Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.1202601 on May 22, 2003

Published online before print May 22, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1202601v1
74/1/95    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tonks, A. J.
Right arrow Articles by Jackson, S. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tonks, A. J.
Right arrow Articles by Jackson, S. K.
(Journal of Leukocyte Biology. 2003;74:95-101.)
© 2003 by Society for Leukocyte Biology

Regulation of platelet-activating factor synthesis in human monocytes by dipalmitoyl phosphatidylcholine

Amanda J. Tonks*, Alex Tonks*, Roger H. K. Morris{dagger}, Kenneth P. Jones{dagger} and Simon K. Jackson{ddagger}

Departments of
* Haematology and
{ddagger} Medical Microbiology, University of Wales College of Medicine, Cardiff, United Kingdom; and
{dagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Platelet-activating factor (PAF) has a major role in inflammatory responses within the lung. This study investigates the effect of pulmonary surfactant on the synthesis of PAF in human monocytic cells. The pulmonary surfactant preparation Curosurf® significantly inhibited lipopolysaccharide (LPS)-stimulated PAF biosynthesis (P<0.01) in a human monocytic cell line, Mono mac-6 (MM6), as determined by 3H PAF scintillation-proximity assay. The inhibitory properties of surfactant were determined to be associated, at least in part, with the 1,2-dipalmitoyl phosphatidylcholine (DPPC) component of surfactant. DPPC alone also inhibited LPS-stimulated PAF biosynthesis in human peripheral blood monocytes. DPPC treatment did not affect LPS-stimulated phospholipase A2 activity in MM6 cell lysates. However, DPPC significantly inhibited LPS-stimulated coenzyme A (CoA)-independent transacylase and acetyl CoA:lyso-PAF acetyltransferase activity. DPPC treatment of MM6 cells decreased plasma membrane fluidity as demonstrated by electron paramagnetic resonance spectroscopy coupled with spin labeling. Taken together, these findings indicate that pulmonary surfactant, particularly the DPPC component, can inhibit LPS-stimulated PAF production via perturbation of the cell membrane, which inhibits the activity of specific membrane-associated enzymes involved in PAF biosynthesis.

Key Words: inflammation • lung • lipid • immune modulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute respiratory distress syndrome (ARDS) is a condition associated with high mortality [1 ], occurring most frequently in the setting of sepsis, trauma, gastric aspiration, or multiple transfusions [2 ]. ARDS is characterized by acute lung injury, hypoxia, and pulmonary edema. Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycerophosphocholine) plays a pivotal role in the pathophysiology of ARDS [3 ], with levels of PAF elevated in the serum and bronchoalveolar lavage fluid (BALF) of these patients [4 , 5 ] and undetectable in control subjects. This potent vasoactive lipid mediator has diverse biological activities and cellular origins. Virtually all of these activities are mediated via a ubiquitously expressed, specific receptor (PAF-R) [6 ]. Biosynthesis of PAF can occur via two distinct pathways, the de novo pathway and the remodeling pathway. The de novo pathway regulates physiological levels of PAF and is most active within cells of the kidney, brain, liver, spleen, and reproductive organs [7 ], and the remodeling pathway is responsible for the formation of PAF in response to inflammatory and immune stimuli [8 ]. The two biosynthetic pathways are associated with distinct enzyme activities. This distinction has allowed the study of the different pathways under physiological and pathological conditions and offers a possible means of targeting PAF biosynthesis in response to inflammatory stimuli. The remodeling pathway involves two processes: first, the generation of a precursor of PAF, alkyl-lyso-PAF, and then the acetylation of this molecule to form PAF. The generation of lyso-PAF involves two enzyme activities, namely phospholipase A2 (PLA2) and a coenzyme A-independent transacylase (CoA-IT). The final step in the sequence is catalyzed by acetyl CoA:lyso-PAF acetyltransferase (AT).

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).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All cell culture reagents were purchased from Invitrogen (Paisley, UK) unless otherwise stated. 1,2-DPPC, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine (type III from egg yolk), sphingomyelin (from chicken egg yolk), cholesterol, LPS from Escherichia coli O111:B4, bovine serum albumin (BSA), 1-alkyl-2-lyso-PC, 1-O-alkyl-L-{alpha}-lyso-PC (lyso-PAF), and 1-O-alkyl-2-acetyl-L-{alpha}-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{perp})/25G, where A|| and A{perp} 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Using MM6 cells as a model for human monocyte/macrophages, the effect of incubation with pulmonary surfactant and phospholipids on LPS-stimulated PAF biosynthesis was investigated, and optimal stimulatory conditions were determined before examination of the effects of surfactant and its phospholipid components on PAF synthesis.

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).



View larger version (29K):
[in this window]
[in a new window]
  		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; {dagger}, P<0.01; {ddagger}, P<0.001, analyzed by ANOVA and Tukey’s pair-wise comparisons.)

 
DPPC inhibits PAF biosynthesis in MM6 cells and PBMs
As DPPC is the major constituent of pulmonary surfactant, its effect on PAF biosynthesis was investigated. In cells incubated with DPPC, dose-dependent inhibition of PAF biosynthesis was observed. Maximal inhibition of PAF production was observed in cells incubated with DPPC at a concentration of 500 µg/ml. At this concentration, LPS-stimulated PAF synthesis was inhibited by 77% (Fig. 2 ) (P<0.05), again reducing PAF production to near basal levels. When human PBMs, the precursors of alveolar macrophages, were incubated with DPPC at 250 µg/ml for 2 h before stimulation of PAF synthesis with LPS, synthesis was significantly inhibited (P<0.05; results not shown). This suggests that DPPC inhibits PAF biosynthesis in PBMs in a similar manner to that seen in MM6 cells.



View larger version (24K):
[in this window]
[in a new window]
  		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.)

 
DPPC inhibits PAF biosynthesis via enzymes of the remodeling pathway
To investigate possible mechanisms by which DPPC could inhibit PAF production, the activities of the enzymes concerned with PAF generation in monocytes were investigated. Incubation of MM6 cells in the presence of LPS significantly increased the activity of PAF biosynthetic enzymes compared with unstimulated cells, as determined by ANOVA and Tukey’s pair-wise comparison. PLA2 activity in MM6 cell lysates was found to be unaffected by DPPC incubation (results not shown). However, the activities of CoA-IT and AT were inhibited in a dose-dependent manner following incubation with DPPC. Incubation of cells with 100 or 500 µg/ml DPPC resulted in complete inhibition of LPS-stimulated CoA-IT activity, resulting in enzyme activity lower than that observed in unstimulated cells, indicating that incubation with DPPC effectively abolished the CoA-IT activity associated with LPS stimulation (Fig. 3 ).



View larger version (27K):
[in this window]
[in a new window]
  		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; {dagger}, P<0.01, analyzed by ANOVA and Tukey’s pair-wise comparisons.)

 
In addition, DPPC inhibited acetyl CoA:lyso-PAF acetyl transferase activity in LPS-stimulated MM6 cells. Again, this inhibition was dose-dependent, and maximal inhibition occurred when cells were incubated with 500 µg/ml (P<0.01; Fig. 4 ), resulting in complete inhibition of acetyl CoA:lyso-PAF acetyltransferase activity. When DPPC was added directly to a cell-free preparation containing these enzymes, it had no effect on either enzyme activity, indicating that an intact cell was necessary for the action of DPPC.



View larger version (29K):
[in this window]
[in a new window]
  		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; {dagger}, P<0.01, analyzed by ANOVA and Tukey’s pair-wise comparisons.)

 
DPPC incubation reduces plasma membrane fluidity
Incubation of MM6 cells with DPPC for 2 h resulted in an increase in the order parameter (S) of spin-labeled cells. Maximal increase was seen in cells incubated with 500 µg/ml DPPC, where S increased to 0.7904 compared with 0.7064 in cells incubated in the absence of DPPC (Table 1 ). This increase in S (~11%) equates to a significant decrease in membrane fluidity [34 ].


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of Phospholipid Incubation on Membrane Fluidity

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PAF has been shown to play a major role in the development of lung injury in ARDS, independent of the underlying etiology of the disease [1 , 9 , 35 ]. PAF is undetectable within the healthy lung but elevated in BALF of patients with ARDS. The presence of PAF in ARDS is accompanied by derangements in pulmonary surfactant composition. Clinical studies have reported a significant decrease in phospholipid content and alterations in the distribution of phospholipid classes, specifically a decrease in the relative amount of DPPC present in ARDS patients [19 ]. This, together with the absence of PAF in healthy subjects, suggest that under physiological conditions, PAF synthesis may be inhibited [4 , 5 ]. Alterations in the quantity and composition of surfactant present within the lung in acute inflammatory episodes are a consequence of damage to alveolar type II cells, the cellular source of surfactant [19 ] and a source of PAF-AH, the enzyme responsible for inactivation of PAF [36 ]. Pulmonary surfactant phospholipids have been shown previously to modulate immune responses [15 , 37 ], but their effect on PAF biosynthesis has not been previously characterized. The present study provides evidence that at physiologically relevant concentrations, pulmonary surfactant phospholipid, specifically DPPC, modulates the biosynthesis of PAF in human monocytic cells stimulated with LPS; this effect is dose-dependent. This suggests a role for surfactant in the healthy lung. Further, DPPC affects PAF production via inhibition of two specific enzymes of the remodeling pathway: CoA-independent transacylase and acetyl CoA:lyso-PAF acetyltransferase. These enzymes are membrane-associated and important in the production of PAF in response to inflammatory stimuli [8 ]. The present study also demonstrates that MM6 cells synthesize significant quantities of PAF in response to LPS stimulation with a pattern of synthesis similar to that previously reported in the murine macrophage cell line IC-21 [23 ] and in human monocytes [38 ]. These cells are a relevant model of human PBMs, the direct precursors of alveolar macrophages, as they exhibit phenotypic, morphological, and functional characteristics of mature monocytes [39 ].

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.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nagase, T., Ishii, S., Kume, K., Uozumi, N., Izumi, T., Ouchi, Y., Shimizu, T. (1999) Platelet-activating factor mediates acid-induced lung injury in genetically engineered mice J. Clin. Invest. 104,1071-1076[Medline]
  2. Mortelliti, M. P., Manning, H. L. (2002) Acute respiratory distress syndrome Am. Fam. Physician 65,1823-1830[Medline]
  3. Miotla, J. M., Jeffery, P. K., Hellewell, P. G. (1998) Platelet-activating factor plays a pivotal role in the induction of experimental lung injury Am. J. Respir. Cell Mol. Biol. 18,197-204[Abstract/Free Full Text]
  4. Nakos, G., Kitsiouli, E. I., Tsangaris, I., Lekka, M. E. (1998) Bronchoalveolar lavage fluid characteristics of early intermediate and late phases of ARDS. Alterations in leukocytes, proteins, PAF and surfactant components Intensive Care Med. 24,296-303[CrossRef][Medline]
  5. Matsumoto, K., Taki, F., Kondoh, Y., Taniguchi, H., Takagi, K. (1992) Platelet-activating factor in bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome Clin. Exp. Pharmacol. Physiol. 19,509-515[Medline]
  6. Kuijpers, T. W., van der Poll, T. (1999) The role of platelet-activating factor in endotoxin related disease Brade, H.et al eds. Endotoxin in Health and Disease ,449-579 Marcel Dekker New York, NY.
  7. Snyder, F. (1995) Platelet-activating factor and its analogs: metabolic pathways and related intracellular processes Biochim. Biophys. Acta 1254,231-249[Medline]
  8. Yamashita, A., Sugiura, T., Waku, K. (1997) Acyltransferases and transacylases involved in fatty acid remodeling of phospholipids and metabolism of bioactive lipids in mammalian cells J. Biochem. (Tokyo) 122,1-16[Abstract/Free Full Text]
  9. Held, H. D., Uhlig, S. (2000) Mechanisms of endotoxin-induced airway and pulmonary vascular hyperreactivity in mice Am. J. Respir. Crit. Care Med. 162,1547-1552[Abstract/Free Full Text]
  10. Braquet, P., Hosford, D. (1989) The potential role of platelet-activating factor (PAF) in shock, sepsis and adult respiratory distress syndrome (ARDS) Prog. Clin. Biol. Res. 308,425-439[Medline]
  11. Clavijo, L. C., Carter, M. B., Matheson, P. J., Wilson, M. A., Wead, W. B., Garrison, R. N. (2001) PAF increases vascular permeability without increasing pulmonary arterial pressure in the rat J. Appl. Physiol. 90,261-268[Abstract/Free Full Text]
  12. Prevost, M. C., Cariven, C., Chap, H. (1988) Possible origins of PAF-acether and lyso-PAF-acether in rat lung alveoli secondary to hypoxia Biochim. Biophys. Acta 962,354-361[Medline]
  13. Walti, H., Polla, B. S., Bachelet, M. (1997) Modified natural porcine surfactant inhibits superoxide anions and proinflammatory mediators released by resting and stimulated human monocytes Pediatr. Res. 41,114-119[Medline]
  14. Wilsher, M. L., Hughes, D. A., Haslam, P. L. (1988) Immunoregulatory properties of pulmonary surfactant: influence of variations in the phospholipid profile Clin. Exp. Immunol. 73,117-122[Medline]
  15. Tonks, A., Morris, R. H. K., Price, A. J., Thomas, A. W., Jones, K. P., Jackson, S. K. (2001) Dipalmitoyl phosphatidylcholine modulates inflammatory functions of monocytic cells independently of mitogen activated protein kinases Clin. Exp. Immunol. 124,86-94[CrossRef][Medline]
  16. Morris, R. H., Price, A. J., Tonks, A., Jackson, S. K., Jones, K. P. (2000) Prostaglandin E(2) and tumour necrosis factor-alpha release by monocytes are modulated by phospholipids Cytokine 12,1717-1719[CrossRef][Medline]
  17. Speer, C. P., Gotze, B., Curstedt, T., Robertson, B. (1991) Phagocytic functions and tumor necrosis factor secretion of human monocytes exposed to natural porcine surfactant (Curosurf) Pediatr. Res. 30,69-74[Medline]
  18. Veldhuizen, R., Nag, K., Orgeig, S., Possmayer, F. (1998) The role of lipids in pulmonary surfactant Biochim. Biophys. Acta 1408,90-108[Medline]
  19. Gunther, A., Ruppert, C., Schmidt, R., Markart, P., Grimminger, F., Walmrath, D., Seeger, W. (2001) Surfactant alteration and replacement in acute respiratory distress syndrome Respir. Res. 2,353-364[CrossRef][Medline]
  20. Stafforini, D. M., Elstad, M. R., McIntyre, T. M., Zimmerman, G. A., Prescott, S. M. (1990) Human macrophages secret platelet-activating factor acetylhydrolase J. Biol. Chem. 265,9682-9687[Abstract/Free Full Text]
  21. Altman, S. A., Randers, L., Rao, G. (1993) Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations Biotechnol. Prog. 9,671-674[CrossRef][Medline]
  22. Pinot, F., Bachelet, M., Francois, D., Polla, B. S., Walti, H. (1999) Modified natural porcine surfactant modulates tobacco smoke-induced stress response in human monocytes Life Sci. 64,125-134[CrossRef][Medline]
  23. Svetlov, S. I., Liu, H., Chao, W., Olson, M. S. (1997) Regulation of platelet-activating factor (PAF) biosynthesis via coenzyme A-independent transacylase in the macrophage cell line IC-21 stimulated with lipopolysaccharide Biochim. Biophys. Acta 1346,120-130[Medline]
  24. Strieter, R. M., Lynch, J. P., Basha, M. A., Standiford, T. J., Kasahara, K., Kunkel, S. L. (1990) Host responses in mediating sepsis and adult respiratory distress syndrome Semin. Respir. Infect. 5,233-247[Medline]
  25. Fink, M. P. (1998) Therapeutic options directed against platelet activating factor, eicosanoids and bradykinin in sepsis J. Antimicrob. Chemother. 41(Suppl. A),81-94[Abstract/Free Full Text]
  26. Hayakawa, H., Myrvik, Q. N., St.Clair, R. W. (1989) Pulmonary surfactant inhibits priming of rabbit alveolar macrophage. Evidence that surfactant suppresses the oxidative burst of alveolar macrophage in infant rabbits Am. Rev. Respir. Dis. 140,1390-1397[Medline]
  27. Rooney, S. A., Canavan, P. M., Motoyama, E. K. (1974) The identification of phosphatidylglycerol in the rat, rabbit, monkey and human lung Biochim. Biophys. Acta 360,56-67[Medline]
  28. Bligh, E. G., Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37,911-917
  29. Sugatani, J., Lee, D. Y., Hughes, K. T., Saito, K. (1990) Development of a novel scintillation proximity radioimmunoassay for platelet-activating factor measurement: comparison with bioassay and GC/MS techniques Life Sci. 46,1443-1450[CrossRef][Medline]
  30. Sun, X., Caplan, M. S., Hsueh, W. (1994) Tumour necrosis factor and endotoxin synergistically activate intestinal phospholipase A2 in mice. Role of endogenous platelet activating factor and the role of exogenous PAF Gut 35,215-219[Abstract/Free Full Text]
  31. Winkler, J. D., Sung, C. M., Bennett, C. F., Chilton, F. H. (1991) Characterization of CoA-independent transacylase activity in U937 cells Biochim. Biophys. Acta 1081,339-346[Medline]
  32. Darmani, H., Harwood, J. L., Jackson, S. K. (1993) Effect of interferon-gamma on membrane conformation in the macrophage-like cell line P388D J. Interferon Res. 13,427-431[Medline]
  33. Harwood, J. L. (1987) Lung surfactant Prog. Lipid Res. 26,211-256[CrossRef][Medline]
  34. Glover, R. E. (1996) Mechanisms of Endotoxin-Monocyte Interactions: An Electron Paramagnetic Resonance Study. Ph.D. thesis University of Wales College of Medicine Cardiff, UK.
  35. Rabinovici, R., Bugelski, P. J., Esser, K. M., Hillegass, L. M., Vernick, J., Feuerstein, G. (1993) ARDS-like lung injury produced by endotoxin in platelet-activating factor-primed rats J. Appl. Physiol. 74,1791-1802[Abstract/Free Full Text]
  36. Jehle, R., Schlame, M., Buttner, C., Frey, B., Sinha, P., Rustow, B. (2001) Platelet-activating factor (PAF)-acetylhydrolase and PAF-like compounds in the lung: effects of hyperoxia Biochim. Biophys. Acta 1532,60-66[Medline]
  37. Wilsher, M. L., Parker, D. J., Haslam, P. L. (1990) Immunosuppression by pulmonary surfactant: mechanisms of action Thorax 45,3-8[Abstract]
  38. Camussi, G., Mariano, F., Biancone, L., de Martino, A., Bussolati, B., Montrucchio, G., Tobias, P. S. (1995) Lipopolysaccharide binding protein and CD14 modulate the synthesis of platelet-activating factor by human monocytes and mesangial and endothelial cells stimulated with lipopolysaccharide J. Immunol. 155,316-324[Abstract]
  39. Geertsma, M. F., Broos, H. R., van den Barselaar, M. T., Nibbering, P. H., van Furth, R. (1993) Lung surfactant suppresses oxygen-dependent bactericidal functions of human blood monocytes by inhibiting the assembly of the NADPH oxidase J. Immunol. 150,2391-2400[Abstract]
  40. Redenti, E., Peveri, T., Ventura, P., Zanol, M., Selva, A. (1994) Characterization of phospholipidic components of the natural pulmonary surfactant Curosurf Farmaco 49,285-289[Medline]
  41. Hidi, R., Vial, D., Havet, N., Berger, A., Vargaftig, B. B., Touqui, L. (1997) Inhibition by pulmonary surfactant Curosurf of secretory phospholipase A2 expression in guinea-pig alveolar macrophages Biochem. Pharmacol. 54,1055-1058[CrossRef][Medline]
  42. Thomassen, M. J., Meeker, D. P., Antal, J. M., Connors, M. J., Wiedemann, H. P. (1992) Synthetic surfactant (Exosurf) inhibits endotoxin-stimulated cytokine secretion by human alveolar macrophages Am. J. Respir. Cell Mol. Biol. 7,257-260
  43. Moya, F. R., Hoffman, D. R., Zhao, B., Johnston, J. M. (1993) Platelet-activating factor in surfactant preparations Lancet 341,858-860[CrossRef][Medline]
  44. Yanoshita, R., Chang, H. W., Son, K. H., Kudo, I., Samejima, Y. (1996) Inhibition of lysoPAF acetyltransferase activity by flavonoids Inflamm. Res. 45,546-549[CrossRef][Medline]
  45. Winkler, J. D., Fonteh, A. N., Sung, C. M., Heravi, J. D., Nixon, A. B., Chabot-Fletcher, M., Griswold, D., Marshall, L. A., Chilton, F. H. (1995) Effects of CoA-independent transacylase inhibitors on the production of lipid inflammatory mediators J. Pharmacol. Exp. Ther. 274,1338-1347[Abstract/Free Full Text]
  46. Baker, P., Owen, J., Nixon, A., Thomas, L., Wooten, R., Daniel, L., O’Flaherty, J., Wykle, R. (2002) Regulation of platelet-activating factor synthesis in human neutrophils by MAP kinases Biochim. Biophys. Acta 1592,175-184[Medline]
  47. Holland, M. R., Venable, M. E., Whatley, R. E., Zimmerman, G. A., McIntyre, T. M., Prescott, S. M. (1992) Activation of the acetyl-coenzyme A: lysoplatelet-activating factor acetyltransferase regulates platelet-activating factor synthesis in human endothelial cells J. Biol. Chem. 267,22883-22890[Abstract/Free Full Text]
  48. Wiseman, H. (1996) Dietary influences on membrane function: importance in protection against oxidative damage and disease J. Nutr. Biochem. 7,2-15[CrossRef]
  49. Peck, M. D. (1994) Interaction of lipids with immune function I: biochemical effects of dietary lipids on plasma membrane J. Nutr. Biochem. 5,466-478[CrossRef]
  50. Hidalgo, C. (1985) Membrane fluidity and the function of the Ca2+-ATPase of sarcoplasmic reticulum Aloia, R. C. Boggs, J. M. eds. Cellular Aspects 4,51-96 Academic London, UK.



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
S. K. Jackson, W. Abate, J. Parton, S. Jones, and J. L. Harwood
Lysophospholipid metabolism facilitates Toll-like receptor 4 membrane translocation to regulate the inflammatory response
J. Leukoc. Biol., July 1, 2008; 84(1): 86 - 92.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. H. Han, J. H. Kim, H. S. Seo, M. H. Martin, G.-H. Chung, S. M. Michalek, and M. H. Nahm
Lipoteichoic Acid-Induced Nitric Oxide Production Depends on the Activation of Platelet-Activating Factor Receptor and Jak2
J. Immunol., January 1, 2006; 176(1): 573 - 579.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1202601v1
74/1/95    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tonks, A. J.
Right arrow Articles by Jackson, S. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tonks, A. J.
Right arrow Articles by Jackson, S. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS