Perfluorocarbon attenuates response of concanavalin A-stimulated mononuclear blood cells without altering ligand-receptor interaction
Dirk Haufe,1
Thomas Luther,2
Matthias Kotzsch,2
Lilla Knels,1 and
Thea Koch1
1Department of Anesthesiology and Intensive Care Medicine, University Hospital Carl Gustav Carus; and 2Institute of Pathology, Technical University Dresden, D-01307 Dresden, Germany
Submitted 9 February 2003
; accepted in final form 29 February 2004
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ABSTRACT
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Intrapulmonary application of perfluorocarbons (PFC) in acute lung injury is associated with anti-inflammatory effects. A direct impact on leukocytic function may be involved. To further elucidate PFC effects on cellular activation, we compared in an in vitro model the response of concanavalin A (ConA)-stimulated lymphocytes and monocytes exposed to perfluorohexane. We hypothesized that perfluorohexane attenuates the action of the lectin ConA by altering stimulant-receptor interaction on the cell surface. Mononuclear blood cells were stimulated by incubation with ConA in the presence of different amounts of perfluorohexane. The response of lymphocytes and monocytes was determined by means of IL-2 secretion and tissue factor (TF) expression, respectively. The influence of perfluorohexane on cell-surface binding of fluorescence-labeled ConA was studied using flow cytofluorometry and fluorescence microscopy. Perfluorohexane itself did not induce a cellular activation but significantly inhibited both monocytic TF expression and, to a far greater extent, IL-2 secretion of ConA-stimulated mononuclear blood cells. The effect of perfluorohexane was due neither to an alteration of cell viability nor to a binding of the stimulant. The amount of cell surface-bound ConA was not altered by perfluorohexane, and the overall pattern of ConA receptor rearrangement did not differ between controls and treated cells. In the present study, we provide further evidence for an anti-inflammatory effect of PFC that might be beneficial in states of pulmonary hyperinflammation. A PFC-induced alteration of stimulant-receptor interaction on the surface membrane does not seem to be the cause of attenuated cell activation.
acute lung injury; anti-inflammatory; immune cells; cell-surface receptor
IN NUMEROUS STUDIES of experimental and human acute lung injury, beneficial effects of liquid ventilation with perfluorocarbons (PFC) have been demonstrated (16, 17, 21, 22). Application of the biochemically inert liquids resulted in improved gas exchange, lung mechanics, and ventilation/perfusion matching. In part, these observations may be due to several unique properties of PFC such as their high density, an impressive oxygen-carrying capacity, and low surface tension. Besides a positive influence on pulmonary function, animals treated with liquid ventilation also exhibited histological evidence of attenuated lung injury and concomitantly reduced leucocytic infiltration (7, 17, 27, 28). Whereas some authors (3, 14, 35) attributed this reduced pulmonary inflammation exclusively to mechanical phenomena (e.g., lavage of alveolar fluid, water-insoluble PFC forming a barrier between lung epithelium and inflammatory cells and mediators), PFC may also directly influence immune cell function. In support of the latter suggestion, our group as well as other investigators found a compromised responsiveness of phagocytic cell populations in terms of cytokine secretion, chemotaxis, adherence, and release of reactive oxygen species in vitro (4, 19, 26, 32, 34). Previous in vivo studies using liquid and aerosolized PFC in a surfactant-washout model confirmed this observation by showing a decreased mRNA expression of proinflammatory cytokines and adhesion molecules in lung tissue samples as well as in microdissected alveolar macrophages and pulmonary parenchymal cells (29, 36, 37). Liquid ventilation with PFC was not associated with an improved survival or a reduced need for ventilatory support in a recent multicenter trial of patients with acute respiratory distress syndrome (ARDS) (15). However, intrinsic anti-inflammatory properties of PFC might be an effective mechanism, introducing PFC as a therapeutical strategy in other illnesses aside from acute lung injury.
Although there is evidence that PFC are ingested by monocytic cells and are, in relatively small quantities, incorporated into cellular membranes, the mechanisms underlying the hyporesponsiveness upon proinflammatory stimulation remain unclear. Several groups have hypothesized that PFC partitioning into lipid bilayers of cellular membranes can alter conformation and function of membrane components thus compromising specific receptor-ligand interactions (19, 33). To further elucidate PFC effects on cellular activation, we analyzed in an in vitro model whether perfluorohexane affects concanavalin A (ConA)-induced stimulation of human mononuclear blood cells (MBC). ConA is a plant lectin that binds to mannosylated glycoproteins and glycolipids on the surface of a wide range of cell populations. It represents an established tool to induce cell activation and to study cell membrane structure and dynamics such as the mobility and redistribution of membrane protein receptors (30, 31, 38). Response of stimulated lymphocytes was determined by means of characteristic IL-2 release. To compare the magnitude of perfluorohexane effects on both nonphagocytosing and phagocytic cells, monocytic tissue factor (TF) expression was analyzed. TF is a transmembrane glycoprotein that functions as the primary cellular initiator of blood coagulation and is described to be a proinflammatory marker of cellular activation of monocytes and macrophages (25). Additional experiments were performed to analyze whether perfluorohexane interferes with the degree and pattern of ConA binding to the cell surface of MBC.
In the present study, we examined whether PFC/perfluorohexane affect lectin-induced activation of MBC in vitro and, thereby, to what extent PFC can influence respective receptor-ligand interaction on cellular membrane surface.
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MATERIALS AND METHODS
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Perfluorohexane.
Perfluorohexane [CF3(CF2)4CF3; ABCR, Karlsruhe, Germany] with a purity of 99% was used for all in vitro studies on MBC. Perfluorohexane is a clear, radiolucent, colorless liquid with a molecular mass of 338 g/mol, a density of 1.672 g/ml, a boiling point of 57°C, a vapor pressure of 177 mmHg (20°C), and low viscosity and surface tension (0.66 cP and 11.4 dyn/cm3, respectively). The liquid can solve 57 ml O2/100 ml perfluorohexane.
Isolation of MBC.
After approval by the local ethics committee and obtaining informed consent, we collected venous citrated blood from healthy volunteers and diluted it with isotonic NaCl. MBC were separated from erythrocytes and granulocytes by density gradient centrifugation (275 g, 25 min, 4°C) using Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) at a density of 1.077 g/ml. After being washed, cells were resuspended in RPMI 1640 cell culture medium (Sigma, Deisenhofen, Germany) supplemented with 5% FCS (Biochrom, Berlin, Germany) yielding a concentration of 1 x 106 cells/ml. Mean cell viability after isolation was >98% as assessed by trypan blue exclusion.
Preparation of MBC for the analysis of IL-2, TF protein, and cell viability.
One-milliliter aliquots of the MBC suspension were stimulated with 5 µg/ml ConA (Sigma), immediately followed by addition of perfluorohexane at a final volume concentration of 5, 10, and 30%, respectively. Samples were vortexed and incubated with gentle and continuous agitation for 6 or 24 h at standard culture conditions (37°C, 5% CO2). As perfluorohexane is a dense and water-immiscible liquid, its presence does not result in a dilution of the cell suspensions. Therefore, controls consisted of stimulated MBC without perfluorohexane. After incubation, probes were centrifuged and 600 µl of cell- and perfluorohexane-free supernatant were removed from perfluorohexane-exposed samples and frozen at 20°C for IL-2 analysis. For comparability, controls were spiked with corresponding doses of perfluorohexane immediately after the incubation period and treated similarly otherwise. The pelleted MBC were resuspended in the remaining supernatants (containing perfluorohexane) and stored frozen until determination of TF antigen by ELISA.
Viability of stimulated MBC was determined after the same experimental procedure as described for IL-2 and TF analysis. After incubation for 0.5, 6, or 24 h, percentage of cell death was assessed by either trypan blue exclusion or the well-established 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. For trypan blue exclusion, 30-µl aliquots of cell suspensions were added to an equal volume of 0.5% trypan blue solution (Biochrom), and at least 200 cells were evaluated on a hemacytometer after 5 min. Alternatively, samples were exposed to a final concentration of 0.5 mg/ml MTT (Roche, Mannheim, Germany), cleaved to its dark blue formazan derivate by mitochondrial dehydrogenases. After 4 h, the reaction was stopped by addition of 10% SDS in 0.01 M HCl and samples were incubated overnight at 37°C to solubilize formazan crystals. Aliquots of 300 µl were transferred to 96-well plates, and optical density (OD) at 570 nm was determined in duplicate using an automated multiplate reader (Sunrise, Tecan, Crailsheim, Germany) and Magellan software (Version 2.5, Tecan). The influence of perfluorohexane on MBC viability was assessed by relating the OD of perfluorohexane-treated cell suspensions to the corresponding controls.
A possible interference of perfluorohexane with ConA such as a binding or inactivation of the stimulant was determined by means of ConAs ability to induce TF generation after being preexposed to perfluorohexane. Cell-free RPMI medium containing 5% FCS and 5 µg/ml of ConA was preincubated with 5, 10, or 30% (vol/vol) perfluorohexane for 90 min at 37°C and continuous agitation in a rotating shaker. After incubation, samples were centrifuged (275 g, 10 min) to layer out perfluorohexane. MBC pellets (1 x 106 cells) were resuspended in either 1 ml of perfluorohexane-free supernatant or an equal volume of culture medium containing 5 µg/ml of ConA (controls). After incubation for 24 h (37°C, 5% CO2), samples were prepared for TF determination as described above.
IL-2 and TF ELISA.
Release of IL-2 by ConA-stimulated MBC was quantified by means of a commercially available ELISA (R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions. All samples were measured in duplicate using an automated multichannel ELISA reader (Titertek MS2, ICN, Eschwege, Germany).
TF antigen was determined using a sandwich-type ELISA with two monoclonal antibodies as described previously (2). Briefly, cells were disrupted by two cycles of freezing and thawing followed by solubilization of TF in 100 µl of lysis buffer [50 mM Tris·HCl, 100 mM NaCl, 1% (vol/vol) Triton X-100, pH 7.6] for 20 min (final volume of 0.5 ml). Microtiter plates (Maxisorp, Nunc, Wiesbaden, Germany) coated with purified anti-TF monoclonal antibody VIC7 (2.5 µg/ml) were incubated for 2 h at 37°C with cell lysates, macroscopically free of perfluorohexane, diluted 1:1 in sample buffer [50 mM Tris·HCl, 100 mM NaCl, 0.2% (vol/vol) Triton X-100, 1% (wt/vol) bovine serum albumin, pH 7.6]. Twofold serial dilutions of standard recombinant TF (American Diagnostica, Greenwich, CT) in sample buffer were added as a reference standard. After incubation with peroxidase-labeled anti-TF monoclonal antibody IIID8 (90 min, 37°C) and subsequent substrate reaction with 3,3'5,5'-tetramethylbenzidine (K&P Laboratories, Gaithersburg, MD) for 20 min, the absorbance was measured at 450 nm with the ELISA reader. TF values were expressed as nanograms TF per 106 cells. In preceding experiments with TF standards and perfluorohexane, an interference of the agent with the TF assay could be excluded.
Interaction of perfluorohexane with the binding of ConA to MBC.
To study a potential interaction between perfluorohexane and lectin binding to cellular membranes, experiments with fluorescence-labeled ConA were performed using flow cytofluorometry and fluorescence microscopy. For flow cytofluorometry, MBC (1 x 106 cells suspended in 1 ml RPMI containing 5% FCS) were stimulated with FITC-conjugated ConA (Sigma) at a final concentration of 5 µg/ml, immediately followed by addition of 5, 10, or 30% (vol/vol) perfluorohexane. Samples were vortexed and incubated for 15 min under standard culture conditions. Additional experiments quantified cell-bound FITC-ConA on MBC that had been stimulated for 15 min after 4-h preexposure to perfluorohexane. Due to water insolubility of perfluorohexane, respective controls consisted of FITC-ConA-stimulated cells without further treatment. After incubation, samples were vortexed and 600 µl of the cell suspensions were removed after perfluorohexane had sedimented spontaneously due to its high density. Cells were washed twice in PBS, fixed, and permeabilized using Cytofix/Cytoperm solution (BD Pharmingen, Heidelberg, Germany). To exclude cell debris or microscopic perfluorohexane particle interference, DNA staining was performed with propidium iodide (red fluorescence). Cell-bound FITC-ConA was determined by flow cytofluorometry (FACScan, Becton Dickinson, Heidelberg, Germany) at an excitation wavelength of 488 nm. Before data acquisition, a gate was set in the red fluorescence channel (FL-3) dot plot to count only events that had at least the DNA content of human diploid cells. Green fluorescence of 20,000 cells was measured in FL-1, and data were processed using WinMDI 2.8 (by J. Trotter, Scripps Research Institute, La Jolla, CA). Separate analyses were carried out for populations of lymphocytes and monocytes that could be discriminated by their characteristic size-to-granularity ratio in the forward vs. sideward scatter plot.
To elucidate whether perfluorohexane influences the pattern of lectin binding, MBC were exposed to 30% (vol/vol) perfluorohexane while being stimulated with FITC-ConA for 15 min at 37°C and then were examined using fluorescence microscopy. Samples were prepared following the same experimental procedure as described for flow cytofluorometry except that cells were fixed in 2% paraformaldehyde without subsequent propidium iodide staining. After cells were transferred to Lab-Tek coverglass slides (Nunc), fluorescence microscopy was conducted using an inverse microscope (DMIRB, Leica, Bensheim, Germany) equipped with a high-resolution color charge-coupled device camera (arc6000c, Baumer Optronic, Radeberg, Germany). Frames were taken at a 100-fold magnification and transferred to a personal computer with image viewer software provided by the manufacturer of the camera.
Data analysis.
Data are presented as means ± SD. Results of ELISA are expressed as percentage of perfluorohexane-treated cells compared with nonexposed controls. Differences between perfluorohexane-incubated samples and corresponding controls in terms of TF generation and IL-2 expression were evaluated with the paired Student's t-test, respectively. Unless stated otherwise, the Kruskal-Wallis test was applied to detect the statistical association between groups. Calculations were performed using the statistical package SPSS 11.0 (SPSS, Chicago, IL) and significance was accepted at P < 0.05.
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RESULTS
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Biocompatibility of perfluorohexane.
After preparation and resuspension in cell culture medium, MBC had a mean viability of >98%. During incubation for up to 24 h, >95% of the cells remained vital in the presence of ConA and perfluorohexane as assessed by trypan blue exclusion (Table 1). Treatment of samples with perfluorohexane was not associated with a significant impairment of cell viability compared with corresponding controls. Biocompatibility of perfluorohexane could be confirmed in additional experiments measuring the MTT mitochondrial dehydrogenases turnover (n = 7). Mitochondrial activity of perfluorohexane-exposed MBC tended to be higher than that of control cells, although differences did not reach statistical significance for all time points (Table 2).
Effect of perfluorohexane on ConA-induced activation of MBC.
The interaction of perfluorohexane with ConA-induced activation of monocytes and lymphocytes was evaluated by means of TF expression and IL-2 secretion, respectively (n = 12). After 6-h incubation, TF analysis by ELISA revealed a significantly decreased monocytic TF content in samples containing 30% (vol/vol) perfluorohexane (0.61 ng/106 cells) compared with corresponding ConA-stimulated controls without perfluorohexane (0.86 ng/106 cells; Fig. 1A). Similarly, perfluorohexane significantly reduced cellular TF expression after 24-h ConA stimulation at all tested concentrations (Fig. 1A). In contrast to the moderately inhibited response of monocytes to lectin stimulation, ConA-induced activation of lymphocytes was substantially diminished by perfluorohexane. Exposure of MBC to perfluorohexane significantly decreased lymphocyte IL-2 secretion after 6 and 24 h by 3966% and 5271%, respectively (Fig. 1B). For both time points, this effect was dependent on the perfluorohexane concentration that was applied (Kruskal-Wallis test, P < 0.05). However, perfluorohexane itself did not induce an activation of MBC as confirmed by very low TF content of control cells and cells treated with different concentrations of perfluorohexane in the absence of ConA (TF content < 0.03 ng/106 cells in all groups). In addition, IL-2 concentrations were below the detection limit of the sensitive ELISA assay in unstimulated probes both with and without perfluorohexane exposure.

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Fig. 1. Bar graph depicting response of concanavalin A (ConA; 5 µg/ml)-stimulated mononuclear blood cells in terms of monocytic tissue factor (TF) expression (A) and lymphocytic IL-2 secretion (B) after incubation with different concentrations of perfluorohexane (PFH). Bars show values of PFH-treated probes normalized to respective controls, set as 100%. Data represent means ± SD of 12 independent experiments. P < 0.05.
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A possible interference of perfluorohexane with ConA such as a binding or sequestering of the stimulant was determined by means of ConA's ability to induce TF generation after being preexposed to perfluorohexane (n = 4). Compared with 5 µg/ml of ConA without prior perfluorohexane exposure, the same ConA concentration that was preconditioned with 5, 10, and 30% (vol/vol) perfluorohexane induced in MBC a normalized TF expression of 100.8 ± 16.9, 97.3 ± 6.3, and 92.3 ± 5.5%, respectively. As individual group values did not differ significantly, an inhibitory effect of perfluorohexane due to nonspecific binding or inactivation of the stimulatory agent ConA could be excluded.
Interaction of perfluorohexane with ConA binding to cellular membranes.
To further elucidate the observed MBC hyporesponsiveness to lectin activation during perfluorohexane exposure, membrane binding of ConA was evaluated with and without perfluorohexane (n = 7). Independent of the treatment and the MBC subpopulations studied, >99.5% of the cells were positive for fluorescence-labeled ConA in flow cytometrical analysis. The amount of FITC-ConA bound to monocytes after 15 min did not differ significantly between perfluorohexane-treated groups and corresponding controls (Fig. 2A). Likewise, additional perfluorohexane preincubation of the cells 4 h before FITC-ConA stimulation did not significantly alter membrane binding of the stimulant (Fig. 2A). Compared with monocytes, the mean fluorescence of lymphocytes was
10 times lower. In this cell population, perfluorohexane again did not induce a significant modulation of ConA binding after 15-min (with and without preincubation for 4 h) incubation (Fig. 2B).

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Fig. 2. Bar graph showing the amount of FITC-conjugated ConA (5 µg/ml) bound to the surface of monocytes (A) and lymphocytes (B). Bars depict arbitrary units of fluorescence intensity (mean channel) in controls and PFH-treated groups after 15-min incubation. Another set of experiments analyzed FITC-ConA binding of mononuclear blood cells preincubated with 0 (control), 5, 10, and 30% (vol/vol) PFH for 4 h and subsequent incubation with FITC-ConA for 15 min (group 15 min, PFH preincubated). Data are means ± SD of 7 independent experiments.
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In experiments using fluorescence microscopy, two subpopulations of MBC could grossly be distinguished. Whereas one set of smaller cells had a comparably weak fluorescence, the other exhibited an overall higher FITC intensity (Fig. 3). According to data from flow cytofluorometry, visualized cells corresponded in terms of size and fluorescence intensity to lymphocytes and monocytes, respectively. ConA is known to induce a typical redistribution of its receptors known as clustering and capping (31). After 15-min incubation, analysis of fixed MBC revealed cells in different stages of this process ranging from homogenously distributed FITC-ConA receptor complexes and clusters of different size to nearly complete cap formation. However, no different pattern of ConA binding could be observed in controls (Fig. 3A) and perfluorohexane-treated cells (Fig. 3B).

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Fig. 3. Fluorescence microscopy images of fixed mononuclear blood cells incubated with FITC-ConA (5 µg/ml) for 15 min. Overall, monocytes (Mo) and lymphocytes (Ly) can be distinguished by means of their size and total fluorescence intensity. Typical ConA-induced rearrangement of ligand-receptor complexes such as clustering (arrows) and cap formation (arrowheads) can be observed. Pattern of ConA binding did not differ between controls (A) and cells concomitantly exposed to 30% (vol/vol) PFH (B). Images (100-fold magnification) are representative of 4 independent experiments.
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DISCUSSION
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Liquid ventilation with PFC represents one experimental approach to improve pulmonary function and to reduce mortality in acute lung injury. In this hyperinflammatory condition, application of PFC reduced pulmonary neutrophil sequestration and concomitantly attenuated lung tissue damage (7, 27, 28). Furthermore, animals and patients treated with liquid or aerosolized PFC exhibited decreased markers of inflammation in bronchoalveolar lavage fluid, alveolar macrophages, and pulmonary parenchymal cells (8, 29, 36, 37). As one explanation for such beneficial effects, direct anti-inflammatory properties of these substances have been assumed. Accordingly, several studies could show decreased chemotaxis, adherence, release of reactive oxygen species, and cytokine secretion of activated phagocytic cells after exposure to PFC (4, 19, 26, 32, 34). However, the cellular mechanism of this inhibition is not known. To further elucidate the interaction between PFC and inflammatory cells, we examined the effects of liquid perfluorohexane on lectin-stimulated MBC using an in vitro model. In the present study, we observed that perfluorohexane exposure substantially decreased the response of lymphocytes to ConA activation in terms of IL-2 secretion. In contrast, the inhibition of monocytic TF expression by perfluorohexane was less pronounced, similar to a previous study using LPS (19). The perfluorohexane effect was due neither to ConA binding or sequestering nor to cellular viability impairment as could be demonstrated by established tests of cell integrity and mitochondrial dehydrogenases activity. The obviously good biocompatibility of the substance in our study is in line with other reports that showed the ingestion of PFC by monocytic cells and neutrophils without any signs of cellular damage, both in the phagocytes themselves and in surrounding tissues (5, 6, 19).
As noted before, previous publications reported a compromised function of predominantly phagocytic cells such as neutrophils, peripheral blood monocytes, and macrophages after treatment with different PFC. In some papers, the inhibitory effect was explained by an uptake of considerable amounts of PFC particles by phagocytes leading to cell activation and a subsequent refractory state to further stimulation (12, 18). However, perfluorohexane added to MBC suspensions in the absence of ConA did not activate monocytes in our present experiments. These results confirm observations of Thomassen et al. (34), who found no induction of TNF-
, IL-1, and IL-6 in human alveolar macrophages that were incubated for 24 h with perfluorooctylbromide, the most extensively studied PFC. In a preliminary analysis of our group, video-enhanced microscopy as well as electron microscopy did not reveal any visible interaction between perfluorohexane and lymphocytes, i.e., adherence or uptake of substance particles. Therefore, we were surprised to notice that after lectin stimulation the response of lymphocytes was influenced by perfluorohexane to a far greater extent than that of monocytic phagocytes. Although PFC moderately inhibited TF expression in the latter population, IL-2 secretion of treated lymphocytes decreased substantially in a dose-dependent manner. Therefore, it could be deduced from our findings that ingestion of PFC is not mandatory for its biological action. Studies showing anti-inflammatory effects of perfluoroocytylbromide on stimulated endothelial cells corroborate this assumption (40).
In addition to an uptake of PFC by phagocytic cells, a partitioning into biological membranes has been demonstrated recently (24). In an in vitro study with different PFC, this was associated with a decreased agonist-induced aggregation of platelets and osmotic hemolysis of erythrocytes. Such nonspecific effects on anucleate cells virtually exclude an involvement of nucleus-dependent processes (transcription, translation) and suggest a direct membrane-mediated mechanism of action. In these experiments, PFC had similar gross physical properties but differed in their respective lipid solubility by a factor of 800. Thereby, Obraztsov and co-workers (24) noted a linear relationship between the magnitude of inhibition and the lipid solubility of the individual PFC. Accordingly, other investigators also found cellular PFC effects that correlated with the agent's lipophilicity but were independent of density and vapor pressure otherwise (23, 39). Perfluorohexane has relatively poor lipid solubility, about four times less than that of perfluorooctylbromide. Considering the data of Obraztsov and co-workers, one would expect only slight to moderate anti-inflammatory effects of perfluorohexane plateauing at an inhibition by
20%. Indeed, this is the case with respect to attenuation of monocytic TF expression. However, perfluorohexane exposure strikingly decreased IL-2 secretion by
4070%, pointing toward another mechanism of PFC action independent of lipid solubility. The same conclusion can be drawn from the dose dependency of IL-2 inhibition at 530% (vol/vol) perfluorohexane because PFC partitioning into cellular membranes saturates at remarkably low concentrations.
We and other investigators speculated that PFC exert their cellular effects by altering the properties of lipid and protein membrane components (13, 19, 33). As can be assumed from their hydrophobic nature, PFC were shown by nuclear magnetic resonance (NMR) spectroscopy to be localized in the bilayer acyl chain region near the membrane center (10). In that study, no effect of PFC membrane intercalation on lipid chain dynamics and lipid headgroup conformation was observed. However, PFC may interact with surface proteins responsible for the initiation of stimulus-induced cell activation. To address this assumption, we examined binding characteristics of FITC-conjugated ConA to MBC using flow cytofluorometry and fluorescence microscopy. ConA, a plant lectin with an affinity to a wide range of mannosylated glycoproteins on the cell surface, represents an established tool to study cell membrane structure and dynamics such as the mobility and redistribution of membrane protein receptors (30). Perfluorohexane decreased the response of lymphocytes and monocytes to ConA stimulation. In contrast, the magnitude of lectin surface binding in both populations did not differ significantly between cells treated with perfluorohexane and respective controls. Similarly, additional preincubation of MBC with perfluorohexane for 4 h before lectin stimulation did not influence ConA binding compared with a concomitant application of both agents. Depending on cell population and culture conditions, ConA has been described to induce definite rearrangements of its receptor molecules known as clustering and cap formation (31, 38). These processes reflect lateral mobility of membrane proteins and are actively influenced by anchoring elements of the cytoskeleton (1). In accordance with other studies, in fluorescence microscopy we observed ConA ligand-receptor complexes aggregating to clusters of different size and forming large caps in some of the cells. Because cap formation is time dependent (1030 min) and described to be present in a variable portion of phagocytic cells and lymphocytes (9, 20, 31), fixation of samples after 15 min would explain why cells were in different stages of ConA receptor distribution in our experiment. However, characteristic lectin-induced clustering and capping did not differ morphologically between probes exposed to 30% (vol/vol) perfluorohexane and controls. It can be concluded from these findings that inhibition of MBC activation does not result from a major alteration of receptor density, mobility, or ConA affinity to glycosylated surface proteins. Correspondingly, in formyl-Met-Leu-Phe-stimulated neutrophils the number of respective receptors and ligand-receptor affinity was not found to be altered by PFC (11). On the other hand, our findings are not in accordance with results from Gunnarson and co-workers (13), who reported an impaired binding of anti-CD45 antibodies to the neutrophil surface, reflecting decreased receptor density or binding affinity.
During intrapulmonary application of PFC, different mechanisms may contribute to the observed anti-inflammatory effect. On the one hand, liquid ventilation could prevent ventilator-associated lung injury and thus diminish secondary lung inflammation. On the other hand, instillation of dense, water-insoluble PFC in a dose adequate to lung functional residual capacity is likely to lavage alveolar edema, leukocytes, and debris and to establish a barrier between these proinflammatory components and the alveolar epithelium. Whereas some authors exclusively hold such a mechanical approach responsible for protective PFC effects (3, 14, 35), attenuation of inflammatory lung injury after aerosolized and vapoprized PFC points toward another mechanism. In the present in vitro study, we provide further evidence that PFC can directly influence cellular activation. Therefore, this is the first report demonstrating an attenuated response of stimulated lymphocytes after exposure to a PFC. Even though the particular mechanism of PFC-cell interaction could not be determined, the data contradict our primary hypothesis of an altered surface receptor conformation with inhibited stimulant binding. Considering previous observations of decreased cell activation at the mRNA level (19, 36), PFC may interfere with surface receptor function or signal transmission, for example, by modifying membrane ion permeability. A study by Fernandez and co-workers (11) similarly localized the probable point of PFC effects to this step of cellular activation. Therein the authors demonstrated in human neutrophils that perfluorooctylbromide inhibits both total cytosolic tyrosine phosphorylation and phosphorylation of an intracellular signaling protein necessary for chemotaxis and phagocytosis. In contrast, respective ligand binding was not influenced by PFC, suggesting an interference with transmembrane signal transduction as the underlying mechanism.
In conclusion, in the present study we for the first time observed a decreased response of in vitro stimulated lymphocytes after exposure to different concentrations of perfluorohexane. Additionally, perfluorohexane significantly inhibited monocyte activation, although compared with lymphocytes to a lower degree. Thus further evidence is provided for PFC having intrinsic anti-inflammatory properties. The immune-modulating effects of PFC might be beneficial in pulmonary hyperinflammation, e.g., ARDS, and may introduce PFC as a therapeutical strategy in other illnesses. The mechanism of PFC attenuating cell activation remains unclear. Our data, however, argue against a PFC-altered stimulant-receptor interaction on the surface membrane.
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GRANTS
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This work was supported by a grant from the Roland Ernst Stiftung für Gesundheitswesen.
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ACKNOWLEDGMENTS
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The authors thank S. Thieme, A. Zobjack (Institute of Pathology, Technical University Dresden), and S. Boettcher (Clinic of Anesthesiology) for excellent technical assistance.
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FOOTNOTES
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Address for reprint requests and other correspondence: D. Haufe, Dept. of Anesthesiology and Intensive Care Medicine, Univ. Hospital Carl Gustav Carus, Technical Univ. Dresden, Fetscherstrasse 74, 01307 Dresden, Germany (E-mail: Dirk.Haufe{at}uniklinikum-dresden.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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