Published online before print May 22, 2003
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Departments of
* Haematology and
Medical Microbiology, University of Wales College of Medicine, Cardiff, United Kingdom; and
School of Applied Science, University of Wales Institute Cardiff, United Kingdom
Correspondence: Dr. Amanda J. Tonks, Department of Haematology, 7th Floor A–B Link, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK. E-mail: tonksaj{at}cf.ac.uk
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Key Words: inflammation • lung • lipid • immune modulation
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimentally, ARDS has been induced in animals by intravenous (i.v.) administration of lipopolysaccharide (LPS; an outer membrane constituent of gram-negative bacteria) or by direct administration of hydrochloric acid to the lung. A similar clinical picture is seen when PAF is administered i.v.; PAF-R inhibitors can block many of these effects, significantly attenuating responses [3 , 9 , 10 ]. In addition, the activity of PAF has been suggested to be a prerequisite for pulmonary vascular constriction during gram-negative bacteraemia [11 ]. The alveolar macrophage is believed to act as the initial activator of the inflammatory cascade, being the main source of PAF and other inflammatory mediators within the lung [12 ].
There is increasing evidence to indicate that under normal circumstances, the alveolar environment attenuates immune responses within the lung. Pulmonary surfactant is the lipid-rich material that lines the alveoli and has been shown to affect the functions of immune cells in vitro [13
, 14
]. Specifically, our group and others have determined that the phospholipid component of surfactant attenuates inflammatory responses [15
16
17
]. Reduced responsiveness may be of benefit to the host, as alveolar macrophages continually encounter inhaled foreign matter, and an excessive inflammatory response may result in serious lung-tissue damage. Phospholipids are the predominant components of pulmonary surfactant, and phosphatidylcholine (PC) accounts for up to 70–80% of the total lipid content by weight, 
50% of which is in the form of dipalmitoyl PC (DPPC) [18
]. Pulmonary surfactant function and composition can be deranged in pulmonary disease and are features of ARDS. Commonly, phospholipid content is reduced, and relative distribution of species is altered, leading to an impairment of the surface tension-lowering and perhaps the immunomodulatory properties of surfactant [19
]. As surfactant derangements are associated with inflammatory episodes, and PAF is detectable in such situations, the role of this material in modulating PAF synthesis is worthy of investigation. The present study examines the possible regulatory role of the DPPC component of pulmonary surfactant on the synthesis PAF in the human monocytic cell line Mono mac 6 (MM6).
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-lyso-PC (lyso-PAF), and 1-O-alkyl-2-acetyl-L--PC (PAF) were all purchased from Sigma Aldrich Co. (Gillingham, Dorset, UK). All radioisotopes were purchased from Amersham Biosciences (Little Chalfont, Buckingham, UK). The commercial pulmonary surfactant preparation Curosurf® was obtained from Serono Pharmaceuticals (Feltham, Middlesex, UK). Chloroform, methanol, acetic acid, hexane, and propan-2-ol were analysis grade and purchased from BDH (Poole, Dorset, UK).
Cell culture
The human monocytic cell line MM6 was obtained from the German Collection of Microorganisms and Cell Cultures (DSM ACC 124; Braunschweig, Germany). MM6 cells were maintained in RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum (FBS; Labtech International, Ringmer, East Sussex, UK), 1% 2 mM L-glutamine, 1% nonessential amino acids, 1% penicillin (50 IU/ml)/streptomycin (100 µg/ml), and 1% sodium pyruvate at 37°C in 5% CO2 humidified atmosphere. Cells were subcultured every 3 days at a density of 4 x 105 cells/ml. In experiments determining PAF production, cells were weaned onto serum-free medium, UltracultureTM (BioWhittaker UK, Wokingham, Berkshire; supplemented as for RPMI-1640 media without FBS), in accordance with the manufacturer’s instructions. This media was used, as fetal calf serum is a source of serum PAF-acetylhydrolase (PAF-AH), which metabolizes and inactivates PAF [20
]. Cell viability was routinely assessed by trypan blue exclusion [21
]. Human peripheral blood monocytes (PBMc) were isolated, purified, and cultured as described previously [22
].
Stimulation of PAF synthesis in monocytic cells
LPS was chosen as a stimulus to induce the production of PAF in the present study, as LPS is a natural component of the outer cell wall of gram-negative bacteria, which has previously been shown to induce the synthesis of PAF in monocytes and neutrophils via the remodeling pathway [23
]. In addition, LPS induces the synthesis of PAF and a wide array of other mediators in inflammatory conditions such as sepsis and ARDS [24
, 25
].
Preparation of surfactant and phospholipid-supplemented medium
The concentration of phospholipids in pulmonary surfactant has previously been estimated to be 10–500 µg/ml [26
, 27
]. The effect of incubating MM6 cells with surfactant preparations or surfactant phospholipids, normalized by phospholipid content at physiologically relevant concentrations on PAF biosynthesis, was investigated. Curosurf® was diluted in supplemented RPMI as described above to give the required phospholipid concentrations (10–500 µg/ml). In the case of phospholipids, the desired amount of lipid was dissolved in chloroform (ClCH3), dried as a thin film under nitrogen on ice in sterile, acid-washed bijoux tubes treated with "RepelCote" (BDH), and stored in the dark at -70°C until required. Lipid preparations were hydrated in supplemented medium and sonicated on ice to minimize potential oxidation and breakdown of the lipid and ensure a homogenous solution. In addition to determining the effect of surfactant preparations on PAF biosynthesis in MM6 cells, the effect of PC subclasses and a mixture of phospholipids in the proportion in which they occur in natural pulmonary surfactant on PAF synthesis was examined. In all experiments, monocytic cells were incubated for 2 h with or without phospholipids/surfactant preparation, and cells were then washed (x3) in phosphate-buffered saline (PBS) and resuspended in fresh media before LPS stimulation and assessment of PAF synthesis or enzyme activity.
Determination of PAF biosynthesis
Following appropriate treatment, PAF was extracted from cells and media by a modification of the method of total lipid extraction described by Bligh and Dyer [28
] and was separated by thin-layer chromatography (TLC) on a 20G silica glass plate, against known standards with a mobile phase consisting of chloroform/methanol/water (65:35:7). Bands were visualized with iodine vapor, and those comigrating with authentic PAF were scraped and eluted with a hexane/propan-2-ol/water mixture (30:70:5) in 1% acetic acid. The eluted samples were evaporated to dryness under nitrogen. PAF was quantified using a scintillation proximity radioimmunoassay (SPA; Amersham Biosciences, Little Chalfont, Buckingham, UK). This assay has been reported to be more specific and sensitive than platelet-based bioassays [29
] and does not suffer from the inherent variability associated with platelet-based bioassays. Samples were resuspended in 500 µl assay buffer. An aliquot of 100 µl was placed into a polypropylene scintillation vial, and SPA reagents were added in accordance with the manufacturer’s instructions. Samples were mixed for 18 h on a rotary mixer before scintillation counting on a ß-counter (Rackbeta1211, LKB Wallac, Milton Keynes, Buckinghamshire, UK). A standard curve was constructed using known standards, and PAF concentrations were determined.
Determination of the effects of phospholipid on the biosynthetic enzymes of the remodeling pathway in MM6 cells
The effect of phospholipids on these biosynthetic enzymes was determined to elucidate whether phospholipid modulation of PAF biosynthesis occurs via inhibition of enzymes in the remodeling pathway.
Determination of PLA2 activity in MM6 cell lysates
PLA2 activity potentially associated with the synthesis of PAF was assessed directly in MM6 cell lysates, as described previously [30
]. Briefly, appropriately treated cells were harvested, and cell lysates were prepared in HEPES buffer. Protein was assessed by Coomassie blue G-250 dye-binding assay, and lysates were stored at -80°C until required. A volume equivalent to 50 µg protein was incubated with 1-stearoyl-2-[14C]-arachidonoyl PC for 30 min. Total phospholipids were extracted by the method of Bligh and Dyer [28
] and separated by TLC on a 20G silica glass plate, against known standards, with a mobile phase consisting of chloroform:methanol:ammonium hydroxide (70:30:5). Bands comigrating with authentic 1-stearoyl-2-arachidonoyl PC and arachidonic acid were scraped and assessed by liquid scintillation counting.
Determination of CoA-IT activity
The activity of microsomal CoA-IT was assessed in MM6 cells by a modification of the method of Winkler and co-workers [31
]. Microsomal fractions were prepared from appropriately treated cells, and a volume equivalent to 50 µg protein was incubated in the presence of 0.1 µCi [3H]1-alkyl-2-lyso-PC (Amersham Biosciences) and 1 µM unlabeled 1-alkyl-2-lyso-PC (in assay buffer with 0.25 mg/ml fatty acid-free BSA; Sigma Aldrich Co.), and total volume was 100 µl. The reaction mixture was incubated for 10 min at 37°C; the reaction was terminated by extraction of phospholipids by the method of Bligh and Dyer [28
]. The extracted phospholipids were separated by TLC on K6 silica gel plates (Fisher Scientific, Loughborough, Leicestershire, UK) with a mobile phase of chloroform:methanol:water (65:35:7). Samples were dissolved in 30 µl chloroform and were spiked with 2 µl authentic PC and lyso-PC, which acted as carriers and internal standards. Plates were air-dried before development with iodine vapor. The band corresponding to authentic PC was scraped off, and the radioactivity associated was determined in 5 ml scintillant (LKB Wallac) by liquid scintillation counting on a ß-counter (Rackbeta1211, LKB Wallac). CoA-IT enzyme activity was determined by the comparison of radioactivity of samples with that associated with a known amount of radioactive substrate.
Determination of acetyl CoA:lyso-PAF acetyltransferase activity
The activity of microsomal acetyl CoA:lyso-PAF acetyltransferase was assessed by a modification of the method of Svetlov and co-workers [23
]. Microsomal fractions were prepared from appropriately treated cells, and a volume equivalent to 50 µg protein was incubated in the presence of 2 nmol 14C-acetyl CoA and 2 nmol lyso-PAF in assay buffer containing 0.25 M Tris-HCl and 25 mM BSA at pH 6.9, and total volume was 100 µl. The reaction mixture was incubated for 20 min at 37°C; the reaction was terminated by the extraction and separation of the phospholipids as outlined above. The band corresponding to authentic PAF was scraped off, and the radioactivity associated with it was determined by liquid scintillation counting as described above. Acetyl CoA:lyso-PAF acetyltransferase enzyme activity was determined by the comparison of radioactivity of samples with that associated with a known amount of radioactive substrate.
Measurement of membrane fluidity by electron paramagnetic resonance (EPR) spectroscopy
MM6 cells were incubated with 0, 10, 100, or 500 µg/ml DPPC for 2 h. After washing (x3) in PBS, cells were resuspended in PBS at a density of 5 x 106/ml to which 50 µg/ml 5-doxyl stearic acid [2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy; Sigma Aldrich Co.] in ethanol was added. The concentration of ethanol in the final suspension was less than 1%. The cells were incubated for 15 min at room temperature, followed by washing in PBS (x3) to remove free spin label. Cells were resuspended in 100 µl PBS, and membrane fluidity was measured by EPR spectroscopy as described previously [32
]. The order parameter (S) was calculated from the relationship, S = (A||–A
)/25G, where A|| and A are the maximal- and minimal-coupling constants, respectively, obtained from the EPR spectra of the spin-labeled cells. S can have a value between 0 and 1, where 0 represents unrestricted motion of the probe, and 1 is from an immobilized probe. EPR spectra were obtained using a BrukerEMX EPR spectrometer (Bruker Ltd., Karlsruke, Germany), using 10 m Watts microwave power and a magnetic field of 3383 Gauss at a frequency of 9.54 GHz over a 100G scan range. Alterations in membrane fluidity were assessed by comparing differences in S.
Statistics
For multiple group comparisons, the data were subjected to one-way ANOVA to determine the overall difference between the group means and Tukey’s honestly significant difference for pair-wise differences within group comparisons. Minitab software Version 12.0 (Minitab, State College, PA) was used for all analyses.
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Preincubation of cells with surfactant-inhibited PAF biosynthesis
Incubation of MM6 cells with the surfactant preparation Curosurf® significantly reduced PAF synthesis in a dose-dependent manner. Maximal inhibition (91%) was seen when the cells were incubated with an equivalent of 500 µg/ml phospholipid (P<0.001). This resulted in PAF levels similar to those seen in unstimulated MM6 cells, effectively inhibiting the production of PAF induced by LPS stimulation (Fig. 1
). Phospholipids and neutral lipids make up 99% of Curosurf® (w/w), suggesting that the inhibitory properties of this preparation may be a result of the lipid component of the surfactant. Thus, the role of phospholipids in the modulation of PAF biosynthesis was investigated further. An artificial surfactant mixture was prepared containing only the phospholipid component of surfactant in the ratios in which they appear in human surfactant, as outlined by Harwood [33
]. Incubation of MM6 cells with this "artificial" surfactant preparation significantly inhibited the synthesis of PAF (P<0.05) in response to LPS in MM6 cells in a dose-dependent manner with maximal inhibition when cells were incubated with artificial surfactant at a concentration of 500 µg/ml (results not shown).
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Figure 1. Effect of Curosurf® on LPS-stimulated PAF biosynthesis. MM6 cells were preincubated with Curosurf® at 0, 20, 100, or 500 µg/ml and then incubated with or without LPS (10 µg/ml) for 30 min. PAF production was determined by 3H PAF SPA. Results are mean ± SD of three independent experiments. (*, P<0.05; , P<0.01; , P<0.001, analyzed by ANOVA and Tukey’s pair-wise comparisons.)
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Figure 2. Effect of DPPC species on LPS-stimulated PAF biosynthesis. MM6 cells were preincubated with DPPC at 0, 10, 100, or 500 µg/ml and then incubated with or without LPS (10 µg/ml) for 30 min. PAF production was determined by 3H PAF SPA. Results are mean ± SD of three independent experiments. (*, P<0.05, analyzed by ANOVA and Tukey’s pair-wise comparisons.)
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Figure 3. Effect of DPPC on CoA-IT activity. MM6 cells were incubated with DPPC at 0, 10, 100, or 500 µg/ml for 2 h before incubation with or without LPS (10 µg/ml) for 30 min. Microsomal fractions were prepared, and enzyme activity was assessed. Results are mean ± SD of three independent experiments. (*, P<0.05; , P<0.01, analyzed by ANOVA and Tukey’s pair-wise comparisons.)
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Figure 4. Effect of DPPC on acetyl CoA:lyso PAF acetyltransferase activity. MM6 cells were incubated with DPPC at 0, 10, 100, or 500 µg/ml for 2 h before incubation with or without LPS (10 µg/ml) for 30 min. Microsomal fractions were prepared, and enzyme activity was assessed. Results are mean ± SD of three independent experiments. (*, P<0.05; , P<0.01, analyzed by ANOVA and Tukey’s pair-wise comparisons.)
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11%) equates to a significant decrease in membrane fluidity [34
]. |
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Table 1. Effect of Phospholipid Incubation on Membrane Fluidity
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Incubation of MM6 cells with the surfactant preparation Curosurf® for 2 h before stimulation with LPS (10 µg/ml) resulted in a dose-dependent inhibition of PAF biosynthesis. Curosurf® is a well-defined surfactant preparation [40 ] containing 99% phospholipids and 1% surfactant proteins SP-B and SP-C [17 ]. Curosurf® has been shown previously to inhibit the synthesis of cyclooxygenase and 5-lipoxygenase products including prostaglandin E2 (PGE2), thromboxane B2 (TxB2), and leukotriene C4 (LTC4) in response to a variety of stimuli in human monocytes [13 ] and TxB2, in alveolar macrophages [41 ]. As the major lipid component of surfactant is DPPC, we investigated the role of this phospholipid in modulating PAF synthesis. The current study demonstrated that DPPC significantly inhibited PAF synthesis in a dose-dependent manner. Further, incubation of cells with surfactant or DPPC had no effect on cell viability and has previously been shown to have no effect on morphology or cellular adhesion [42 ]. It is possible that surfactant does not affect the functions of resting macrophages but attenuates the responses of cells activated by environmental stimuli within the alveoli. Curosurf® and all other natural surfactant preparations contain PAF [43 ], and it is possible that a small amount of exogenous PAF contributed to the final measurement, in which case the PAF levels measured may in fact be an overestimate of true PAF synthesis in these treated cells. Thus, the PAF production in cells treated with surfactant are even lower than those measured, supporting the evidence for the anti-inflammatory nature of the material.
Under inflammatory conditions, PAF is synthesized from membrane phospholipids via the remodeling pathway by the actions of three major enzyme activities, PLA2, CoA-IT, and acetyl CoA:lyso-PAF acetyltransferase. DPPC had no effect on PLA2 activity in MM6 cell lysates but inhibited the activity of CoA-IT and acetyl CoA:lyso-PAF acetyltransferase in MM6 cells. Inhibition of either enzyme has been shown previously to inhibit PAF biosynthesis [44 , 45 ]. The coordination and regulation of activation of the enzymes involved in PAF synthesis are poorly understood [46 ]. These enzymes are activated and inactivated by covalent modifications. The activation of acetyl CoA:lyso-PAF acetyltransferase involves phosporylation/dephosphorylation events believed to be initiated via protein kinase C (PKC) [47 ]. Both enzyme activities inhibited by DPPC are associated with cellular membranes and are in the vicinity of their respective substrates, whereas the location of a specific PLA2 associated with PAF synthesis is uncertain. DPPC incubation resulted in a significant decrease in membrane fluidity in MM6 cells. Alterations in membrane fluidity can have profound effects on the conformation and therefore the function of membrane-associated enzymes, such as CoA-IT and acetyl CoA:lyso-PAF acetyltransferase, [48 ]. The activation of cells via numerous receptor-dependent mechanisms is believed to be dependent on clustering receptors on the plasma membrane. Decreasing membrane fluidity retards the formation of such clusters and inhibits cellular activation [49 ] and the activity of membrane-associated enzymes [50 ]. It is possible that the decrease in membrane fluidity induced by DPPC in the current study is associated with inhibition of PAF biosynthesis via inhibition of the activity of CoA-IT and acetyl CoA:lyso-PAF acetyltransferase.
Pulmonary surfactant and specifically, the DPPC component have been shown previously to have immunomodulatory properties, affecting a range of responses of immune-competent cells. In addition, this modulation appears to be independent of stimuli. It is possible that DPPC is inhibiting some common signaling pathways in LPS-treated cells. PKC is a possible candidate; this molecule has been implicated as having a role in PAF synthesis, but the inhibition of reactive oxygen intermediate production, LTC4, and PGE2 synthesis by Curosurf® has been shown to be initiated via a PKC-independent mechanism [13 ], and we have previously shown that DPPC incubation does not affect activity of p38 and p42/p44 mitogen-activated protein kinases in MM6 cells [15 ]. It may be that DPPC has its effects via downstream targets common to a number of signaling pathways. Elucidation of the mechanisms involved in the attenuation of immune-cell responses in the presence of surfactant will improve our understanding of innate-immune responses within the lung. This in turn will enhance our ability to appropriately treat a range of pulmonary diseases, targeting specific inflammatory responses, and may lead to the development of improved therapies.
Received December 10, 2002; revised February 26, 2003; accepted February 27, 2003.
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