RU2794568C1 - Method for suppressing excessive neutrophil activation in an ex vivo model - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to means of therapeutic effect on the respiratory system of a patient, the main component of which is sevoflurane volatile anesthetic, the invention can be used to suppress excessive activation of neutrophils. The method includes exposure to a gas mixture, while a decrease in inflammatory hyperactivation of neutrophils in an ex vivo model is achieved by exposure to sevoflurane at a concentration of 3 vol.% corresponding to 1.5 MAC within 30 minutes. The invention can be used to treat patients with pathological conditions in which there is a systemic inflammatory response syndrome (SIRS) after major operations, surgical interventions using a heart-lung machine, concomitant trauma, severe sepsis and septic shock, tissue ischemia-reperfusion syndrome in stroke and myocardial infarction, diabetes mellitus, tumor metastasis, pulmonary edema and acute respiratory distress syndrome, burn disease, etc.

EFFECT: method allows to reduce the activity of neutrophils, reduce inflammation, prevent or reduce the severity of multiple organ failure, reduce the duration of treatment and increase the survival of patients in critical conditions.

1 cl, 3 tbl

Description

SUBSTANCE: invention relates to medicine, namely to means of therapeutic effect on the patient's respiratory system, the main component of which is the volatile anesthetic sevoflurane. The invention can be used to treat patients with pathological conditions in which there is a systemic inflammatory response syndrome (SIRS) after major operations, surgical interventions using a heart-lung machine, concomitant trauma, severe sepsis and septic shock, tissue ischemia-reperfusion syndrome in stroke and myocardial infarction, diabetes mellitus, tumor metastasis, pulmonary edema and acute respiratory distress syndrome, burn disease, etc. critical states.

State of the art

Inflammation is a typical pathological process aimed at protecting the body from infection. However, in critically ill patients, inflammation may also be a key pathogenic factor in tissue damage and progression of multiorgan dysfunction. Neutrophils are one of the main participants in the inflammatory process, and therefore their dysfunction may be of great importance in the pathogenesis of critical conditions. Thus, a number of studies have demonstrated the role of neutrophils in their progression due to additional tissue damage in sepsis, acute respiratory distress syndrome (ARDS), coagulopathy, and other pathological processes and conditions. Thus, therapy aimed at normalizing neutrophil function may be appropriate in these patients.

Currently, a large number of neutrophilic factors are known that affect infectious agents, components of the immune system, and body tissues. A number of therapeutic methods are known that affect certain links in the functioning of neutrophils: the effect on the formation of neutrophils, their release from the bone marrow, recruitment and migration, chemotaxis, activation, as well as on the main enzymes of neutrophils [Bartaula-Brevik S, Lindstad Brattas M.K. ., Tvedt T.N.A. et al. Splenic tyrosine kinase (SYK) inhibitors and their possible use in acute myeloid leukemia. Expert Opin Investig Drugs. 2018;27(4):377-387. doi:10.1080/13543784.2018.1459562; Westhovens R., Taylor P.C., Alten R. et al. Filgotinib (GLPG0634/GS-6034), an oral JAK1 selective inhibitor, is effective in combination with methotrexate (MTX) in patients with active rheumatoid arthritis and insufficient response to MTX: results from a randomised, dose-finding study (DARWIN 1). Ann Rheum Dis. 2017;76(6):998-1008. doi: 10.1136/annrheumdis-2016-210104].

However, most of the targeted drugs are characterized by certain disadvantages: insufficient selectivity of action, neutropenia and immunosuppression due to excessive suppression of neutrophil function, and others [Jonsson H., Allen P., Peng S.L. Inflammatory arthritis requires Foxo3a to prevent Fas ligand-induced neutrophil apoptosis. Nat Med. 2005;11(6):666-671. doi:10.1038/nml248, Serhan C.N., Levy B.D. Resolvins in inflammation: emergence of the pro-resolving superfamily of mediators. J Clin Invest. 2018;128(7):2657-2669. doi:10.1172/JCI97943].

There are also several therapeutic agents and methods aimed at solving the problem of eliminating excessive inflammatory activation of neutrophils.

In particular, a neutrophil activation regulator is known, including batroxobin as an active ingredient: for inhibiting neutrophil degranulation, for inhibiting Mac-1 expression by neutrophils, for inhibiting the formation of NETs by neutrophils, for inhibiting transendothelial migration of neutrophils, and also for treating diseases caused by neutrophil activation, which are selected from the group consisting of sepsis, ARDS, acute pancreatitis and acute pulmonary disorder (RU 2742417). This method involves the introduction of batroxobin - a drug with fibrinolytic properties. Contraindications to its administration are: organ dysfunction, coagulopathy, old age. The use of the known method does not give the technical results of the proposed method, since the syndrome of excessive inflammation in various pathological conditions is usually accompanied by organ dysfunction and coagulopathy.

There is also known a method for regulating the activation of neutrophils in an in vitro model by using lithium chloride (LiCl), which allows to reduce the level of neutrophil activation by reducing the level of expression of CD11b and CD66b molecules (RU 2716492). The known method is based on the molecular mechanism of action, which consists in the fact that LiCl phosphorylates (inactivates) GSK-3 beta and causes not only the inactivation of the CD-11b integrin, which is also responsible for binding to ICAM-1 cell adhesion molecules, but also the inactivation of CD- 66b, which regulates the release of granules with proteolytic enzymes from the cytosol to the surface of neutrophils, which can damage tissues during inflammation.

The drug containing LiCl belongs to substances with pronounced cytoprotective properties due to the ability to phosphorylate Ser9 molecules of glycogen synthetase kinase 3-beta (GSK3beta), leading to inhibition of enzyme activity [Klein, P.S., and Melton, D.A. (1996) A molecular mechanism for the effect of lithium on development, Proc. Natl. Acad. sci. USA, 93, 8455-8459].

The authors of the known method have shown that LiCl has an anti-inflammatory effect on human neutrophils. Such a protective effect limits the hyperproduction of pro-inflammatory cytokines, their penetration through histohematological barriers, followed by infiltration of target organ tissues by leukocytes and cytokines, which leads to the development of multiple organ failure in critical conditions, including, but not limited to, SIRS after heart and vascular surgery, SIRS in severe comorbid trauma, sepsis, pulmonary edema and ARDS, and severe burns. The use of the known method for regulating the activation of neutrophils by using LiCl does not give the technical results of the proposed method, since the pharmacological substance of the LiCl salt for intravenous administration has not been developed to date, and the clinical use of lithium salts in critical conditions seems unlikely, given its toxic effects on the central nervous system , kidneys and liver.

The closest to the claimed method in terms of the totality of matching essential features is a method for reducing inflammatory hyperactivation of neutrophils, including inhalation with a gas mixture: xenon 30 vol.%, oxygen not more than 40 vol.%, the rest is nitrogen, in a closed circuit lasting 60 minutes (RU 2758536) . EFFECT: method provides a decrease in neutrophil activity, a decrease in inflammatory processes, prevents or reduces the severity of multiple organ failure, and also reduces the duration of treatment and increases the survival rate of patients in critical conditions. adopted as a prototype.

The use of a known method for regulating the activation of neutrophils, including inhalation with a gas mixture: xenon 30 vol.%, does not give the technical results of the proposed method, since a method for the clinical use of xenon in critical conditions accompanied by the development of a hyperinflammatory response has not been developed to date. Xenon is used only intraoperatively, as an agent for anesthesia, while the inflammatory reaction develops in the postoperative period.

The claimed invention is aimed at solving the problem of expanding the arsenal of technical means for suppressing excessive neutrophil activation, namely: the possibility of using sevoflurane to correct the inflammatory process in critically ill patients, as well as determining the optimal concentrations of sevoflurane in the process of human neutrophil activation in an ex vivo model.

The task of expanding the arsenal of technical means of suppressing excessive activation of neutrophils is achieved due to the fact that, as in the known method, the reduction of inflammatory hyperactivation of neutrophils is achieved by exposure to a gas mixture.

The peculiarity of the proposed method lies in the fact that the reduction of inflammatory hyperactivation of neutrophils is achieved by exposure to sevoflurane at a dose of 3.0 vol.%, which corresponds to 1.5 minimum alveolar concentration (MAC), which is widely used in clinical practice and has no toxic effects. 1 MAC is the concentration of the anesthetic in volume percent that prevents the patient's motor response to the skin incision, for sevoflurane = 2 vol.%. [Inhalation induction and maintenance of anesthesia / V.V. Likhvantsev, K.Yu. Borisov, M.V. Gabitov and others; Under the editorship of Professor V.V. Likhvantseva. - Moscow: Publishing House "Medical Information Agency", 2013. - 320 p. - ISBN 978-5-9986-0107-1. - EDN RTMISB. https://www.elibrary.ru/item.asp?id=23431414].

Disclosure of the essence of the invention;

In view of the important role of neutrophils in the regulation of inflammation in infectious and non-infectious diseases, the search for optimal methods for determining the activation of neutrophils is an urgent issue. It is known to use CD11b and CD66b proteins as markers of neutrophil activation [Grebenchikov O.A., Kasatkina I.S., Kadantseva K.K., Meshkov M.A., Baeva A.A. Effect of LiCl on neutrophil activation by serum from patients with septic shock. General resuscitation. 2020; 16(5):45-55. https://doi.org/10.15360/1813-9779-2020-5-45-55]. These proteins are integrins that are rapidly expressed on the cytoplasmic membrane of neutrophils upon their activation, which has been demonstrated in a number of studies [Nicholson G.C., Tennant R.C., Carpenter D.C. et al. A novel flow cytometric assay of human whole blood neutrophil and monocyte CD11b levels: Upregulation by chemokines is related to receptor expression, comparison with neutrophil shape change, and effects of a chemokine receptor (CXCR2) antagonist. Pulm Pharmacol Ther. 2007; 20(1):52-59. doi:10.1016/j.pupt.2005.11.009, Schmidt T., Brodesser A., Schnitzler N. et al. CD66b Overexpression and Loss of C5a Receptors as Surface Markers for Staphylococcus aureus-Induced Neutrophil Dysfunction. Akiyama T, ed. PLOS ONE. 2015; 10(7):e0132703. doi: 10.137l/journal.pone.0132703 and others].

Among other markers of neutrophil activation, phosphorylated glycogen synthase kinase-3β (phospho-HSC-3β) can be noted. GSK-3 is a serine protease that is involved in the regulation of various cellular processes, and this enzyme regulates not only the synthesis of glycogen, but also demonstrated its role in the regulation of the cell cycle (cell division) and a large number of different signaling pathways. Both isoforms of this enzyme, GSK-3α and GSK-3β, are found in neutrophils, but their functions in these cells are not well understood. Nevertheless, phosphorylation of both isoenzymes has been shown to be an important regulatory mechanism in neutrophils. Thus, the amount of phospho-HSC-3β in neutrophils may reflect their activity [Ostroumova O.D., Kochetkov A.I., Pavleeva E.E., Kravchenko E.V. Drug-induced neutropenia and agranulocytosis. Safety and risk of pharmacotherapy. 2020;8(3): 109-122. https://doi.org/10.30895/2312-7821-2020-8-3-109-122, Borgquist J.D., Quinn M.T., Swain S.D. Adhesion to extracellular matrix proteins modulates bovine neutrophil responses to inflammatory mediators. J Leukoc Biol. 2002;71(5):764-774].

R., Baluja A., Veiras Del Rio S. et al. Effects of sevoflurane postconditioning on cell death, inflammation and TLR expression in human endothelial cells exposed to LPS. J Transl Med. 2013; 11(1):87. doi: 10.1186/1479-5876-11-87].Sevoflurane is an inhalation anesthetic whose positive effect on critically ill neutrophil function has been demonstrated in a number of in vitro and in vivo experiments [Beck-Schimmer B., Baumann L., Restin T. et al. Sevoflurane attenuates systemic inflammation compared with propofol, but does not modulate neuro-inflammation: A laboratory rat study. Eur J Anaesthesiol. 2017; 34(11):764-775. doi: 10.1097/EJA.0000000000000668, Koutsogiannaki S., Hou L., Babazada H. et al. The volatile anesthetic sevoflurane reduces neutrophil apoptosis via Fas death domain-Fas-associated death domain interaction. FASEB J. 2019; 33(11): 12668-12679. doi:10.1096/fj.201901360R,

R., Baluja A., Veiras Del Rio S. et al. Effects of sevoflurane postconditioning on cell death, inflammation and TLR expression in human endothelial cells exposed to LPS. J Transl Med. 2013; 11(1):87. doi: 10.1186/1479-5876-11-87].

It is also known to reduce the severity of the inflammatory process and oxidative stress in patients after surgery using sevoflurane as an anesthetic [De Conno E., Steurer M.R., Wittlinger M. et al. Anesthetic-induced Improvement of the Inflammatory Response to One-lung Ventilation. Anesthesiology.2009; 110(6): 1316-1326. oi:10.1097/ALN.0b013e3181al0731, Schilling T., Kozian A., Senturk M. et al. Effects of Volatile and Intravenous Anesthesia on the Alveolar and Systemic Inflammatory Response in Thoracic Surgical Patients. Anest].

Currently, in the databases available to the authors, there are no results of studies on the use of sevoflurane to correct the inflammatory process in critically ill patients.

Description of the experiment procedure

The study was carried out on a cell culture of neutrophils isolated from the venous blood of 5 healthy men aged 25-34 years. Erythrocytes were precipitated in heparinized venous blood, and the plasma was layered on ficoll (PanEco, Russia) with a density of 1.077 g/ml and centrifuged at room temperature at 400g 30 minutes. Then the supernatant was removed and all further procedures were carried out on ice using chilled solutions. Contaminated erythrocytes were removed by resuspension of the sediment in 2 ml of deionized sterile water for 45 sec, and then 2 ml of 2x PBS was added to restore tonicity and then centrifuged at 200g, +4°C for 10 minutes. Precipitated neutrophils were washed with PBS and resuspended in culture medium (RPMI-1640+10% FBS). Then, the isolated neutrophils were placed in an incubator for 30 min, where an air mixture of room temperature was supplied, containing various concentrations of sevoflurane in the air: 0.5 MAC (1 vol.%), 1.0 MAC (2.0 vol.%), 1, 5 MAC (3.0 vol.%).

After that, the cells were placed in a humidified CO ₂ incubator (37°C, 5% CO ₂ ) for 30 min, after which further manipulations were performed. After incubation with an activator (LPS), neutrophils at a concentration of 5 million/ml were lysed in hot buffer (62.5 mM Tris-HC1, pH 6.8; 2% SDS; 10% glycerol; 50 mM DTT, 0.01% bromophenol blue ) for 4 min at 94°C. Proteins were separated in 12% polyacrylamide gel and transferred to PVDF membranes (Amersham, USA). Further, 5% BSA in TBST buffer (25 mM Tris pH 7.4.0.15 M NaCl, 0.1% Tween20) blocked non-specific binding sites. The membranes were then incubated overnight at 4°C with antibodies in 5% BSA in TBST (anti-phospho-p38 (T180/Y182), Cell Signaling, USA). With the second antibodies (against mouse immunoglobulins conjugated with horseradish peroxidase and diluted in 5% BSA/TBST solution), the membranes were incubated for 1 hour. Visualization was performed with the SuperSignal West Pico kit (ThermoFisher, USA) using the Bio-Rad ChemiDocTM Touch gel-documenting system.

Neutrophil apoptosis was assessed by flow cytometry. Neutrophils were incubated with lipopolysaccharides (LPS) and fMLP for 22 hours at 37°C in a humidified CO ₂ incubator. The cells were then centrifuged at 400 g for 5 min and the pellet was resuspended in 70 μl of buffer (10 mM HEPES, 120 mM NaCl, 2.5 mM CaCl, pH=7.4). 2.5 µl of annexin V conjugated with FITC fluorescent dye (ThermoFisher, USA) was added to each sample and left for 25 min at 37°C. Then PI was added to a final concentration of 10 µg/ml, incubated for another 5 min, after which 50,000 cells were analyzed using a Beckman Coulter CytoFLEX flow cytometer. Annexations of V-positive and PI-negative cells were considered apoptotic.

Enzyme immunoassay (ELISA) was performed using reagents for enzyme immunoassay for determining the concentration of the analyzed cytokines in the neutrophil cultivation medium (Vector-Best, Russia), according to the manufacturer's protocol. Blood serum samples containing sediment were purified by centrifugation at 400 g for 5 min at room temperature. For analysis, 100 μl of sample dilution solution (SDS) was added to all wells of the strips of the plates used. Then, 100 µl of each calibration sample and 100 µl of the control sample were added to the corresponding wells in duplicates, and 100 µl of the analyzed blood serum samples were added to the remaining wells in duplicates. The plates were taped and incubated for 2 hours with shaking on a shaker at 37°C and 700 rpm. After removing the wash film and removing residual liquid, 100 μl of conjugate No. 1 was added to all wells. After repeated incubation and washing, 100 μl of conjugate No. 2 was added to all wells. After the third incubation and washing, 100 µl of TMB was added to all wells and incubated in a place protected from light for 25 min at room temperature, after which 100 µl of stop reagent was added to all wells.

The optical density (OD) of the solutions in the wells of the strips was measured on a BioRad iMark vertical scanning spectrophotometer at the main wavelength of 450 nm and the reference wavelength in the range of 620–655 nm. For each pair of wells, the arithmetic mean OD value was calculated, after which a calibration plot of the dependence of the arithmetic mean OD value on the cytokine concentration in the calibration samples (pg/ml) was plotted in linear coordinates. Next, according to the calibration curve, the concentration of cytokines in the control and analyzed samples was determined.

To determine the level of activation of neutrophils, their degranulation was assessed by an increase in CD11b molecules (a component of membranes of specific, gelatinous and quaternary granules) and CD66b (a component of membranes of specific granules) on the cell surface using flow cytometry. Cell samples were incubated with LPS for 30 min at 37°C, then 2.5 µl of antibodies to CD11b conjugated with fluorescent dye FITC (ThermoFisher, USA) and 0.5 µl of antibodies to CD66b conjugated with fluorescent dye AlexaFluor647 ( ThermoFisher, USA) and incubated on ice for 30 minutes. Then, 50,000 cells were analyzed using a Beckman Coulter CytoFLEX flow cytometer.

Statistica 10.0 (StatSoft, Inc.) and MedCalc 12.5.0.0 (MedCalc Software bvba) were used for statistical analysis. The results are presented by the median with an interquartile interval. Intergroup differences were assessed using the Wilcoxon W test for linked samples and were considered statistically significant at p<0.05.

When studying the pro-inflammatory activation of neutrophils, it was found that the expression levels of CD11b and CD66b on the surface of intact neutrophils were 3898.0 [3340.0-4200.0] and 8786.0 [8112.0-10100.0] conventional fluorescence units, respectively. Incubation of neutrophils with LPS and fMLP significantly increased the expression of these molecules:

- when treated with LPS at a dose of 200 ng/ml, the expression of CD11b and CD66b increased by 2.3 and 2.2 times (p=0.002 and p=0.001, respectively);

- when fMLP was treated at a dose of 100 nM, the expression of CD11b and CD66b increased by 1.7 and 2.0 times (p=0.025 and p=0.03, respectively).

These results are consistent with literature data on increased expression of CD11b and CD66b upon activation of neutrophils under the influence of various factors initiating inflammation.

When neutrophils were incubated with the same concentration of LPS after exposure to sevoflurane for 30 minutes at a dose of 1 vol.% (0.5 MAC) and a dose of 2 vol.% (1 MAC), the CD11b expression level also significantly increased compared to intact neutrophils (table 1). Thus, it was found that exposure to sevoflurane for 30 minutes at a dose of 0.5-1.0 MAC under ex vivo conditions does not affect the pro-inflammatory activation of neutrophils.

When neutrophils were incubated with the same concentration of LPS after exposure for 30 minutes to sevoflurane at a dose of 1.5 MAC, the expression level of CD11b and CD66b increased compared to intact neutrophils. The change in CD11b expression in this experiment was not statistically significant (p=0.055), the change in CD66b expression was statistically significant (p=0.007). Thus, it was found that exposure to sevoflurane at a dose of 1.5 MAC reduces the pro-inflammatory activation of neutrophils under the action of LPS.

Similar data were obtained when neutrophils were incubated with fMLP and exposed to sevoflurane for 30 minutes: the use of sevoflurane at a dose of 0.5 MAC and 1 MAC did not affect the pro-inflammatory activation of neutrophils. The increase in CD11b and CD66b expression with fMLP was statistically significant and did not differ significantly from that without sevoflurane. When neutrophils were incubated with the same fMLP concentration after exposure to sevoflurane at a dose of 1.5 MAC for 30 minutes, the expression level of CD11b and CD66b increased compared to intact neutrophils by 1.2 times and 1.4 times. The change in the expression of CD11b and CD66b in this experiment was not statistically significant. Thus, a decrease in the pro-inflammatory activation of neutrophils under the influence of sevoflurane at a concentration of 1.5 MAC is also noted with the activation caused by fMLP at a dose of 100 nM.

The revealed decrease in the expression of CD11b and CD66b when exposed to sevoflurane at a dose of 1.5 MAC confirms the possibility of suppressing excessive activation of neutrophils under the action of this anesthetic.

Annex V and propidium iodide were used to study the effect of sevoflurane on the level of apoptosis and necrosis of human neutrophils 22 hours after their isolation. It was found that the level of spontaneous necrosis and apoptosis of neutrophils after 22 hours of incubation was 57.0 [56.8-57.9]%, and exposure to sevoflurane 0.5 and 1.5 MAC did not affect the ability of neutrophils to spontaneous apoptosis: the level of apoptosis amounted to 52.9 [50.1-54.5]%, (p>0.05) for 0.5 MAC; 53[51.5-54.7]%, (p>0.05) for 1.0 MAC and 57.3[53.0-58.8]%, (p>0.05) for 1.5 POPPY. Therefore, sevoflurane at these concentrations did not affect the level of neutrophil apoptosis.

The authors of the proposed method evaluated the effect of sevoflurane on the change in the expression of pro-inflammatory cytokines IL-1 p, IL-6 and IL-8 by neutrophils under the influence of LPS and fMLP. In intact neutrophils, the level of IL-6 and IL-8 was determined (table 2). It was found that the effect of sevoflurane at a concentration of 0.5 MAC and 1.0 MAC on intact neutrophils did not affect the level of expression of these cytokines. When exposed to sevoflurane at a concentration of 1.5 MAC, there was a tendency to reduce the level of expression of these cytokines by intact neutrophils (table 2).

The addition of LPS to the incubation medium at a dose (200 ng/mL) significantly increased the level of pro-inflammatory cytokines in neutrophils. Similarly, the addition of fMLP at a dose of 100 nM to the incubation medium also statistically significantly increased the level of pro-inflammatory cytokines in neutrophils.

Exposure to sevoflurane at a concentration of 1.5 MAC for 30 min abolished the activation of IL-6 and IL-8 under the influence of LPS, but significantly increased the level of the pro-inflammatory cytokine IL-ip in neutrophils (Table 2).

In a similar experiment performed with neutrophil activation by 100 nM fMLP, exposure to sevoflurane at a concentration of 0.5-1.0 MAC also did not prevent an increase in the expression of pro-inflammatory cytokines. At the same time, when exposed to sevoflurane at a concentration of 1.5 MAC, the addition of fMLP at a dose of 100 nM to the incubation medium significantly increased the level of expression of IL-6 by neutrophils activated by LPS or fMLP and the expression of IL-8 by neutrophils activated by LPS.

The final stage of the study was the study of the effect of sevoflurane on the level of GSK-3β phosphorylation in neutrophils after exposure to LPS. It turned out that LPS stimulation leads to dephosphorylation of GSK-3P kinase in neutrophils, significantly reducing the level of the phosphorylated form compared to the basal one. Exposure to 0.5 MAC of sevoflurane did not affect the level of GSK-3βP phosphorylation in stimulated neutrophils. When exposed to 1.0 MAC of sevoflurane, there was a trend towards an increase in the level of GSK-3β phosphorylation in stimulated neutrophils (p=0.06). At the same time, exposure to 1.5 MAC of sevoflurane significantly increased the level of GSK-3β phosphorylation in stimulated neutrophils (p=0.008) (Table 3).

The results obtained allow us to state the effect of sevoflurane at a dose of 1.5 MAC on GSK-3P phosphorylation in LPS-stimulated neutrophils.

Thus, the observed decrease in neutrophil activation under the action of LPS and fMLP, expressed in the expression level of CD11b and CD66b, when exposed to sevoflurane at a dose of 1.5 MAC confirms the possibility of suppressing excessive activation of human neutrophils during inflammation under the action of sevoflurane. It has been established that sevoflurane has a pronounced anti-inflammatory effect due to the suppression of neutrophil hyperactivation, which was demonstrated on human neutrophils in an ex vivo model.

Claims (1)

A method for suppressing excessive activation of neutrophils, including exposure to a gas mixture, characterized in that a decrease in inflammatory hyperactivation of neutrophils in an ex vivo model is achieved by exposure to sevoflurane at a concentration of 3 vol.%, which corresponds to 1.5 MAC, for 30 minutes.