutrophils (PMNs) which represent a pivotal component of the neutrophil machinery involved in the clearance of host damaged tissues and exogenous invading microorganisms (Deby-Dupont et al., 1999). During neutrophil
∗ Corresponding author. Tel.: +32 4 366 71 80. E-mail address: [email protected] (G. Minguet).
phagocytosis, most of these processes are confined to the phagolysosome, leading to effective intracellular destruction of tissue debris and microbial pathogens. However, activated PMNs also release MPO in the extracellular space, which results in collateral damages to healthy host structures. The cytotoxic potential of MPO is attributable to its dual peroxidase and chlorination activities able to inactivate multiple biological molecules including proteins, lipids and nucleic acids. It is now recognized that extracellular MPO not only represents a marker of neutrophil activation but also plays a role in collateral injuries encountered during the course of inflammatory diseases (Davies
http://dx.doi.org/10.1016/j.vetimm.2015.02.010 0165-2427/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Minguet, G., et al., Effects of isoflurane and sevoflurane on the neutrophil myeloperoxidase system of horses. Vet. Immunol. Immunopathol. (2015), http://dx.doi.org/10.1016/j.vetimm.2015.02.010
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et al., 2008; Klebanoff, 2005). Hence, our group demonstrated that MPO has a pathophysiological significance in various clinical conditions associated with acute PMNs stimulation like i.e. intestinal ischaemia (Grulke et al., 2008), laminitis (de la Rebiere et al., 2008) or sustained physical exercise (Serteyn et al., 2010). Volatile anaesthetics such as sevoflurane and isoflurane are commonly used in veterinary anaesthesiology to provide anaesthesia during surgical procedures. Numerous studies have shown that these volatile anaesthetics have anti-inflammatory and organ protective effects partly attributable to an inhibition of neutrophil activation (Hu et al., 2005). We developed in vitro experimental models to study the effect of volatile anaesthetics on the degranulation of total and active MPO in whole blood and their interaction with the activity of pure MPO. We previously demonstrated that sevoflurane inhibits the degranulation and the activity of equine MPO (Minguet et al., 2013). However, recent investigations have suggested that the immunomodulatory properties of volatile anaesthetics are pharmacological class effects imputable to their common halogenated carbon groups (Urner et al., 2011). Therefore, we hypothesized that isoflurane, a halogenated anaesthetic chemically and pharmacologically related to sevoflurane, could similarly inhibit the horse MPO system. The objective of this study was to evaluate the effects of isoflurane and sevoflurane on the degranulation of total and active MPO from unstimulated and stimulated horse PMNs in a whole blood model. In addition, we evaluated the potential interaction of the volatile agents with the peroxidase and chlorination cycles of pure solubilized MPO. 2. Material and methods 2.1. Blood samples Samples of whole blood were collected from adult healthy horses (n = 14) by jugular venipuncture in EDTA tubes in compliance with institutional ethical guidelines for animal investigations. Blood samples were put in a six-well (2 mL per well) cell culture plate (Nunclon delta surface; Nunc, Denmark) before exposure to isoflurane or sevoflurane. 2.2. Exposure to volatile anaesthetics Isoflurane and sevoflurane (Forene® and Sevorane® , Abbott Laboratories, Wavre, Belgium) were administered to reproduce duration and doses susceptible to be used in the practice of clinical anaesthesia. According to a minimal alveolar concentration (MAC) of 1.3% for isoflurane and 2.3% for sevoflurane, the volatile anaesthetics were administered at 1 and 2 MAC for 1 h at 37 ◦ C as previously described (Minguet et al., 2013). Briefly, treated blood samples were placed in a tight incubator chamber (Billups-Rothenberg, Del Mar, CA) and isoflurane or sevoflurane was delivered via a specific calibrated vaporizer (Dräger, Lübeck, Germany) supplied with air as the carrier gas and anaesthestic concentrations were measured and adjusted with a Capnomac Ultima® multigas analyzer (Datex Ohmeda, Helsinki, Finland). Untreated control blood
samples were placed in a tight incubator filled with air at 37 ◦ C and without volatile anaesthetics. 2.3. Activation of neutrophils in whole blood Polymorphonuclear neutrophils were stimulated as already described with solutions of cytochalasin B (CB) in 100% dimethylsulfoxide (DMSO) and N-formylmethionylleucyl-phenylalanine (fMLP) in 10% DMSO to reach the final concentration of 5 g/mL CB and 10−6 mol/L fMLP in whole blood (Ceusters et al., 2012). The effects of the vehicle DMSO solutions were also studied as controls. Our method for PMNs activation consisted of an incubation of whole blood with CB (5 g/mL) as a priming agent under 1-h exposure to isoflurane, sevoflurane or air, followed by an additional activation of 30 min with fMLP (10−6 mol/L). Control tests were performed in parallel with unstimulated blood samples. Samples were poured in culture plates with large well diameter (5 cm) and all experiments were conducted on a rotating base allowing a slight stirring of the content to improve the diffusion of the volatile anaesthetics and stimulants, and to avoid cell sedimentation. 2.4. Measurement of total and active MPO released in plasma After the 90-min anaesthetic incubation and neutrophil stimulation, blood samples were centrifuged (450 × g, 10 min) and plasma was collected to measure the total and active MPO. An ELISA assay (Equine MPO ELISA, BioPtis, Belgium) was used to measure total equine MPO in plasma (Franck et al., 2005). Before the ELISA assays, the plasma was diluted 100-fold with 20 mmol/L PBS buffer at pH 7.4 and added with 5 g/L BSA and 0.1% Tween 20. The active MPO released by neutrophils in plasma was measured with a Specific Immunological Extraction Followed by Enzymatic Detection (SIEFED) developed for the specific quantification of active equine neutrophil MPO (Franck et al., 2006). Briefly, this technique consists of the extraction of MPO from plasma by its capture on specific immobilized antibodies, followed by a washing step to eliminate non-specifically bound compounds and interfering substances. Finally, the peroxidase activity of MPO is detected in situ by Amplex red as a fluorogenic substrate in the presence of a nitrite-based amplifier system. For the SIEFED assay, plasma samples were not diluted. 2.5. Measurement of the activity of purified MPO To study the direct effect of the volatile anaesthetics on the peroxidase and chlorination activity of MPO, we added the specific substrates required to detect enzymatic reactions immediately after exposure of a purified MPO solution to isoflurane or sevoflurane. Purified equine MPO was diluted in phosphate buffer (50 mmol/L, pH 7.5) to a final concentration of 230 ng/mL and incubated in microplate wells (100 L per well) in air or in air plus 1 and 2 MAC of isoflurane or sevoflurane for 1 h at 37 ◦ C and under a slight stirring. Thereafter, the peroxidase activity of MPO was directly measured by adding 100 L of a 40 mmol/L Amplex red solution freshly prepared
Please cite this article in press as: Minguet, G., et al., Effects of isoflurane and sevoflurane on the neutrophil myeloperoxidase system of horses. Vet. Immunol. Immunopathol. (2015), http://dx.doi.org/10.1016/j.vetimm.2015.02.010
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in phosphate buffer (50 mmol/L at pH 7.5) containing 10 mol/L H2 O2 . The chlorination activity of MPO was measured directly by adding 100 L of a 20 mmol/L solution of 3 -(p-aminophenyl) fluorescein (APF) freshly prepared in sodium acetate buffer (25 mmol/L, pH 5.5) containing 137 mmol/L NaCl and 20 mol/L H2 O2 . To assess the interaction of isoflurane and sevoflurane with MPO, we used the SIEFED as a pharmacological tool (Kohnen et al., 2007; Franck et al., 2008). This method allows the washout of the tested pharmacological compound from the milieu before the measurement of the residual peroxidase activity of MPO. Purified equine MPO (120 ng/mL) diluted in 20 mmol/L PBS at pH 7.4, added with 5 g/L BSA and 0.1% Tween 20 was put in microplate wells (100 L per well) coated with anti-equine MPO antibodies. Then, microplates were incubated in air or air plus 1 and 2 MAC of isoflurane or sevoflurane for 1 h at 37 ◦ C and under a slight stirring. After incubation, the MPO solution was eliminated by four washes to eliminate unbound MPO and the anaesthetic in solution, then 100 L of a 40 mmol/L Amplex red solution freshly prepared in phosphate buffer (50 mmol/L at pH 7.5) containing 10 mol/L H2 O2 was added to the wells to measure the residual peroxidase activity of MPO. 2.6. Statistical analysis Data are presented as relative values (%) in reference to the corresponding control group in air defined as 100%. All data are expressed as medians and quartiles of at least three independent experiments carried out at least in triplicate with different blood batches from healthy horses (n > 9). For the quantification of MPO activity, the experiments were replicated at least 14 times with the same batch of purified equine MPO. Comparison between groups was performed with the software GraphPad Prism 6 (GraphPad software, San Diego, CA, USA) by a Kruskal–Wallis test with Dunn’s post hoc test for multiple comparisons. A p-value 0.05) nor in active (+7.3%, p > 0.05) MPO release from PMNs. As compared with the corresponding conditions in air, 1 MAC isoflurane increased by 9.3% (p < 0.05) the release of total MPO by unstimulated PMNs but had no effect on total MPO release by stimulated neutrophils. At 2 MAC, isoflurane did not change the total MPO release by both unstimulated and stimulated neutrophils. As compared with the corresponding conditions in air, 1 MAC sevoflurane had no effect on the release of total MPO by unstimulated neutrophils but increased by 42.0% (p < 0.05) the total MPO release by stimulated neutrophils. At 2 MAC, sevoflurane reduced by 27.9% (p < 0.05) the total MPO release by unstimulated neutrophils but had no effect when neutrophils were stimulated (Fig. 1). The opportunity to measure the active fraction of MPO after neutrophil activation is important on a pathophysiological and therapeutic perspective because during inflammation, it is the active fraction and not the total one which is potentially responsible for tissue injuries. As compared with the corresponding conditions in air, 1 MAC isoflurane reduced by 45.8% (p < 0.05) and by 38.5% (p < 0.05) the release of active MPO by unstimulated and stimulated neutrophils respectively. However, 2 MAC isoflurane had no effect on active MPO release either by unstimulated and stimulated neutrophils. Sevoflurane, at 1 MAC, reduced by 66.8% (p < 0.05) the release of active MPO by unstimulated
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Fig. 1. Effect of isoflurane (Iso) and sevoflurane (Sevo) on the release of total MPO by unstimulated (UNST) and CB + fMLP-stimulated neutrophils. Results are expressed as relative values (%) of the corresponding control group in air considered as 100%. Data are presented as medians and quartiles (interquartile range, min.–max.) of at least three independent experiments carried out at least in triplicate (n > 9). *p < 0.05 versus the same treatment group in air.
neutrophils but had no effect when neutrophils were stimulated. Furthermore, 2 MAC sevoflurane reduced by respectively 49.5% (p < 0.05) and 63.0% (p < 0.05) the release of active MPO by unstimulated and stimulated cells (Fig. 2). These results are consistent with previous studies conducted with isolated PMNs that demonstrated a direct and reversible inhibition of neutrophil functions by volatile anaesthetics acting at specific levels of the cellular transduction pathway (Fröhlich et al., 1997; Saad et al., 2010). In this work, volatile anaesthetics mainly showed a reduction of the active MPO fraction rather than the total one. Particularly, isoflurane at 1 MAC and sevoflurane at 2 MAC decreased active MPO degranulation not only by non-stimulated but also by stimulated neutrophils. Such prominent effect on active MPO could be attributable to a direct interaction of volatile anaesthetics with the enzyme activity. In the second part of our study, we tested the hypothesis that isoflurane and sevoflurane can directly inhibit the
Fig. 2. Effect of isoflurane (Iso) and sevoflurane (Sevo) on the release of active MPO by unstimulated (UNST) and CB + fMLP-stimulated neutrophils. Results are expressed as relative values (%) of the corresponding control group in air considered as 100%. Data are presented as medians and quartiles (interquartile range, min.–max.) of at least three independent experiments carried out at least in triplicate (n > 9). *p < 0.05 versus the same treatment group in air.
Fig. 3. Effect of isoflurane (Iso) and sevoflurane (Sevo) on the peroxidase and chlorination activity of equine purified MPO. Results are expressed as relative values (%) of the corresponding control group in air considered as 100%. Data are presented as medians and quartiles (interquartile range, min.–max.) of at least 14 independent experiments (n > 14). *p < 0.05 versus air.
peroxidase or chlorination activity of purified equine MPO. As compared with the controls in air, isoflurane reduced the peroxidase activity of MPO by 13.0% (p < 0.05) at 1 MAC and by 9.8% (p < 0.05) at 2 MAC. In addition, the reduction of chlorination activity by isoflurane was 5.8% (p < 0.05) at 1 MAC but non-significant at 2 MAC. As compared with the controls in air, sevoflurane reduced the peroxidase activity of MPO by 24.0% (p < 0.05) at 1 MAC and by 38.7% (p < 0.05) at 2 MAC. Sevoflurane had no significant effect on the chlorination activity at 1 MAC, but it reduced this activity by 18.4% (p < 0.05) at 2 MAC (Fig. 3). To specify the interaction between MPO and the anaesthetics, we conducted a SIEFED assay with pure MPO consisting of its capture by specific antibodies followed by washing to eliminate isoflurane and sevoflurane from the MPO suspension before enzymatic revelation. No persistent residual effect of the volatile anaesthetics was observed on the peroxidase activity of the immunocaptured MPO, except for isoflurane at 2 MAC that exhibited a slight but significant (5.3%, p < 0.05) inhibition (Fig. 4). From these observations, it is unlikely that inhibition of MPO activity by anaesthetics is related to an irreversible alteration (i.e. by covalent chemical bonds) of the active site of the enzyme following isoflurane or sevoflurane exposure. However, volatile anaesthetics can establish non-covalent electrostatic bonds (amphiphilic interactions) with specific functional domains of certain proteins and enzymes involved in the immune response, leading to a reversible biological inactivation (Yuki et al., 2008, 2010). It is plausible that isoflurane and sevoflurane induce similar conformational changes within the active site of MPO and alter electron transfer of the native ferric state, compound I and compound II involved in peroxidase and chlorination reactions. Pharmacodynamic studies have emphasized the importance of halogen moieties to explain volatile anaesthetics properties including immunomodulation (Eckenhoff and Johansson, 1997; Urner et al., 2011). Isoflurane and sevoflurane share identical trifluorinated groups (CF3 ) consistent with common pharmacological class effects. However, slight structural differences between both molecules may explain for their bit different dose effects on MPO degranulation and on MPO activity observed in the present study. Concentrations of
Please cite this article in press as: Minguet, G., et al., Effects of isoflurane and sevoflurane on the neutrophil myeloperoxidase system of horses. Vet. Immunol. Immunopathol. (2015), http://dx.doi.org/10.1016/j.vetimm.2015.02.010
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Fig. 4. Effect of isoflurane (Iso) and sevoflurane (Sevo) on the residual peroxidase activity of equine purified MPO. Results are expressed as relative values (%) of the corresponding control group in air considered as 100%. Data are presented as medians and quartiles (interquartile range, min.–max.) of at least 14 independent experiments (n > 14). *p < 0.05 versus air.
volatile anaesthetics are important issues when investigating their pharmacological activity. This study was not designed to assess a dose-dependent effect of isoflurane and sevoflurane which would have required evaluation of a wide range of anaesthetic concentrations. We investigated the effects of isoflurane and sevoflurane at 1 and 2 MAC because these concentrations are clinically pertinent and may be representative of the potential impact of the volatile anaesthetics on horse MPO degranulation and activity in the daily practice of anaesthesia. However, in vivo studies are warranted to examine the clinical significance of these findings. In summary, we demonstrated that isoflurane and sevoflurane, two commonly used volatile agents for inhalational anaesthesia, comparably interact with the neutrophil MPO system of horses. Although isoflurane and sevoflurane did not show a straightforward effect on total MPO release, both anaesthestics reduced active MPO degranulation. These volatile agents directly inhibited the peroxidase and, to a lower extent, the chlorination activity of the enzyme, although the interaction with MPO was more persistent with isoflurane. Further studies are warranted to precise the mechanistic basis as well as the clinical relevance of these observations. Conflict of interest The authors declare no conflict of interest. References Ceusters, J.D., Serteyn, D.A., Minguet, G., de la Rebière de Pouyade, G., Romainville, J., Deby-Dupont, G.P., Mouithys-Mickalad, A.A., Franck, T.J., 2012. An in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils. Vet. Immunol. Immunopathol. 150, 221–227. Davies, M.J., Hawkins, C.L., Pattison, D.I., Rees, M.D., 2008. Mammalian heme peroxidases: from molecular mechanisms to health implications. Antioxid. Redox Signal. 10, 1199–1234.
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Deby-Dupont, G., Deby, C., Lamy, M., 1999. Neutrophil myeloperoxidase revisited: its role in health and disease. Intensivmed. Notfallmed. 36, 500–513. de la Rebiere, G., Franck, T., Deby-Dupont, G., Salciccia, A., Grulke, S., Péters, F., Serteyn, D., 2008. Effects of unfractionated and fractionated heparins on myeloperoxidase activity and interactions with endothelial cells: possible effects on the pathophysiology of equine laminitis. Vet. J. 178, 62–69. Eckenhoff, R.G., Johansson, J.S., 1997. Molecular interactions between inhaled anesthetics and proteins. Pharmacol. Rev. 49, 343–367. Franck, T., Grulke, S., Deby-Dupont, G., Deby, C., Duvivier, H., Peters, F., Serteyn, D., 2005. Development of an enzyme-linked immunosorbent assay for specific equine neutrophil myeloperoxidase measurement in blood. J. Vet. Diagn. Invest. 17, 412–419. Franck, T., Kohnen, S., Deby-Dupont, G., Grülke, S., Deby, C., Serteyn, D., 2006. A specific method for measurement of equine active myeloperoxidase in biological samples and in in vitro tests. J. Vet. Diagn. Invest. 18, 326–334. Franck, T., Kohnen, S., Grulke, S., Neven, P., Goutman, Y., Peters, F., Pirotte, B., Deby-Dupont, G., Serteyn, D., 2008. Inhibitory effect of curcuminoids and tetrahydrocurcuminoids on equine activated neutrophils and myeloperoxidase activity. Physiol. Res. 57, 577–587. Fröhlich, D., Rothe, G., Schwall, B., Schmid, P., Schmitz, G., Taeger, K., Hobbhahn, J., 1997. Effects of volatile anaesthetics on human neutrophil oxidative response to the bacterial peptide FMLP. Br. J. Anaesth. 78, 718–723. Grulke, S., Franck, T., Gangl, M., Péters, F., Salciccia, A., Deby-Dupont, G., Serteyn, D., 2008. Myeloperoxidase assay in plasma and peritoneal fluid of horses with gastrointestinal disease. Can. J. Vet. Res. 72, 37–42. Hu, G., Salem, M.R., Crystal, G.J., 2005. Role of adenosine receptors in volatile anesthetic preconditioning against neutrophil-induced contractile dysfunction in isolated rat hearts. Anesthesiology 103, 287–295. Klebanoff, S.J., 2005. Myeloperoxidase: friend and foe. J. Leukoc. Biol. 77, 598–625. Kohnen, S., Franck, T., Van Antwerpen, P., Boudjeltia, K.Z., MouithysMickalad, A., Deby, C., Moguilevsky, N., Deby-Dupont, G., Lamy, M., Serteyn, D., 2007. Resveratrol inhibits the activity of equine neutrophil myeloperoxidase by a direct interaction with the enzyme. J. Agric. Food Chem. 55, 8080–8087. Le, Y., Murphy, P.M., Wang, J.M., 2002. Formyl-peptide receptors revisited. Trends Immunol. 23, 541–548. Malle, E., Furtmüller, P.G., Sattler, W., Obinger, C., 2007. Myeloperoxidase: a target for new drug development? Br. J. Pharmacol. 152, 838–854. Minguet, G., de la Rebière, G., Franck, T., Joris, J., Serteyn, D., Sandersen, C., 2013. Sevoflurane inhibits equine myeloperoxidase release and activity in vitro. Vet. Anaesth. Analg. 40, 166–175. Nakagawara, M., Takeshige, K., Takamatsu, J., Takahashi, S., Yoshitake, J., Minakami, S., 1986. Inhibition of superoxide production and Ca2+ mobilization in human neutrophils by halothane, enflurane, and isoflurane. Anesthesiology 64, 4–12. Saad, M.M., Eom, W., Hu, G., Kim, S.J., Crystal, G.J., 2010. Persistency and pathway of isoflurane-induced inhibition of superoxide production by neutrophils. Can. J. Anaesth. 57, 50–57. Serteyn, D., Sandersen, C., Lejeune, J.P., de la Rebière de Pouyade, G., Ceusters, J., Mouithys-Mickalad, A., Niesten, A., Fraipont, A., van Erck, E., Goachet, A.G., Robert, C., Leclerc, J.L., Votion, D.M., Franck, T., 2010. Effect of a 120 km endurance race on plasma and muscular neutrophil elastase and myeloperoxidase concentrations in horses. Equine Vet. J. 38 (Suppl.), 275–279. Strum, D.P., Eger, E.I., 1987. Partition coefficients for sevoflurane in human blood, saline, and olive oil. Anesth. Analg. 66, 654–656. Tay, S.P., Cheong, S.K., Hamidah, N.H., Ainoon, O., 1998. Flow cytometric analysis of intracellular myeloperoxidase distinguishes lymphocytes, monocytes and granulocytes. Malays. J. Pathol. 20, 91–94. Urner, M., Limbach, L.K., Herrmann, I.K., Müller-Edenborn, B., RothZ’Graggen, B., Schlicker, A., Reyes, L., Booy, C., Hasler, M., Stark, W.J., Beck-Schimmer, B., 2011. Fluorinated groups mediate the immunomodulatory effects of volatile anesthetics in acute cell injury. Am. J. Respir. Cell Mol. Biol. 45, 617–624. Yuki, K., Astrof, N.S., Bracken, C., Yoo, R., Silkworth, W., Soriano, S.G., Shimaoka, M., 2008. The volatile anesthetic isoflurane perturbs conformational activation of integrin LFA-1 by binding to the allosteric regulatory cavity. FASEB J. 22, 4109–4116. Yuki, K., Astrof, N.S., Bracken, C., Soriano, S.G., Shimaoka, M., 2010. Sevoflurane binds and allosterically blocks integrin lymphocyte function-associated antigen-1. Anesthesiology 113, 600–609.
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Short communication
Effects of isoflurane and sevoflurane on the neutrophil myeloperoxidase system of horses Grégory Minguet a,∗ , Thierry Franck b , Jean Joris a , Justine Ceusters b , Ange Mouithys-Mickalad b , Didier Serteyn b,c , Charlotte Sandersen c a
Department of Anaesthesia and Intensive Care Medicine, CHU de Liège, Domaine Universitaire du Sart-Tilman, 4000 Liège, Belgium Center for Oxygen Research and Development, Institute of Chemistry B6a, University of Liège, Domaine Universitaire du Sart-Tilman, 4000 Liège, Belgium c Department of Clinical Sciences, Anaesthesiology and Equine Surgery, Faculty of Veterinary Medicine, University of Liège, Domaine Universitaire du Sart-Tilman, 4000 Liège, Belgium b
a r t i c l e
i n f o
Article history: Received 18 November 2014 Received in revised form 10 February 2015 Accepted 26 February 2015 Keywords: Volatile anaesthetics Isoflurane Sevoflurane Neutrophil Myeloperoxidase Oxidative stress
a b s t r a c t Volatile anaesthestics have shown to modulate the oxidative response of polymorphonuclear neutrophils (PMNs). We investigated the effects of isoflurane and sevoflurane on the degranulation of total and active myeloperoxidase (MPO) from horse PMNs and their direct interaction with MPO activity. Whole blood from horse was incubated in 1 and 2 minimal alveolar concentrations (MAC) of isoflurane or sevoflurane for 1 h and PMNs were stimulated with cytochalasin B (CB) plus N-formyl-méthionyl-leucyl-phenylalanine (fMLP). After stimulation, the plasma was collected to measure total and active MPO by enzymelinked immunosorbent assay (ELISA) and specific immunological extraction followed by enzymatic detection (SIEFED) respectively. The effects of 1 and 2 MAC of isoflurane and sevoflurane on the peroxidase and chlorination activity of pure MPO were assessed by fluorescence using Amplex red and 3 -(p-aminophenyl) fluorescein (APF) respectively and in parallel with a SIEFED assay to estimate the potential interaction of the anaesthetics with the enzyme. Although isoflurane and sevoflurane had inconsistent effects on total MPO release, both volatile agents reduced active MPO release and showed a direct inhibition on the peroxidase and the chlorination activity of the enzyme. A persistent interaction between MPO and anaesthetics was evidenced with isoflurane but not with sevoflurane. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Myeloperoxidase (MPO) is a pro-oxidant haemic enzyme from azurophilic granules of polymorphonuclear neutrophils (PMNs) which represent a pivotal component of the neutrophil machinery involved in the clearance of host damaged tissues and exogenous invading microorganisms (Deby-Dupont et al., 1999). During neutrophil
∗ Corresponding author. Tel.: +32 4 366 71 80. E-mail address: [email protected] (G. Minguet).
phagocytosis, most of these processes are confined to the phagolysosome, leading to effective intracellular destruction of tissue debris and microbial pathogens. However, activated PMNs also release MPO in the extracellular space, which results in collateral damages to healthy host structures. The cytotoxic potential of MPO is attributable to its dual peroxidase and chlorination activities able to inactivate multiple biological molecules including proteins, lipids and nucleic acids. It is now recognized that extracellular MPO not only represents a marker of neutrophil activation but also plays a role in collateral injuries encountered during the course of inflammatory diseases (Davies
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et al., 2008; Klebanoff, 2005). Hence, our group demonstrated that MPO has a pathophysiological significance in various clinical conditions associated with acute PMNs stimulation like i.e. intestinal ischaemia (Grulke et al., 2008), laminitis (de la Rebiere et al., 2008) or sustained physical exercise (Serteyn et al., 2010). Volatile anaesthetics such as sevoflurane and isoflurane are commonly used in veterinary anaesthesiology to provide anaesthesia during surgical procedures. Numerous studies have shown that these volatile anaesthetics have anti-inflammatory and organ protective effects partly attributable to an inhibition of neutrophil activation (Hu et al., 2005). We developed in vitro experimental models to study the effect of volatile anaesthetics on the degranulation of total and active MPO in whole blood and their interaction with the activity of pure MPO. We previously demonstrated that sevoflurane inhibits the degranulation and the activity of equine MPO (Minguet et al., 2013). However, recent investigations have suggested that the immunomodulatory properties of volatile anaesthetics are pharmacological class effects imputable to their common halogenated carbon groups (Urner et al., 2011). Therefore, we hypothesized that isoflurane, a halogenated anaesthetic chemically and pharmacologically related to sevoflurane, could similarly inhibit the horse MPO system. The objective of this study was to evaluate the effects of isoflurane and sevoflurane on the degranulation of total and active MPO from unstimulated and stimulated horse PMNs in a whole blood model. In addition, we evaluated the potential interaction of the volatile agents with the peroxidase and chlorination cycles of pure solubilized MPO. 2. Material and methods 2.1. Blood samples Samples of whole blood were collected from adult healthy horses (n = 14) by jugular venipuncture in EDTA tubes in compliance with institutional ethical guidelines for animal investigations. Blood samples were put in a six-well (2 mL per well) cell culture plate (Nunclon delta surface; Nunc, Denmark) before exposure to isoflurane or sevoflurane. 2.2. Exposure to volatile anaesthetics Isoflurane and sevoflurane (Forene® and Sevorane® , Abbott Laboratories, Wavre, Belgium) were administered to reproduce duration and doses susceptible to be used in the practice of clinical anaesthesia. According to a minimal alveolar concentration (MAC) of 1.3% for isoflurane and 2.3% for sevoflurane, the volatile anaesthetics were administered at 1 and 2 MAC for 1 h at 37 ◦ C as previously described (Minguet et al., 2013). Briefly, treated blood samples were placed in a tight incubator chamber (Billups-Rothenberg, Del Mar, CA) and isoflurane or sevoflurane was delivered via a specific calibrated vaporizer (Dräger, Lübeck, Germany) supplied with air as the carrier gas and anaesthestic concentrations were measured and adjusted with a Capnomac Ultima® multigas analyzer (Datex Ohmeda, Helsinki, Finland). Untreated control blood
samples were placed in a tight incubator filled with air at 37 ◦ C and without volatile anaesthetics. 2.3. Activation of neutrophils in whole blood Polymorphonuclear neutrophils were stimulated as already described with solutions of cytochalasin B (CB) in 100% dimethylsulfoxide (DMSO) and N-formylmethionylleucyl-phenylalanine (fMLP) in 10% DMSO to reach the final concentration of 5 g/mL CB and 10−6 mol/L fMLP in whole blood (Ceusters et al., 2012). The effects of the vehicle DMSO solutions were also studied as controls. Our method for PMNs activation consisted of an incubation of whole blood with CB (5 g/mL) as a priming agent under 1-h exposure to isoflurane, sevoflurane or air, followed by an additional activation of 30 min with fMLP (10−6 mol/L). Control tests were performed in parallel with unstimulated blood samples. Samples were poured in culture plates with large well diameter (5 cm) and all experiments were conducted on a rotating base allowing a slight stirring of the content to improve the diffusion of the volatile anaesthetics and stimulants, and to avoid cell sedimentation. 2.4. Measurement of total and active MPO released in plasma After the 90-min anaesthetic incubation and neutrophil stimulation, blood samples were centrifuged (450 × g, 10 min) and plasma was collected to measure the total and active MPO. An ELISA assay (Equine MPO ELISA, BioPtis, Belgium) was used to measure total equine MPO in plasma (Franck et al., 2005). Before the ELISA assays, the plasma was diluted 100-fold with 20 mmol/L PBS buffer at pH 7.4 and added with 5 g/L BSA and 0.1% Tween 20. The active MPO released by neutrophils in plasma was measured with a Specific Immunological Extraction Followed by Enzymatic Detection (SIEFED) developed for the specific quantification of active equine neutrophil MPO (Franck et al., 2006). Briefly, this technique consists of the extraction of MPO from plasma by its capture on specific immobilized antibodies, followed by a washing step to eliminate non-specifically bound compounds and interfering substances. Finally, the peroxidase activity of MPO is detected in situ by Amplex red as a fluorogenic substrate in the presence of a nitrite-based amplifier system. For the SIEFED assay, plasma samples were not diluted. 2.5. Measurement of the activity of purified MPO To study the direct effect of the volatile anaesthetics on the peroxidase and chlorination activity of MPO, we added the specific substrates required to detect enzymatic reactions immediately after exposure of a purified MPO solution to isoflurane or sevoflurane. Purified equine MPO was diluted in phosphate buffer (50 mmol/L, pH 7.5) to a final concentration of 230 ng/mL and incubated in microplate wells (100 L per well) in air or in air plus 1 and 2 MAC of isoflurane or sevoflurane for 1 h at 37 ◦ C and under a slight stirring. Thereafter, the peroxidase activity of MPO was directly measured by adding 100 L of a 40 mmol/L Amplex red solution freshly prepared
Please cite this article in press as: Minguet, G., et al., Effects of isoflurane and sevoflurane on the neutrophil myeloperoxidase system of horses. Vet. Immunol. Immunopathol. (2015), http://dx.doi.org/10.1016/j.vetimm.2015.02.010
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in phosphate buffer (50 mmol/L at pH 7.5) containing 10 mol/L H2 O2 . The chlorination activity of MPO was measured directly by adding 100 L of a 20 mmol/L solution of 3 -(p-aminophenyl) fluorescein (APF) freshly prepared in sodium acetate buffer (25 mmol/L, pH 5.5) containing 137 mmol/L NaCl and 20 mol/L H2 O2 . To assess the interaction of isoflurane and sevoflurane with MPO, we used the SIEFED as a pharmacological tool (Kohnen et al., 2007; Franck et al., 2008). This method allows the washout of the tested pharmacological compound from the milieu before the measurement of the residual peroxidase activity of MPO. Purified equine MPO (120 ng/mL) diluted in 20 mmol/L PBS at pH 7.4, added with 5 g/L BSA and 0.1% Tween 20 was put in microplate wells (100 L per well) coated with anti-equine MPO antibodies. Then, microplates were incubated in air or air plus 1 and 2 MAC of isoflurane or sevoflurane for 1 h at 37 ◦ C and under a slight stirring. After incubation, the MPO solution was eliminated by four washes to eliminate unbound MPO and the anaesthetic in solution, then 100 L of a 40 mmol/L Amplex red solution freshly prepared in phosphate buffer (50 mmol/L at pH 7.5) containing 10 mol/L H2 O2 was added to the wells to measure the residual peroxidase activity of MPO. 2.6. Statistical analysis Data are presented as relative values (%) in reference to the corresponding control group in air defined as 100%. All data are expressed as medians and quartiles of at least three independent experiments carried out at least in triplicate with different blood batches from healthy horses (n > 9). For the quantification of MPO activity, the experiments were replicated at least 14 times with the same batch of purified equine MPO. Comparison between groups was performed with the software GraphPad Prism 6 (GraphPad software, San Diego, CA, USA) by a Kruskal–Wallis test with Dunn’s post hoc test for multiple comparisons. A p-value 0.05) nor in active (+7.3%, p > 0.05) MPO release from PMNs. As compared with the corresponding conditions in air, 1 MAC isoflurane increased by 9.3% (p < 0.05) the release of total MPO by unstimulated PMNs but had no effect on total MPO release by stimulated neutrophils. At 2 MAC, isoflurane did not change the total MPO release by both unstimulated and stimulated neutrophils. As compared with the corresponding conditions in air, 1 MAC sevoflurane had no effect on the release of total MPO by unstimulated neutrophils but increased by 42.0% (p < 0.05) the total MPO release by stimulated neutrophils. At 2 MAC, sevoflurane reduced by 27.9% (p < 0.05) the total MPO release by unstimulated neutrophils but had no effect when neutrophils were stimulated (Fig. 1). The opportunity to measure the active fraction of MPO after neutrophil activation is important on a pathophysiological and therapeutic perspective because during inflammation, it is the active fraction and not the total one which is potentially responsible for tissue injuries. As compared with the corresponding conditions in air, 1 MAC isoflurane reduced by 45.8% (p < 0.05) and by 38.5% (p < 0.05) the release of active MPO by unstimulated and stimulated neutrophils respectively. However, 2 MAC isoflurane had no effect on active MPO release either by unstimulated and stimulated neutrophils. Sevoflurane, at 1 MAC, reduced by 66.8% (p < 0.05) the release of active MPO by unstimulated
Please cite this article in press as: Minguet, G., et al., Effects of isoflurane and sevoflurane on the neutrophil myeloperoxidase system of horses. Vet. Immunol. Immunopathol. (2015), http://dx.doi.org/10.1016/j.vetimm.2015.02.010
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Fig. 1. Effect of isoflurane (Iso) and sevoflurane (Sevo) on the release of total MPO by unstimulated (UNST) and CB + fMLP-stimulated neutrophils. Results are expressed as relative values (%) of the corresponding control group in air considered as 100%. Data are presented as medians and quartiles (interquartile range, min.–max.) of at least three independent experiments carried out at least in triplicate (n > 9). *p < 0.05 versus the same treatment group in air.
neutrophils but had no effect when neutrophils were stimulated. Furthermore, 2 MAC sevoflurane reduced by respectively 49.5% (p < 0.05) and 63.0% (p < 0.05) the release of active MPO by unstimulated and stimulated cells (Fig. 2). These results are consistent with previous studies conducted with isolated PMNs that demonstrated a direct and reversible inhibition of neutrophil functions by volatile anaesthetics acting at specific levels of the cellular transduction pathway (Fröhlich et al., 1997; Saad et al., 2010). In this work, volatile anaesthetics mainly showed a reduction of the active MPO fraction rather than the total one. Particularly, isoflurane at 1 MAC and sevoflurane at 2 MAC decreased active MPO degranulation not only by non-stimulated but also by stimulated neutrophils. Such prominent effect on active MPO could be attributable to a direct interaction of volatile anaesthetics with the enzyme activity. In the second part of our study, we tested the hypothesis that isoflurane and sevoflurane can directly inhibit the
Fig. 2. Effect of isoflurane (Iso) and sevoflurane (Sevo) on the release of active MPO by unstimulated (UNST) and CB + fMLP-stimulated neutrophils. Results are expressed as relative values (%) of the corresponding control group in air considered as 100%. Data are presented as medians and quartiles (interquartile range, min.–max.) of at least three independent experiments carried out at least in triplicate (n > 9). *p < 0.05 versus the same treatment group in air.
Fig. 3. Effect of isoflurane (Iso) and sevoflurane (Sevo) on the peroxidase and chlorination activity of equine purified MPO. Results are expressed as relative values (%) of the corresponding control group in air considered as 100%. Data are presented as medians and quartiles (interquartile range, min.–max.) of at least 14 independent experiments (n > 14). *p < 0.05 versus air.
peroxidase or chlorination activity of purified equine MPO. As compared with the controls in air, isoflurane reduced the peroxidase activity of MPO by 13.0% (p < 0.05) at 1 MAC and by 9.8% (p < 0.05) at 2 MAC. In addition, the reduction of chlorination activity by isoflurane was 5.8% (p < 0.05) at 1 MAC but non-significant at 2 MAC. As compared with the controls in air, sevoflurane reduced the peroxidase activity of MPO by 24.0% (p < 0.05) at 1 MAC and by 38.7% (p < 0.05) at 2 MAC. Sevoflurane had no significant effect on the chlorination activity at 1 MAC, but it reduced this activity by 18.4% (p < 0.05) at 2 MAC (Fig. 3). To specify the interaction between MPO and the anaesthetics, we conducted a SIEFED assay with pure MPO consisting of its capture by specific antibodies followed by washing to eliminate isoflurane and sevoflurane from the MPO suspension before enzymatic revelation. No persistent residual effect of the volatile anaesthetics was observed on the peroxidase activity of the immunocaptured MPO, except for isoflurane at 2 MAC that exhibited a slight but significant (5.3%, p < 0.05) inhibition (Fig. 4). From these observations, it is unlikely that inhibition of MPO activity by anaesthetics is related to an irreversible alteration (i.e. by covalent chemical bonds) of the active site of the enzyme following isoflurane or sevoflurane exposure. However, volatile anaesthetics can establish non-covalent electrostatic bonds (amphiphilic interactions) with specific functional domains of certain proteins and enzymes involved in the immune response, leading to a reversible biological inactivation (Yuki et al., 2008, 2010). It is plausible that isoflurane and sevoflurane induce similar conformational changes within the active site of MPO and alter electron transfer of the native ferric state, compound I and compound II involved in peroxidase and chlorination reactions. Pharmacodynamic studies have emphasized the importance of halogen moieties to explain volatile anaesthetics properties including immunomodulation (Eckenhoff and Johansson, 1997; Urner et al., 2011). Isoflurane and sevoflurane share identical trifluorinated groups (CF3 ) consistent with common pharmacological class effects. However, slight structural differences between both molecules may explain for their bit different dose effects on MPO degranulation and on MPO activity observed in the present study. Concentrations of
Please cite this article in press as: Minguet, G., et al., Effects of isoflurane and sevoflurane on the neutrophil myeloperoxidase system of horses. Vet. Immunol. Immunopathol. (2015), http://dx.doi.org/10.1016/j.vetimm.2015.02.010
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Fig. 4. Effect of isoflurane (Iso) and sevoflurane (Sevo) on the residual peroxidase activity of equine purified MPO. Results are expressed as relative values (%) of the corresponding control group in air considered as 100%. Data are presented as medians and quartiles (interquartile range, min.–max.) of at least 14 independent experiments (n > 14). *p < 0.05 versus air.
volatile anaesthetics are important issues when investigating their pharmacological activity. This study was not designed to assess a dose-dependent effect of isoflurane and sevoflurane which would have required evaluation of a wide range of anaesthetic concentrations. We investigated the effects of isoflurane and sevoflurane at 1 and 2 MAC because these concentrations are clinically pertinent and may be representative of the potential impact of the volatile anaesthetics on horse MPO degranulation and activity in the daily practice of anaesthesia. However, in vivo studies are warranted to examine the clinical significance of these findings. In summary, we demonstrated that isoflurane and sevoflurane, two commonly used volatile agents for inhalational anaesthesia, comparably interact with the neutrophil MPO system of horses. Although isoflurane and sevoflurane did not show a straightforward effect on total MPO release, both anaesthestics reduced active MPO degranulation. These volatile agents directly inhibited the peroxidase and, to a lower extent, the chlorination activity of the enzyme, although the interaction with MPO was more persistent with isoflurane. Further studies are warranted to precise the mechanistic basis as well as the clinical relevance of these observations. Conflict of interest The authors declare no conflict of interest. References Ceusters, J.D., Serteyn, D.A., Minguet, G., de la Rebière de Pouyade, G., Romainville, J., Deby-Dupont, G.P., Mouithys-Mickalad, A.A., Franck, T.J., 2012. An in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils. Vet. Immunol. Immunopathol. 150, 221–227. Davies, M.J., Hawkins, C.L., Pattison, D.I., Rees, M.D., 2008. Mammalian heme peroxidases: from molecular mechanisms to health implications. Antioxid. Redox Signal. 10, 1199–1234.
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