Recruitment of neutrophils to an inflammatory site in response to invading bacteria or non-infectious processes is a crucial step in the physiology of the acute inflammatory response. Adherence of neutrophils to endothelial cells followed by transmigration through the endothelial cells depends on a network of several events involving neutrophil surface adhesion molecules, inflammatory cytokines and chemoattractant chemokines. However, transmigration of neutrophils through endothelial cells to surrounding organ tissues is not always beneficial. In ischaemia/reperfusion injury, activated neutrophils contribute to organ damage by releasing toxic reactive oxidative species and increased cytokine release . Isoflurane and sevoflurane are known to protect against ischaemia/reperfusion injury of the heart . Proposed mechanisms include inhibition of leuccocyte transmigration and activation , as well as several direct effects on intracellular components, also termed as anaesthetic-induced preconditioning .
Recently, it was shown that xenon reduces the infarct size after regional ischaemia in the rabbit heart in vivo, but the underlying mechanisms are unknown. Since adhesion molecule receptors on neutrophils are closely involved in the pathophysiology of ischaemia/reperfusion injury and in the modulation of the neutrophil function, we were interested to examine the effect of xenon on neutrophil adhesion molecule expression in an established in vitro model. Selectin and β2-integrin expression on unstimulated neutrophils and after stimulation with either N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol-12-myristate-13-acetate (PMA) was measured using two-colour flow cytometry.
The protocol of this study was approved by our institutional review board. After informed consent, venous blood was collected from 16 healthy volunteers into sterile sodium citrate blood collection tubes (Sarstedt, Nümbrecht, Germany). Human neutrophils were isolated from citrated whole blood by density gradient centrifugation using Polymorphprep® (Nycomed, Oslo, Norway). Freshly isolated neutrophils were immediately resuspended in 37°C prewarmed modified Hanks'-buffered salt solution (HBSS without Ca2+ and Mg2+; Sigma Chemical, St. Louis, MO, USA).
Incubation of neutrophils was performed in a 5 L airtight box as previously described . In brief, cells were incubated for 60 min with 21% oxygen and 5% carbon dioxide at 37°C. Cells were exposed to either 30% or 60% xenon. Xenon was delivered as a gas/oxygen mixture using a low-ΔP-mass flow meter with control unit (Type F-201C-FA-22-V® and E-7300-AAA®; HI-TEC Bronkhorst, EC Veenendal, The Netherlands). Xenon gas concentrations were monitored using an Ecotec 500® Euro gas analyser and measured with Masterquad V3.2MG® software (both Leybold, Cologne, Germany). Carbon dioxide was measured with a multigas-analyser (Datex Compact®; Datex, Helsinki, Finland). To avoid artificial cell activation, blood samples were not bubbled with fresh gas. The control samples were placed in an identical box and exposed to the same atmospheric conditions. At the end of the incubation time, all samples were immediately processed for stimulation and staining procedures.
Stimulation procedures were performed in sealed polypropylene tubes to avoid evaporation of the anaesthetics. Neutrophils were stimulated with FMLP, 100 nmol (Sigma) and PMA, 100 nmol (Sigma; only 60% xenon) 10 min at 37°C before 200 μL of the neutrophil suspension was transferred to polystyrene tubes (Falcon®; Becton-Dickinson, San Jose, CA, USA) containing saturating concentrations of fluorochrome-conjugated monoclonal antibodies. Staining was performed at 4°C in the dark and stopped after 30 min by adding 1 mL lysing solution (FACS Lysing Solution, Becton-Dickinson). The cells were washed with phosphate-buffered saline supplemented with bovine serum albumin 1%, centrifuged and finally resuspended in phosphate-buffered saline containing bovine serum albumin 1% and paraformaldehyde 2%. Neutrophils were stored at 4°C in the dark until flow cytometric analysis.
The following phycoerythrin (PE)-conjugated antibodies were used in this study: anti-PSGL-1 (clone KPL-1), anti-L-selectin (clone Dreg 56), anti-CD11a (clone HI111), and anti-CD11b (clone ICRF44, all from Pharmingen, San Diego, CA, USA). Neutrophils were also stained with the pan leucocyte marker CD45 (clone HI30, fluorescein isothiocyanate-conjugated (FITC)), and IgG1-PE (clone MOPC-21) was used as negative control.
Flow cytometric measurements were performed on a FACSCalibur® flow cytometer (Becton-Dickinson), which was calibrated daily with CaliBRITE® beads (Becton-Dickinson). Neutrophils were gated by their forward scatter (FSC) and sideward scatter (SSC), as well as their CD45-FITC signal characteristics. Debris was omitted by setting a threshold in FL1. The data of 10 000 neutrophils were stored and the expression of the adhesion molecules PSGL-1, L-selectin, CD11a and CD11b were analysed by measuring the mean fluorescence intensity (MFI) of the specific antibody.
Data are presented as median and range. Differences between xenon-exposed and control samples assessed in parallel were compared using Wilcoxon signed rank sum tests. P < 0.05 was regarded as significant.
Xenon modulates surface expression of PSGL-1 and L-selectin in unstimulated neutrophils
Xenon led to a reduction of the expression of PSGL-1 on unstimulated neutrophils by 10% at both tested concentrations in comparison with control samples. Furthermore, surface molecule expression of L-selectin following exposure of neutrophils to 60% xenon was also reduced by 15% (Tables 1 and 2).
Xenon modulates the FMLP-induced reduction in neutrophil surface expression of L-selectin
Stimulation of neutrophils by the bacterial peptide FMLP leads to an activation-dependent decrease in the surface expression of PSGL-1 and L-selectin. The surface expression of the β2-intergins CD11a and CD11b is increased compared with untreated neutrophils.
On FMLP-activated neutrophils we observed an increased shedding (30% compared to controls) of L-selectin from the neutrophil surface following incubation with xenon. Neutrophil β2-integrin expression was neither altered on unstimulated nor after stimulation of neutrophils with FMLP following the incubation with xenon.
PMA-activated changes in neutrophil selectin and β2-integrin expression is not modulated by xenon
To characterize whether xenon modulates the neutrophil signalling transduction upstream or downstream of protein kinase C (PKC), we also used PMA as a direct activator of PKC. Activation of neutrophils by PMA results in similar changes of the surface expression of both selectins and β2-integrins as FMLP. These changes in adhesion molecule surface expression were not modulated by 60% xenon.
The main finding of the present study was that xenon reduced the expression of both selectins PSGL-1 and L-selectin on unstimulated neutrophils and increased the activation-dependent shedding of L-selectin from the neutrophil membrane surface.
Restoration of blood flow is essential to prevent tissue necrosis and regain organ function. Paradoxically, restoration of blood flow also initiates a cascade of events that may augment tissue damage by promoting endothelial and microvascular dysfunction, cell damage through oxygen-free radicals, intracellular calcium overload, and altered cell metabolism . Isoflurane and sevoflurane have been shown to protect the heart against reperfusion-induced cell damage [2-4]. Several mechanisms may contribute to these beneficial properties including inhibition of leucocyte transmigration and activation [3,7], as well as direct effects on intracellular organ components [2,4,8]. Effects on intracellular components are also called anaesthetic-induced preconditioning, because it shares several cellular mechanisms as seen in ischaemic preconditioning .
Recently, Preckel and colleagues showed that xenon administration during early reperfusion reduces regional ischaemia in the rabbit heart . However, the underlying mechanism is unknown. As there is a close relation between neutrophil function and ischaemia/reperfusion tissue damage, we investigated the effect of xenon on neutrophil selectin and β2-integrin activation in an established in vitro model. During ischaemia/reperfusion, neutrophils adhere to endothelial cell in a series of well-defined steps, involving neutrophil cell surface adhesion receptors and their endothelial counterparts . Initial neutrophil tethering and rolling on endothelial cells is mediated by PSGL-1 and L-selectin. Neutrophil arrest and transmigration through endothelial cells involves the β2-integrins CD11a and CD11b.
Xenon reduced the expression of both selectins on unstimulated neutrophils, and augmented the FMLP-induced shedding of L-selectin. In vitro studies showed that even moderate decreases in the cell surface expression of PSGL-1 and L-selectin reduced adhesion of neutrophils to endothelium [10,11]. In a rat arthritis model, L-selectin shedding induced by painful stimuli reduced neutrophil migration into inflamed joints . Furthermore, a reduction of the L-selectin expression by 30% significantly reduced adhesion of neutrophils to endothelial cells in vitro. Since xenon increased PSGL-1 and L-selectin shedding, we suggest that xenon might attenuate neutrophil adhesion to endothelium cells.
L-selectin downregulation from the neutrophil surface occurs spontaneously, upon ligand binding or following neutrophil activation by cytokines, chemoattractants or phorbol esters [13,14].
After FMLP-induced stimulation, L-selectin is rapidly activated by phosphorylation in a PKC-dependent pathway . Phosphorylation of L-selectin occurs in conjunction with the dissociation of calmodulin [15,16], a calcium-binding protein bound to the cytoplasmatic domain of L-selectin, followed by proteolytic shedding of L-selectin from the neutrophil cell surface .
Interestingly, Kahn and colleagues  recently showed that calmodulin inhibitors directly induced proteolytic L-selectin shedding without leading to general neutrophil activation and enhance FMLP-induced L-selectin shedding. We observed that xenon decreased the expression of L-selectin on unstimulated neutrophils without affecting the expression of CD11b, indicating that neutrophils were not activated. In addition, xenon also enhanced the downregulation of L-selectin from FMLP-activated neutrophils. This suggests that xenon may affect the expression of L-selectin by calmodulin inhibition. However, we were unable to demonstrate any effects of xenon on the PMA-induced L-selectin shedding. This could be explained with the used high concentration of PMA, which induced a nearly 90% reduction in L-selectin expression.
In contrast to L-selectin, the mechanism for the activation-induced downregulation of PSGL-1 are poorly understood. One described mechanism is that neutrophil activation by FMLP results in surface redistribution of PSGL-1 from the tips of the microvilli to the uropods . Recently, Davenpeck and colleagues  provided evidence that under certain conditions PSGL-1 is shed from the neutrophil surface by proteolytic cleavage within minutes after exposure to PMA and platelet activating factor (PAF). Shedding of PSGL-1 was sensitive to EDTA but unaffected by metalloproteinase inhibitors known to inhibit L-selectin shedding . In addition, a potential autocrine mechanism for downregulation of neutrophil PSGL-1 expression and adhesion to P-selectin was described by Gardiner and colleagues . In this study, purified and neutrophil-released elastase and cathepsin G mediated proteolytic cleavage of PSGL-1 in vitro. Paradoxically, we have found that xenon downregulated the expression of PSGL-1 on unstimulated neutrophils, but not on activated neutrophils. Interestingly, xenon seems to directly induce downregulation of PSGL-1 without causing general neutrophil activation, but the underlying mechanism remains unclear. Although the data presented in this paper demonstrate in vitro alterations of PSGL-1 expression, the in vivo functional consequences remain to be determined.
The β2-integrins CD11a and CD11b mediate tight adhesion and transmigration by binding to endothelial ICAM-1 . Recently, it was shown that blocking CD11a or CD11b alone by using monoclonal antibodies did not inhibit leucocyte transmigration, but blockade of both receptors significantly attenuated leukocyte migration across human umbilical vein endothelial cells . In the present study, administration of xenon did not modulate the unstimulated and FMLP-stimulated expression and activation of both β2-integrins on the neutrophil cell surface membrane. In contrast, we have demonstrated that isoflurane inhibits the activation of CD11b and CD11a , which could partially explain the inhibiting effect of isoflurane on neutrophil accumulation during ischaemia/reperfusion injury .
In conclusion, this study demonstrated that xenon augments the shedding of the selectin PSGL-1 and L-selectin from the neutrophil cell surface. Since both selectins are involved in the initial contact between neutrophils and endothelial cells, xenon may affect neutrophil adhesion to endothelium during ischaemia/reperfusion injury. However, because the β2-integrin expression was not affected by xenon, further investigations are required to clarify whether xenon may modulate neutrophil transmigration and accumulation during ischaemia/reperfusion injury.
This study was supported by START, a research grant of the Rheinisch-Westfälische Technische Hochschule Aachen, Germany. Xenon was donated by Messer GmbH, Krefeld, Germany.
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