Inhibition of Neutrophil Activation by Volatile Anesthetics Decreases Adhesion to Cultured Human Endothelial Cells 

Halothane (Halothan Hoechst) was obtained from Hoechst (Frankfurt, Germany), isoflurane (Forene) was obtained from Abbott (Wiesbaden, Germany), and sevoflurane (Sevofrane) was obtained from Maruishi Pharmaceuticals (Kobe, Japan). The volatile anesthetics were added to a mixture of room air plus 5% carbon dioxide using calibrated vaporizers (Vapor 19.1; Drager, Lubeck, Germany for halothane and isoflurane; Vapor 19.3 for sevoflurane). The concentration (0.5, 1, and 2 MAC) in the gas phase was monitored using a piezo electric gas detector (Drager). All MAC values refer to human use; that is, 0.375, 0.75, and 1.5 vol% for halothane; 0.575, 1.15, and 2.3 vol% for isoflurane; and 0.855, 1.71, and 3.4 vol% for sevoflurane.

Human umbilical vein endothelial cells (HUVECs) were isolated according to a previously published method, [14] which is explained briefly herein. Umbilical cords were taken, with parents' previous consent, just after birth and stored at 4 [degree sign]C in phosphate-buffered saline (PBS) containing 50 U/ml penicillin and 50 [micro sign]g/ml streptomycin. Cords were rinsed with 40–60 ml PBS with added antibiotics at the concentrations previously noted and incubated for 5 min at 37 [degree sign]C with 0.1% collagenase A (Boehringer-Mannheim, Mannheim, Germany) in PBS without calcium. Subsequently, umbilical veins were rinsed with medium 199 supplemented with 10% heat-inactivated fetal calf serum (Seromed; Biochrom KG, Berlin, Germany), 1 mM sodium pyruvate (Seromed), and antibiotics (50 U/ml penicillin and 50 [micro sign]g/ml streptomycin). Cells were collected in 50-ml tubes and centrifuged for 8 min at 124g. After this washing procedure was repeated, the cell pellet was resuspended initially in 5 ml endothelial growth medium and seeded into 25-cm²flasks (Costar, Cambridge, MA) coated with 0.03% collagen G (Seromed). The cell cultures were incubated at 37 [degree sign]C and 95% humidity in room air with added 5% carbon dioxide. Unless otherwise indicated, all tissue culture reagents and additives were obtained from Sigma (Deisenhofen, Germany).

The day after seeding, cells were washed with PBS to eliminate blood cell contamination. When cells had formed a confluent monolayer (after 2–4 days), they were dispersed by brief trypsination (2.5 g/l). Trypsin inhibitor (from soybeans) was added afterwards because the endothelial growth medium contained only a small amount of serum. The detached HUVECs were passaged into 12- or 96-well culture plates coated with collagen G, and cultured with the tissue culture medium noted previously until they were again confluent (2–4 days). The confluent cells were identified as endothelial cells by their cobblestone appearance and by positive labeling with mouse anti-human Factor VIII (Dianova-Immunotech, Hamburg, Germany) according to the method of Jaffe et al. [14] via indirect immunofluorescence microscopic analysis performed on one plate per batch. The HUVECs were used for experiments on the second day after confluence.

Peripheral venous blood samples (20 ml filled into polypropylene syringes containing 2% EDTA as the final concentration per milliliter of blood) were obtained from healthy volunteers. The PMNs were immediately isolated with a magnetic antibody separation technique, as described in detail previously in reports from our laboratory. [15] Briefly, after centrifugation at 400g for 15 min, the platelet-rich plasma was discarded. The leukocyte and erythrocyte-rich buffy coats (500–800 [micro sign]l) were incubated for 15 min at 4 [degree sign]C with 20 ml monoclonal antibody against human CD15 conjugated with colloidal "superparamagnetic" microbeads (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany). These beads are irregularly shaped and have a mean diameter of 30–60 nm (based on the supplier's information). A separation column filled with steel wool (Miltenyi Biotech GmbH) was washed at room temperature with 500 [micro sign]l PBS containing 0.5% bovine albumin, placed in a magnetic field (Mini-MACS separation unit; Miltenyi, Biotech), and a flow resistor was attached. The column was filled with the magnetically tagged buffy coat and, after disconnecting the flow resistor, flushed four times with 500-[micro sign]l aliquots of PBS to remove unlabeled cells (erythrocytes, monocytes, lymphocytes, and platelets). The column was taken out of the magnetic field and flushed with 1 ml PBS. Subsequently, the effluent was diluted with 9 ml PBS and centrifuged at 350g for 10 min and the resulting pellet was suspended in Tyrode's solution (1 ml). The cells were counted using a Coulter counter (Coulter Electronics, Krefeld, Germany) and adjusted to a final cell count of 4 x 10⁵cells/ml with Tyrode's solution. All steps after incubation with the antibody were performed at room temperature. The total time taken for the isolation was approximately 1 h.

This magnetic antibody cell separation may be regarded as a relatively simple, fast, and reliable method to isolate neutrophils. The PMNs obtained in this manner are pure (99%), display a low basal activation, and perform functionally at least as well as PMNs obtained by conventional density gradient methods. [15]

Freshly isolated nonactivated PMNs (40,000/well) were added to unstimulated HUVECs (control) and to HUVECs that were exposed for 10 min to 1 mM H²O²for activation. In another set of experiments, PMNs were prestimulated with 10 nM fMLP for 15 min before application to HUVECs. After a 2-min centrifugation of the culture dishes (14g) to allow for rapid contact of the neutrophils with the endothelial layer and subsequent 15 min of contact, unbound PMNs were removed by three washes with warm PBS (200 [micro sign]l/well).

Adherent PMNs were counted using the method of Bradley et al., [16] as modified by Mullane et al., [17] which relies on measurement of the myeloperoxidase activity, an enzyme that occurs virtually exclusively in neutrophils. The endothelial cells and the adhering neutrophils were lysed with 100 [micro sign]l hexadecyl-trimethyl ammonium bromide (Sigma) and subjected to freeze-thawing to liberate myeloperoxidase. The lysates were reacted with 100 [micro sign]l substrate (160 [micro sign]M o-dianisidine dihydrochloride [Sigma] plus 0.001% H²O²) at pH 6. The rate of change of extinction of the resulting orange dye was measured spectrophotometrically using an enzyme-linked immunosorbent assay reader (MR 7000; Dynatech, Guernsey, UK) at 450 nm in samples (wells) and standards of varying PMN numbers. A standard curve for PMN-myeloperoxidase was established to correlate the optical density with the number of adherent PMNs, with a detection limit of 500 PMNs.

In another set of experiments, HUVEC monolayers, grown in 2 x 96 well plates as before, PMNs, or both were pretreated with gas at 37 [degree sign]C for 40 min with 0.5, 1, and 2 MAC of one of the volatile anesthetics (halothane, isoflurane, or sevoflurane). A flow-over method of gas equilibration was chosen, because bubbling of culture wells is not feasible (because of the danger of nonspecific activation or damage to cells).

There were four different groups of treatment as follows:

1. HUVECs plus PMNs not treated with volatile anesthetics,

2. only HUVECs pretreated with gas for 40 min to 0.5, 1, and 2 MAC volatile anesthetic and then exposed to volatile anesthetic throughout the experiment,

3. only PMNs preexposed for 40 min to 0.5, 1, and 2 MAC volatile anesthetic and exposed to volatile anesthetic throughout the experiment, and

4. both HUVECs plus PMNs preexposed for 40 min to 0.5, 1, and 2 MAC volatile anesthetic and then exposed to volatile anesthetic throughout the experiment.

In all these groups, the following experiments were performed:

- adherence of PMNs on HUVECs during a control condition (no stimulation of HUVECs or PMNs),

- adherence of (primarily) unstimulated PMNs on stimulated HUVECs (10 min with 1 mM H²O²), and

- adherence of stimulated PMNs (15 min with 10 nM fMLP) on unstimulated HUVEC.

In addition, the expression of P-selectin on HUVECs and of CD11b on PMNs were assessed.

The effect of the volatile anesthetics on P-selectin expression on HUVECs with or without stimulation by 1 mM H²O²for 10 min was determined by flow cytometric analysis. The HUVEC monolayers in 12-well plastic plates (in one set of experiments not pretreated, in another pretreated with gas for 40 min with 1 MAC halothane) were stained directly in the culture dishes with 20 [micro sign]l of a phycoerythrin-labeled monoclonal antibody against human P-selectin (clone AK6; Biozol, Eching, Germany). After washing with PBS without calcium and exposure to collagenase A for only a few seconds, the cells were removed from the culture dish using a cell scraper. Immediately after detachment, the cells were fixed with 3% formaldehyde and then analyzed using the flow cytometer. Isotype controls were performed with a matched nonspecific antibody.

The expression of CD11b on cells identified as neutrophils after staining with a phycoerythrin-labeled CD11b antibody (obtained from Guildhay, Guildford, UK) was measured in samples of freshly isolated PMNs (the method was described already) by analyzing the mean fluorescence intensity using a FACScan (Becton Dickinson, Heidelberg, Germany). The expression of CD11b on PMNs after exposure to 1 MAC halothane, isoflurane, or sevoflurane, respectively, for 40 min at 37 [degree sign]C was compared with that of PMNs in time-matched controls (gassed with room air plus 5% carbon dioxide). Aliquots of each group were stimulated with fMLP (10 nM) for 15 min at 37 [degree sign]C to test for differences in the upward regulation of CD11b. To preclude further stimulation of the cells as a result of staining procedures, the neutrophils were fixed routinely with FACS lysing solution (Becton Dickinson), a fixative containing approximately 1.5% formaldehyde, before antibody application. Isotype controls were performed using a matched nonspecific antibody for each experiment, so that nonspecific monoclonal antibody binding could be ruled out.

If not indicated otherwise, all data shown in the text are presented as the mean +/- SD. Statistical evaluation was performed using one-way analysis of variance (ANOVA) and, when appropriate, by multiple comparison tests between the groups using the Student-Newman-Keul's test. Differences between groups were considered significant at P <or= to 0.05.

During control conditions, approximately 2,000 neutrophils attrached per well on a confluent monolayer of HUVECs (Figure 1 and Figure 2). Incubation of HUVECs for 10 min with 1 mM H²O², to mimic oxidative stress as after ischemia-reperfusion, induced an approximately two-fold increase in adherence of resting PMNs to HUVECs. Similar to H²O², but by coincidence, a twofold increase in neutrophil adhesion was also observed when PMNs were prestimulated with 10 nM fMLP (Figure 1 and Figure 2).

Pretreatment of HUVECs with 0.5, 1, or 2 MAC of one of the volatile anesthetics did not influence the basal adhesion of neutrophils (for data with 1 MAC, see Figure 1 and Figure 2; other values are not shown). Enhanced adhesion induced by exposure of endothelial cells to 1 mM H²O (²) or by activation of neutrophils with 10 nM fMLP, respectively, also was not modified by preincubation of HUVECs for 40 min with one of the volatile anesthetics at any concentration tested (for the concentration of 1 MAC, data are depicted in Figure 1 and Figure 2; data for 0.5 and 2 MAC are not shown).

Pretreatment of the PMNs with halothane, isoflurane, or sevoflurane, respectively, did not change basal adhesion, whereas fMLP-induced adhesion was reduced to control levels (see Figure 1 and Figure 2for 1 MAC; for 0.5 and 2 MAC, data are not shown). Surprisingly, enhanced adherence of primarily unstimulated neutrophils to H²O^2-stimulatedHUVECs also was abolished completely if only PMN were pretreated with halothane, isoflurane, or sevoflurane (Figure 1 and Figure 2). As shown in Figure 1for 1 MAC halothane, no additive effects could be observed on inhibition of neutrophil adhesion by combined preincubation of HUVECs and PMNs with anesthetic. Similar results were obtained after preexposure of HUVEC plus PMN to either 1 MAC of isoflurane or sevoflurane (data not shown).

Only slight differences in the effectiveness of the volatile anesthetics were apparent in experiments with increasing concentrations of halothane, isoflurane, and sevoflurane, respectively, as shown in Figure 3. The adherence of neutrophils was significantly reduced after treatment of PMNs with 0.5 MAC halothane, both for prestimulation of PMNs with fMLP (Figure 3, top) and prestimulation of HUVECs with H²O²(Figure 3, bottom). In contrast, at this concentration of 0.5 MAC, isoflurane and sevoflurane did not modify the H²O^2-or fMLP-induced enhancement of neutrophil adhesion. Fully inhibitory effects of the three anesthetics were obtained at the concentration of 1 MAC. No additional inhibitory effect was observed at the concentration of 2 MAC of the anesthetic agents (Figure 3).

(Figure 4(top) and Table 1) show changes in the surface expression of the cell adhesion molecule CD11b, a [Greek small letter beta](^2-integrin) member, on PMNs before and after stimulation with 10 nM fMLP. The exposure of PMNs to 1 MAC halothane for 40 min did not affect the baseline expression of CD11b. After incubation of neutrophils for 15 min with 10 nM fMLP, CD11b was significantly upwardly regulated to approximately twice the level. This effect of fMLP could be abolished by pretreating the cells with 1 MAC halothane (from 40 min before and during the 15-min stimulation). Similar results were observed with 1 MAC isoflurane and sevoflurane, respectively (data not shown). The degree of inhibition of CD11b expression appears to be comparable with the extent of inhibition of neutrophil adhesion.

Basal expression of P-selectin on HUVECs did not differ between untreated cells and cells pretreated with gas (40 min) with 1 MAC halothane. Stimulation of endothelial cells with 1 mM H²O²led to a statistically significant increase in P-selectin levels by approximately 200%(Figure 4, Table 2). In agreement with the failure of volatile anesthetics to inhibit PMN adhesion if only endothelial cells were pretreated with gas, we found that 1 MAC halothane could not alter the increase in P-selectin expression induced by 1 mM H²O²(Figure 4[bottom], Table 2).

Restitution of blood and oxygen after ischemia may limit tissue injury, but a large body of evidence has documented that reperfusion itself may induce additional damage, defined as reperfusion injury. Acute reperfusion of ischemic tissue initiates an inflammatory cascade characterized by generation of oxygen-derived free radicals [12] and proinflammatory stimuli. [18] Neutrophils have been implicated as primary mediators of reperfusion injury in the heart [4,19,20] and other organs and tissues. [21,22] In addition, PMNs may be involved in the systemic inflammatory response syndrome, in sepsis, and so forth.

The critical initial step in the pathogenesis of PMN-mediated injury is the adhesion of PMNs to the vascular endothelium. [3,23] These adherent PMNs can undergo activation with subsequent production of many highly potent reactive species, including H²O². [24] Irritation of the endothelium by H²O²can cause acute and protracted externalization of adhesion molecules, such as P-selectin and platelet-activating factor, leading to further sequestration of inflammatory cells. The provoked end points are capillary plugging, cellular membrane disruption, intracellular and interstitial edema, impairment of metabolic activity, and cell necrosis.

Many therapeutic regimens are designed with the goal of interrupting or modulating the early stage of this inflammatory cascade: the process of adhesion.

Various drugs have been or are being evaluated with respect to their ability to reduce PMN adhesion. [5,25] Surprisingly, among other drugs, commonly used therapeutic agents, such as anesthetics (e.g., halothane, isoflurane, and sevoflurane), have been shown to exert antiadhesive effects at 1 and 2 MAC. [7] In contrast, Morisaki et al. [26] found in the rat mesenteric microcirculation that 2 MAC halothane and sevoflurane increased leukocyte adhesion above the level seen with 1 MAC of the respective agent. The results of the current study confirm those of Kowalski et al., [7] who used an isolated perfused guinea pig heart subjected to 15 min of global ischemia followed by 15 min of reperfusion. We found that these three volatile anesthetics at clinically relevant concentrations also strongly inhibited adhesion of PMN to cultured human endothelial cells, provided there was a foregoing inflammatory process.

We simulated features of the early phase of inflammation by incubating endothelial cells with 1 mM H²O². [27] The chosen concentration has been shown to mimic oxidative stress and rapidly stimulate the adherence of PMNs, but it does not lead to cell damage itself. [27] In another set of experiments, we activated PMNs with 10 nM fMLP. [28] Both interventions led to enhanced PMN adhesion to HUVECs. Separate exposure of PMNs to 1 or 2 MAC halothane, isoflurane, and sevoflurane fully attentuated neutrophil adhesion induced by the aforementioned agents. Although 0.5 MAC halothane also elicited significant inhibition, it is nevertheless likely that the three volatile anesthetics directly prevent neutrophil activation with comparable potency. When only HUVECs were pretreated with 0.5, 1, or 2 MAC of one of the volatile anesthetics, no inhibition of adhesion was found during any of the conditions we tested (stimulation of HUVECs with H²O (²) or activation of PMNs with fMLP).

Volatile anesthetics such as halothane have been shown to alter markedly various functions of neutrophils. They suppressed neutrophil recruitment in an experimental model of inflammation, [29] decreased chemotaxis motaxis in vitro [30] and in vivo, [31] and inhibited superoxide production in human neutrophils. [11] Alteration of neutrophil-associated oxidative metabolism by volatile anesthetics also has been documented by measuring chemiluminescence. [10] These findings are in accordance with our study, showing a depression of activation of PMN by pretreating these cells with halothane, isoflurane, or sevoflurane. This action was apparent in the attenuated upward regulation of the adhesion molecule CD11b in the presence of 1 MAC of any one of the three anesthetics after stimulation with fMLP.

Normally, unstimulated PMNs do not adhere to the endothelium. When stimulated, they become more tightly attached to the endothelial cells, becoming firmly adhesive. This step is mediated by glycoproteins such as CD11b, a neutrophil adhesion molecule, which can be expressed on the cell surface within minutes. [32] Because basal adhesion was not influenced, a direct action of halothane on the receptor itself or on the receptor-ligand interaction seems unlikely. However, receptor upward regulation was prevented. This could be medicated by changes in the physical properties of the membrane, which have been reported to occur with halothane. [33,34] Among the parameters that correlate best with anesthetic properties is lipid solubility. [35] Insertion of anesthetic molecules into lipid membranes results in membrane expansion, and thereby reduces the stability of ionic channels. [36] It is possible that such an expansion could distort the cell membrane configuration, thereby decreasing upward regulation of CD11b.

Endothelial cells respond to stimulation with H²O²with immediate (within minutes) membrane expression of the adhesion molecule P-selectin. [37] P-selectin is normally presynthesized and stored within the Weibel-Palade bodies in resting endothelial cells and can contribute to the initial PMN-endothelial interaction (rolling). [38] Contrary to our expectations, no inhibition of H²O^2-mediatedmobilization of P-selectin was obtained after exposure of HUVECs to 1 MAC halothane for 40 min. Exemplary studies with 1 MAC isoflurane or sevoflurane, respectively, also indicated the same lack of effect for these two agents.

Surprisingly, whereas the volatile anesthetics affected neither H (²) O^2-stimulatedP-selectin expression nor basal CD 11b expression on PMNs at 1 MAC, an attenuation of the enhanced PMN adherence resulting from H (²) O²treatment of HUVECs was observed. A possible explanation could be that the endothelial cell-dependent adhesion process, which requires activation of the PMNs, is disrupted. A known mechanism of endothelial cell-dependent PMN activation is the expression of platelet-activating factor on the endothelial cell surface within minutes of appropriate stimulation. [39] The functional upward regulation of CD 11b/CD18 by endothelial cell-associated platelet-activating factor [40] might be prevented by the pretreatment of PMNs with the volatile anesthetics, just as is the effect of fMLP. However, this remains to be investigated.

Interestingly, Morisaki et al. [26] report an upward regulation of P-selectin in rat mesenteric vessels in situ after elevation of sevoflurane from 1 MAC to 2 MAC. These authors also found an increase in the number of firmly adherent PMNs in the presence of 2 MAC sevoflurane or halothane. However, that particular study compared a situation with a higher level of volatile anesthetic (2 MAC) with the situation in which 1 MAC anesthetic was already present. The basal values (no anesthetic) and the inflammatory status of the animal preparations remain unknown. Furthermore, a different species (rat) and another vascular bed (mesenteric) were examined. Finally, all the current adhesion experiments were conducted during static conditions (i.e., in the absence of shear stress), so P-selectin had no real function. [41] The observed lack of effect of 1 MAC halothane on enhanced P-selectin expression after HUVEC stimulation suggests that, also in vivo during conditions of flow and inflammation, the activation of PMNs will be affected by volatile anesthetics, because of the attenuated upward regulation of neutrophil CD11b, rather than the rolling mediated by endothelial P-selectin.

The model we used in this study allows the in vitro effects of volatile anesthetics on neutrophil-endothelial adhesion to be analyzed. The findings indicate that halothane, isoflurane, and sevoflurane prevent neutrophil adhesion, as previously observed in reperfused heart preparations, [7] by reducing neutrophil activity rather than by affecting the endothelial site.