Surfactant phospholipid DPPC downregulates monocyte respiratory burst via modulation of PKC

Alex Tonks,1 Joan Parton,2 Amanda J. Tonks,2 Roger H. K. Morris,3 Alison Finall,2 Kenneth P. Jones,3 and Simon K. Jackson2

Departments of 1Haematology and 2Medical Microbiology, School of Medicine, Wales College of Medicine, Cardiff University; and 3School of Applied Sciences, University of Wales Institute, Cardiff, United Kingdom

Submitted 13 October 2004 ; accepted in final form 21 January 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pulmonary surfactant phospholipids have been shown previously to regulate inflammatory functions of human monocytes. This study was undertaken to delineate the mechanisms by which pulmonary surfactant modulates the respiratory burst in a human monocytic cell line, MonoMac-6 (MM6). Preincubation of MM6 cells with the surfactant preparations Survanta, Curosurf, or Exosurf Neonatal inhibited the oxidative response to either lipopolysaccharide (LPS) and zymosan or phorbol 12-myristate 13-acetate (PMA) by up to 50% (P < 0.01). Preincubation of MM6 cells and human peripheral blood monocytes with dipalmitoyl phosphatidylcholine (DPPC), the major phospholipid component of surfactant, inhibited the oxidative response to zymosan. DPPC did not directly affect the activity of the NADPH oxidase in a MM6 reconstituted cell system, suggesting that DPPC does not affect the assembly of the individual components of this enzyme into a functional unit. The effects of DPPC were evaluated on both LPS/zymosan and PMA activation of protein kinase C (PKC), a ubiquitous intracellular kinase, in MM6 cells. We found that DPPC significantly inhibited the activity of PKC in stimulated cells by 70% (P < 0.01). Western blotting experiments demonstrated that DPPC was able to attenuate the activation of the PKC{delta} isoform but not PKC{alpha}. These results suggest that DPPC, the major component of pulmonary surfactant, plays a role in modulating leukocyte inflammatory responses in the lung via downregulation of PKC, a mechanism that may involve the PKC{delta} isoform.

superoxide; signal transduction; dipalmitoyl phosphatidylcholine; inflammation; reactive oxygen intermediate


UNDER NORMAL CONDITIONS, the predominant cell type in the alveolar compartment of the lungs is the alveolar macrophage. Alveolar macrophages coexist with pulmonary surfactant in the liquid lining layer that covers the alveolar surface. These phagocytes are thought to play a central role in the inflammatory response by means of phagocytosis and production of a number of specific mediators including reactive oxygen intermediates (ROIs), lipid metabolites, and cytokines (40). The alveolar surface is very vulnerable to damage related to inflammatory changes. These may result from particulate insults, oxidant gases, or damage secondary to infection. The immune response within this region of the lung must carefully balance pro- and anti-inflammatory responses without compromising host defenses.

Pulmonary surfactant is a complex, multifunctional material produced by type II alveolar epithelial cells, consisting of ~90% lipids and 10% proteins by weight (19, 44). The role of pulmonary surfactant in reducing alveolar surface tension was first elucidated nearly 40 years ago; more recently antibacterial and anti-inflammatory activities have been associated with surfactant (24). There is evidence indicating pulmonary surfactant lipids and surfactant proteins (SP-A, -B, -C, and -D) alter the bactericidal activity of the alveolar macrophage (20, 37). Recent reports suggest surfactant proteins (SP-A and SP-D) regulate a variety of immune cell functions in vitro including enhanced chemotaxis and phagocytosis and alterations in the production of ROIs and cytokines (43). The excessive and inappropriate production of these ROIs can lead to local tissue damage associated with inflammatory conditions including acute respiratory distress syndrome (ARDS), which may be related to changes in surfactant components including phospholipid composition (22).

Dipalmitoyl phosphatidylcholine (DPPC) is the major phospholipid constituent of pulmonary surfactant, accounting for ~60% by weight of total lipids (11). The immunomodulatory role of DPPC within the lung is not fully elucidated, but a number of studies have indicated its importance with respect to inflammatory cell function in addition to its primary role in reducing surface tension (8, 27, 34, 38). In previous studies we have demonstrated that phospholipids, in particular phosphatidylcholine (PC), can modulate the production of ROIs and the potent inflammatory cytokine tumor necrosis factor (TNF)-{alpha} in human monocytes, precursors of macrophages (27). Furthermore, human peripheral blood monocytes cocultivated with a purified DPPC preparation released significantly less ROIs than those monocytes that were exposed to media alone (38). Other reports have also demonstrated the inhibitory effect of pulmonary surfactant on ROI generation in neutrophils (1), peripheral blood monocytes (16), and alveolar macrophages (21).

The mechanisms of respiratory burst inhibition by surfactant have not been well characterized in terms of signaling pathways. The multicomponent enzyme NADPH oxidase is responsible for the production of superoxide (and indirectly, other ROIs). It has been shown that mitogen-activated protein kinases (MAPK) participate in the regulation of oxidant production by phosphorylation of p47phox, a component of the NADPH oxidase (13). However, we have previously shown that DPPC does not attenuate the activation of MAPK (38). It may be possible that DPPC mediates its effects through another kinase. One such protein that has been shown to be involved in respiratory burst activation is protein kinase C (PKC); inhibition of ROI production is easily achieved by the use of the PKC inhibitor staurosporine (45). In addition, PKC, a ubiquitous kinase central to many signaling cascades, has been shown to phosphorylate some components of the NADPH oxidase (14). PKC exists as a family of isozymes, which are differentially expressed in various cells and can be divided into three groups on the basis of their biochemical and structural properties, conventional PKC (PKC{alpha}, {beta}I, {beta}II, and PKC{gamma}), novel PKC (PKC{epsilon}, {eta}, {theta}, and PKC{delta}), and atypical PKC (PKC{zeta} and PKC{lambda}) (30). However, little is known about the role of these specific PKC isozymes in ROI production.

The aim of this study was to examine the cellular mechanisms involved in the modulation of ROI production in the human monocytic cell line MonoMac 6 (MM6) by means of surfactant and, in particular, DPPC. We were specifically interested in determining whether DPPC exerts a direct effect on the intracellular signaling processes at the level of PKC activity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Bisindolylmaleimide I (GF-109203X), Bis-tyrphostin, rottlerin, genistein, and Gö-6976 were purchased from Alexis (Nottingham, UK). Each inhibitor was diluted in RPMI 1640 medium to give a final working concentration that inhibits activity of target proteins by 50%. The synthetic phospholipid L-{alpha}-phosphatidylcholine dipalmitoyl (DPPC), aprotinin, 2-mercaptoethanol, leupeptin, phenylmethylsulfonyl fluoride (PMSF), and Triton X-100 were purchased from Sigma-Aldrich (Poole, Dorset, UK).

Cell culture. The human monocytic cell line MM6 was obtained from the German collection of micro-organisms and cell cultures (DSM, Braunschweig, Germany). MM6 cells were maintained in RPMI 1640 without L-glutamine (Sigma) respectively. Media were supplemented with 1% bovine insulin, 10% heat-inactivated FBS, 1% 2 mM L-glutamine, 1% nonessential amino acids, 1% penicillin (50 IU/ml)/streptomycin (100 µg/ml), and 1% sodium pyruvate (purchased from Gibco, Paisley, UK) at 37°C in 5% CO2 humidified atmosphere. Viability of cells was assessed by Trypan blue dye exclusion and the CellTiter AQueous-one solution proliferation assay (Promega).

Isolation of human monocytes from peripheral blood was performed by density centrifugation over Ficoll Paque and negative selection of monocytes using the MACS monocyte isolation kit (MiltenyiBiotec, Camberley, UK). Briefly, EDTA-treated whole blood from healthy donors was diluted with two to four volumes of PBS. The diluted cell suspension was layered over Ficoll Paque (1.077 density) and centrifuged at 400 g for 40 min at 20°C. The interface cells (lymphocytes, monocytes, and thrombocytes) were carefully aspirated, washed, and resuspended in PBS containing 2 mM EDTA. The cells were indirectly magnetically labeled with a cocktail of hapten-conjugated CD3, CD7, CD19, CD45RA, CD56, and anti-IgE antibodies and MACS MicroBeads coupled to an antihapten monoclonal antibody. The magnetically labeled T cells, NK cells, B cells, dendritic cells, and basophils were retained on a MACS column in the magnetic field. The isolated monocytes were resuspended at 1x106cells/ml in supplemented RPMI and used immediately.

Preparation of surfactant and lipid media. The physiological concentrations of surfactant lipids within the lung have been previously estimated to be between 100 and 500 µg/ml (20). The concentrations of surfactant and phospholipid utilized were similar to that present in the normal lung. Survanta, purchased from Abbott Laboratories (Kent, UK), is a sterile, nonpyrogenic bovine lung extract containing DPPC, palmitic acid, and tripalmitin. The solution contains phospholipids (25 mg/ml), neutral lipids (1.15 mg/ml), fatty acids (2.4 mg/ml), and surfactant-associated proteins (<1.0 mg/ml). Curosurf, purchased from Serono Pharmaceuticals (Middlesex, UK), consists of a sterile suspension containing phospholipids (80 mg/ml) from porcine lung. This natural modified surfactant contains 99% polar lipids with >40% DPPC and 1% hydrophobic associated proteins (SP-B, SP-C). Exosurf Neonatal (Exosurf), purchased from GlaxoWellcome (Hertfordshire, UK), was supplied as a vial of white sterile freeze-dried powder containing colfosceril palmitate (125 mg/ml of chemically modified DPPC). Exosurf was reconstituted with preservative and endotoxin-free sterile water. The desired dilutions of each surfactant were made with supplemented RPMI and used within 8 h of reconstitution. The desired amounts of DPPC were prepared as previously described (38).

Preparation of stimulants. An opsonized zymosan (OpZ) suspension was prepared as previously reported (38). OpZ suspensions (2.5 mg/ml) were prepared in advance and stored frozen at –70°C. Phorbol 12-myristate 13-acetate (PMA, Sigma) was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 1 mg/ml and diluted in RPMI 1640 to give a final working concentration of 100 ng/ml.

Chemiluminescence. To investigate the effect of surfactant on ROI production, MM6 cells prepared at a density of 1x106 cells/ml were preincubated with Curosurf, Survanta, or Exosurf at concentrations of 20, 100, or 500 µg/ml for 2 h at 37°C. Cells were washed in PBS (3x) and resuspended in supplemented RPMI and primed for 18 h with 100 ng/ml LPS (serotype 0111:B4, Sigma). To investigate the long-term effects of DPPC on ROI production, MM6 cells (1x106 /ml) were incubated with DPPC-supplemented media (100 µg/ml) for 2 h at 37°C. After this, cells were washed and further cultured in non-DPPC-supplemented RPMI for 0, 24, or 36 h before LPS priming. Alternatively, MM6 cells were continuously cultured with DPPC (100 µg/ml)-supplemented media for up to 36 h before LPS priming as above.

After appropriate experimental treatment we used luminol-enhanced chemiluminescence (LCL) to quantitate the release of ROIs as previously described (38). Experiments were performed in quadruplicate, and each experiment was repeated three times. LCL was initiated by the addition of OpZ (125 µg/ml) or PMA (100 ng/ml). LCL results are expressed as relative light units.

Effect of DPPC on ROI production by human peripheral blood monocytes. To extend the effects seen with DPPC on a continuous cell line, ROI production was assessed in isolated human peripheral blood monocytes by the oxidation of 2'7'-dichlorofluoroscein diacetate (DCFH-DA) to the highly fluorescent 2'7'-dichlorofluoroscein (DCF) by ROIs. ROI production was measured by flow cytometric analysis as basal levels of isolated monocytes were elevated using the luminol chemiluminescent technique. In brief, isolated human peripheral blood monocytes (1 x 106/ml) were preincubated with DPPC (125 µg/ml) for 2 h at 37°C in 5% CO2 atmosphere. Cells were washed in PBS (3x) and resuspended in supplemented RPMI at (2 x 106/ml). DCFH-DA (100 µM) was preincubated with the cells for 15 min at 37°C followed by stimulation with PMA (100 ng/ml) for 15 min at 37°C. Fluorescence (FL1) from single cells was collected using a logarithmic amplifier after gating on the combination of forward light scatter and perpendicular light scatter. A total of 10,000 cells were analyzed per sample, and data was acquired and analyzed using Cellquest (BD Biosciences). The fluorescence distribution was analyzed and displayed as a single histogram. Controls were monocytes incubated without lipids and/or DCFH-DA or PMA.

LCL control experiments. We have previously shown that oxidative responses in DPPC-treated MM6 cells were independent of priming with LPS (100 ng/ml) (38). Control experiments were performed to determine whether surfactant interfered directly with the chemiluminescence assay. MM6 cells prepared at a density of 1 x 106 /ml were primed as described above, followed by washing in PBS (3x). Cells were resuspended in standard buffer containing appropriate surfactant and chemiluminescent assays were performed immediately.

Measurement of NADPH oxidase activity in a reconstituted cell system. To investigate the effects of DPPC directly on the monocyte NADPH oxidase, MM6 cells were separated into membrane-containing and cytoplasmic (soluble) fractions. These fractions contain the NADPH-O2 oxidoreductase (membrane) and all the soluble (cytosolic) components required for full assembly and activation of the enzyme complex (5). Combination of the fractions into a "reconstituted cell system" was used to investigate whether DPPC could directly inhibit the NADPH oxidase activated in the absence of PKC phosphorylation (5). MM6 cells (1 x 108) were fractionated according to Bolscher et al. (5). Unstimulated cells were suspended in sonication buffer (0.34 M sucrose, 10 mM HEPES, 1 mM EGTA, 1 mM PMSF, and 100 µM leupeptin in PBS, pH 7.0) at a concentration of 5 x 107 MM6 cells/ml and were sonicated for 3x 15-s intervals using the Sanyo Soniprep sonicator. The sonicated suspension was centrifuged for 10 min at 160 g to remove unbroken cells and cell nuclei. The postnuclear supernatant was layered on a discontinuous gradient of 5 ml of 30% (wt/vol) sucrose resting on 10 ml of 50% (wt/vol) sucrose. The gradient was centrifuged in a Beckman SW28 rotor (Beckman, High Wycombe, UK) at 90,000 g for 2 h at 4°C in an ultracentrifuge (Beckman L5-65, Beckman). The application zone, containing soluble oxidase-component, was stored at –70°C. The membranes were collected from the interface between the 50 and 30% sucrose layers and stored in small aliquots at –70°C.

NADPH oxidase activity was measured by electron paramagnetic resonance (EPR) oximetry, as the rate of oxygen consumption by the cytosolic and membranous components of the NADPH oxidase that was isolated from MM6 cells. The neutral nitroxide 4-oxo-2,2,6,6-tetramethylpiperidine-d16-1-oxyl (15N PDT; purchased from MSD Isotopes, St. Louis, MO) was used as a spin probe. The EPR spectral line width of this probe is very sensitive to oxygen, and it has been well characterized as a probe for EPR oximetry (23). Oxygen consumption rates were obtained by measuring oxygen concentration in a closed chamber over time and finding the slope of the resulting linear plot. MM6 membrane (10 µl) and cytoplasmic components (10 µl) (which were equivalent to 2 x 106 MM6 cells) were added to 400 µl of assay buffer [10 mM HEPES containing 0.17 M sucrose, 75 mM NaCl, 0.5 mM EGTA, 1 mM MgCl2, 2 mM NaN3 (pH 7.0), and 10 mM guanosine 5'-O-(3-thiotriphosphate)]. DPPC was added to the membrane and cytosol assay to a final concentration of 100 or 500 µg/ml. Assembly of the NADPH oxidase was initiated by the addition of SDS at a final concentration of 100 µM as described by Bromberg and Pick (6). After 3 min, 15N PDT and NADPH were added at final concentrations of 0.5 and 250 mM, respectively. The assay mixture was drawn into a glass capillary tube and sealed at both ends, avoiding entrapment of any air bubbles. The EPR line width was scanned repeatedly at 60-s intervals for 15 min at room temperature using a Varian E104B spectrometer (Palo Alto, CA) operating at 9.5 GHz. Data were acquired using "in house" data acquisition software. Line width measurements were equated with an oxygen concentration (µM) obtained from a standard plot of the 15N PDT probe at various oxygen concentrations between 100% nitrogen and air (210 µM oxygen). Each test was repeated in triplicate. Control samples (lacking DPPC, SDS, and NADPH) were assayed on each day of testing. Results were expressed as the consumption of oxygen with time for each experimental setting.

Pharmacological modulation of ROI production by PKC inhibitors. To investigate the role of PKC in the production of ROIs, LCL experiments were performed with inhibitors of PKC signaling. MM6 cells (1 x 106 /ml) were separately incubated with bisindolylmaleimide I (1 µM), bis-tyrphostin (0.5 µM), genistein (2.5 µM), Gö-6976 (10 nM), or rottlerin (10 µM) and LPS (100 ng/ml) for 18 h followed by three washings in PBS. LCL was performed as previously described (38) using OpZ as a stimulant for ROI production.

Quantitation of PKC activity. The activity of calcium- and phospholipid-dependent PKC was quantified using the SignaTECT PKC Assay System (Promega). MM6 cells (1 x 106 cells/ml) were incubated with DPPC (100 µg/ml) for 2 h at 37°C in a humidified atmosphere with 5% CO2. After lipid incubation, MM6 cells were washed in PBS (3x) and suspended in fresh supplemented RPMI. Cells were primed with LPS (100 ng/ml) for 18 h and stimulated with 125 µg/ml OpZ for 30 min. Stimulation of cells with agonist occurred at peak PKC activity in MM6 cells, as previously determined in our laboratory (data not shown). Control cells were incubated without DPPC and/or stimulant. After appropriate experimental treatment, MM6 cells were washed twice in ice-cold PBS and suspended in 500 µl of ice-cold extraction buffer [25 mM Tris·HCl (pH 7.4) containing 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM 2-mercaptoethanol, 0.5 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin]. The suspended cells were sonicated on ice for 30 s (3x 10-s intervals) using the Sanyo Soniprep sonicator. The lysate was centrifuged at 14,000 g for 5 min at 4°C, and the supernatant was passed over a 1-ml column of DEAE cellulose (Whatmann DE52) that had been pre-equilibrated in extraction buffer. The column was washed with 5 ml of extraction buffer, and the PKC-containing fraction was eluted from the column using 5 ml of elution buffer (extraction buffer containing 200 mM NaCl). The PKC enzyme activities of all crude extracts were determined on day of sample preparation according to the manufacturer's instruction. Results are expressed as PKC enzyme activity in pmol·min–1·µg–1 of protein.

Western blotting for PKC isotype activation. Phorbol esters or membrane-permeable diacylglycerol (DAG) analogs stimulate a shift in location of PKC from the cytosol to the membrane, a process termed PKC translocation and is considered to be an index of PKC activation (31). The effect of DPPC incubation on PKC isotype translocation was investigated by Western blotting on cytosolic and membrane fractions of cells.

MM6 (1 x 106 cells/ml) were preincubated with or without 100–250 µg/ml of DPPC for 2 h. Cells were washed in PBS (3x) and primed with or without LPS (100 ng/ml) for 18 h. Cells were stimulated with 100 ng/ml of PMA for 15 min or OpZ for 30 min. These times were determined as a result of experiments to ascertain the optimal conditions for PKC activity (data not shown).

After stimulation the cells were washed twice in ice-cold PBS and lysed in lysing buffer [containing 100 µl of 100 mm NaCl, 10 mm Tris·HCl (pH 7.2), 2 mm EDTA, 0.5% (wt/vol) deoxycholate, 1% (vol/vol) NP-40, 10 mm MgCl2, 1 mm phenylsulfonyl fluoride, and 100 µm sodium orthovanadate] for 15 min on ice, followed by sonication (Sanyo, Soniprep) for 10 s on ice to reduce sample viscosity. Cells were centrifuged at 800 g for 5 min at 4°C to remove nuclei and unbroken cells. The postnuclear supernatant was further centrifuged at 1,000 g for 60 min at 4°C. The supernatant from this latter centrifugation contained the cytosolic proteins, whereas the pellet that was resuspended in lysing buffer containing 1% Triton consisted of membrane-bound proteins. Protein concentrations of the cytosolic and membrane extracts were determined by a modified Lowry method (Bio-Rad). Samples of equal protein content were loaded onto precast 8% SDS page gels (Life Sciences) and electrophoresed at 200 V for 50 min. Gels were electroblotted on nitrocellulose membranes (Life Sciences), and detection of PKC{alpha}, PKC{delta} protein, or phosphorylated PKC{delta} protein was determined using monoclonal antibodies (1:1,000 dilution) (BD, Cambridge, UK) in conjunction with an anti-mouse WesternBreeze chemiluminescent immmunodetection system (Life Sciences) according to the manufacturer's instruction. Densitometry of the Western blots was performed using the Bio-Rad Gel Doc 2000 and Quantity One analysis software (Bio-Rad).

Statistics. For multiple-group comparisons, the data were subjected to one-way analysis of variance (ANOVA) to determine overall difference between the group means and Tukey's honestly significant difference for pair-wise differences for within-group comparisons. Differences in medians were analyzed by Mann-Whitney. Minitab software version 12.0 (Minitab) was used for all analyses. All results are expressed as means (± SD) of three separate experiments.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of pulmonary surfactants on ROI production. Initial control experiments showed that the surfactant preparations Curosurf, Survanta, and Exosurf did not directly interfere with the chemiluminescent assay under the conditions tested (data not shown). Figure 1, A and B, demonstrates that 100 ng/ml PMA or 125 µg/ml OpZ in MM6 cells primed with LPS for 18 h (control) generated ~8–10 times greater levels of ROIs than unstimulated or unprimed cells (cells alone). Therefore, in this study, MM6 cells were primed with LPS before stimulation with OpZ to generate maximal ROI production. LCL generation peaked at ~15 min after stimulation with PMA and 50 min after stimulation with OpZ. These times of LCL peak light emissions were used to compare surfactant- or DPPC-treated cells with control cells in all subsequent experiments.



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Fig. 1. Dose response of surfactant treatment on respiratory burst activity of MonoMac 6 (MM6) cells primed with LPS and stimulated with PMA (A) or opsonized zymosan (OpZ, B). MM6 cells (1 x 106 cells/ml) were incubated with or without the indicated surfactant for 2 h at 37°C. After incubation, cells were washed in PBS (3x) and resuspended in supplemented RPMI medium and primed with LPS (100 ng/ml) for 18 h. Reactive oxygen intermediate (ROI) production (respiratory burst activity) was measured by luminol-enhanced chemiluminescence using OpZ (125 µg/ml) or PMA (100 ng/ml) as stimulants. Control values (100%) are MM6 cells incubated in the absence of surfactant before priming with LPS. Cells alone represent MM6 cells not treated with LPS or stimulant. *P < 0.05, {dagger}P < 0.01 analyzed by ANOVA and Tukey's pairwise comparisons.

 
We first examined in the human monocytic cell line MM6 the effects of Curosurf, Survanta, or Exosurf on ROI production. Pretreatment of MM6 cells with these surfactants for 2 h at 20 or 100 µg/ml did not influence the production of ROIs stimulated by PMA compared with the control cells incubated without surfactant (Fig. 1A). However, at 500 µg/ml, all surfactant preparations tested significantly reduced ROI production by ~25–30% in PMA-stimulated cells (P < 0.01 analyzed by ANOVA and Tukey's pairwise comparison). When the effect of surfactant was determined on cells stimulated with OpZ, a yeast cell wall extract opsonized in complement factors, a significant dose-dependent decrease in ROI production was observed (Fig. 1B).

The greatest inhibitory effects were seen when each surfactant was preincubated with MM6 cells at a concentration of 500 µg/ml, accounting for approximately a 40% reduction in ROIs (P < 0.01 analyzed by ANOVA and Tukey's pairwise comparisons). The effects of surfactant on ROI production appear to be greater in cells stimulated with OpZ than those cells stimulated with PMA.

Effect of DPPC on ROI production. The major pulmonary surfactant phospholipid is DPPC. We have previously demonstrated that DPPC inhibits ROI production in response to OpZ in a dose- and time-dependent manner (38). However, we do not know whether these effects are irreversible. Using previously described optimal conditions for DPPC inhibition of ROI production (2 h incubation with >100 µg/ml), we next examined the effect of continuous and discontinuous incubation of DPPC on ROI production. After DPPC incubation, MM6 cells either were washed in PBS and further cultured in media alone (discontinuous culture) or were continuously cultured in DPPC-supplemented media for the times indicated before being primed and stimulated with OpZ (Fig. 2). Peak light emission of cells treated with DPPC for 2 h and immediately primed and stimulated with OpZ was significantly inhibited (P < 0.01) from that of control MM6 (–DPPC, +LPS, +OpZ) (Fig. 2A). When DPPC was removed from MM6 cell incubation media by washing and resuspension in non-DPPC supplemented for 24 h before LPS priming and LCL, a significant inhibitory effect was still noted. However, extension of the DPPC-free incubation to 36 h resulted in loss of the inhibitory effects of DPPC treatment (Fig. 2A). Continuous culture with DPPC (100 µg/ml) for up to 9 days still resulted in ~40% inhibition of ROI production (Fig. 2B). Control experiments again showed that DPPC did not interfere with the chemiluminescent assay when DPPC was added to the chemiluminescent buffer at the time of OpZ stimulation and thus excluded a direct interaction of the stimulating agents with DPPC.



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Fig. 2. Reversing the inhibitory effect of dipalmitoyl phosphatidylcholine (DPPC) on respiratory burst activity in MM6 cells primed with LPS and stimulated with OpZ. MM6 cells (1 x 106 cell/ml) were incubated with DPPC (100 µg/ml) for 2 h. Cells were then cultured in the absence (discontinuous, A) or in the presence (continuous, B) of DPPC (100 µg/ml) for time indicated. After incubation, cells were washed in PBS (3x) and resuspended in supplemented RPMI and primed with LPS (100 ng/ml) for 18 h. ROI production (respiratory burst activity) was measured by luminol-enhanced chemiluminescence using OpZ (125 µg/ml) as a stimulant. Control values (100%) are MM6 cells incubated in the absence of DPPC before priming with LPS and stimulation with OpZ. Cells alone represent MM6 cells not treated with LPS or stimulant. {dagger}P < 0.01 analyzed by ANOVA and Tukey's pairwise comparisons.

 
These studies were also extended to include the effects of DPPC on peripheral blood monocyte ROI production. DCFH-DA-treated human peripheral blood monocytes were stimulated with PMA to induce ROI production and analyzed by flow cytometry for the fluorescent oxidized DCF product. Treatment of these cells with DPPC (125 µg/ml) inhibited the ROI response by ~30% (P < 0.05, data not shown). This is a similar inhibition to that obtained from chemiluminescence assays in which DPPC inhibited the LCL response of the human monocytic cell line MM6. In addition, the observed inhibitory effects of DPPC on monocytic ROI production are not due to reduced cell viability since Trypan blue dye exclusion and the CellTiter AQueous-one solution proliferation assay demonstrated >90% viable cells under all experimental conditions tested.

Effect of DPPC on oxygen consumption using a reconstituted cell system. To determine whether DPPC could directly inhibit the NADPH oxidase by impairing the assembly of this enzyme, a reconstituted cell system was used (5). This system allowed us to investigate the effects of DPPC on NADPH oxidase activated without PKC stimulation to determine at which points in the activation pathway this lipid is acting. Unstimulated MM6 cells (100 x 106 cells) were fractionated by sonication and sucrose density centrifugation. Membranous and cytoplasmic components equivalent to 2 x 106 cells, when combined together in an appropriate buffer containing the anionic detergent SDS and NADPH, will consume oxygen and produce ROIs (5). NADPH oxidase activity was measured by consumption of oxygen as detected by EPR coupled with the oxygen-sensitive stable paramagnetic nitroxide probe 15N PDT. The effect of DPPC on oxygen consumption in this cell-free system was studied by measuring the line width of the EPR spectra of the oxygen-sensitive 15N PDT probe with time (17). Membranous and cytoplasmic components were incubated with DPPC (100 or 500 µg/ml) before assembly of the components into a functional enzyme complex by SDS. The change in oxygen concentration for the cell-free NADPH oxidase samples incubated with or without DPPC is shown in Fig. 3. The rate of oxygen consumption for control cell fractions incubated without DPPC was 2.5 ± 0.61 µM/min (slope measured over the first 6 min of consumption). The consumption of cell fractions incubated with DPPC (2.6 ± 0.31 µM/min) was not significantly different from that of the control when compared by Mann-Whitney analysis. Confirmatory chemiluminescent studies demonstrated little difference between control and DPPC-treated ROI responses in this reconstituted cell system (data not shown). Together these results suggest that DPPC does not directly affect the activity or the assembly of the individual components of the NADPH oxidase and its mechanism of action must be proximal to the assembly of the oxidase complex in MM6 cells.



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Fig. 3. Effect of DPPC on oxygen consumption measured in a MM6 cell-free system. Electron paramagnetic resonance (EPR) oximetry was performed with a spin probe [4-oxo-2,2,6,6-tetramethylpiperidine-d16-1-oxyl (15N PDT) 0.5 mM] in 2 x 106 MM6 cell equivalents preincubated with 100 or 500 µg/ml DPPC. Activation of the NADPH oxidase occurred only in the presence of SDS (100 µM) and NADPH (250 mM). Negative control represents cell equivalents incubated without SDS/NADPH/DPPC. Data represents means ± SD of 3 separate experiments.

 
Effect of DPPC on PKC activity. To determine whether DPPC affects PKC activation, MM6 cells were incubated with 100 µg/ml of DPPC. This concentration of DPPC was used since it significantly inhibited ROI production and it also reflects those levels likely to be encountered in human pulmonary surfactant (20). We studied the effects of DPPC in MM6 cells using LPS and OpZ or PMA as a stimulus. Maximal PKC activity in response to OpZ or PMA in MM6 cells were shown to peak at 30 or 15 min, respectively (data not shown). MM6 cells (1 x 106) were preincubated with 100 µg/ml DPPC, followed by washing and priming with LPS. Cells were stimulated with OpZ (125 µg/ml) for 30 min. Controls were incubated without lipid; in addition, controls were also performed with or without LPS and OpZ stimulation. Incubation of MM6 cells with LPS (100 ng/ml) for 18 h alone and not stimulated with OpZ did not significantly activate PKC. On the other hand, cells stimulated with OpZ (125 µg/ml) alone stimulated PKC activity by 66% compared with unstimulated cells. However, when cells were primed with LPS and then stimulated with OpZ, an enhanced or "priming" effect was seen with respect to PKC activity. Under these conditions, there was an approximately sixfold increase in PKC activity compared with untreated cells. LPS priming did not affect the responses when PMA was used as stimulus (not shown). Stimulation of the cells with PMA was therefore done in the absence of LPS priming. These results suggest PKC is likely to be involved in the priming or activation of NADPH oxidase.

Figure 4 demonstrates that the activity of PKC induced by LPS and OpZ is significantly reduced in DPPC-treated cells (P < 0.01) compared with cells incubated without DPPC. In addition, when PMA was used as a stimulus, DPPC still significantly inhibited the activity of PKC (P < 0.05). These results suggest that the phospholipid DPPC has a very significant effect on the activity of PKC when stimulated with the "particulate" zymosan extract or the "soluble" DAG analog PMA.



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Fig. 4. Effect of DPPC on PKC activity in PMA- or LPS-primed and OpZ-stimulated cells. The activity of PKC was quantified using the SignaTECT PKC Assay System (Promega). MM6 cells (1 x 106 cells/ml) were incubated with DPPC (100 µg/ml) for 2 h at 37°C in a humidified atmosphere with 5% CO2. After lipid incubation, cells were washed in PBS (3x) and suspended in fresh supplemented RPMI. Cells were primed with LPS (100 ng/ml) for 18 h and stimulated with 125 µg/ml OpZ for 30 min. After appropriate experimental treatment, the PKC enzyme activities of each sample were determined on day of sample preparation according to the manufacturer's instruction. As controls, MM6 cells were incubated with or without LPS and OpZ. DPPC-treated cells were compared with MM6 cells treated with PMA or LPS and OpZ but without DPPC. *P < 0.05, {dagger}P < 0.01 analyzed by Mann-Whitney.

 
Effect of PKC inhibitors on ROI production. In this study we have examined the role of PKC in ROI production by using selective and specific inhibitors of PKC and determining their effects on ROI production. Figure 5 shows the effects of the specific PKC inhibitors bis-indolylmaleimide I, Gö-6976, and rottlerin and the tyrosine kinase inhibitors genistein and bis-tyrphostin on LPS-primed and OpZ-stimulated ROI production. MM6 cells incubated with the general tyrosine kinase inhibitors had no significant effect on ROI production compared with the control cells incubated with LPS and OpZ. On the other hand, the specific PKC inhibitors had substantial inhibitory effects on ROI production. Gö-6976 is a PKC inhibitor with high selectivity for the A group PKC isotypes ({alpha}, {beta}, {gamma}) (26) and showed 50% inhibition of ROI production in MM6 cells treated with this inhibitor (Fig. 5). The use of rottlerin, previously reported to be a PKC{delta} inhibitor (18), resulted in complete inhibition of ROI production in MM6 cells treated with this inhibitor (Fig. 5). In addition, previous studies utilizing PKC{delta}-null mice (9) or human neutrophils immunodepleted of PKC{delta} demonstrated deficient superoxide production by NADPH oxidase (7). These data suggest that the PKC isoenzymes {alpha}, {beta}, and {gamma} are all involved in activation of the respiratory burst. Further characterization of PKC isoforms involved in the respiratory burst and the effect of DPPC on them was made by translocation and immunoblotting experiments.



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Fig. 5. Effect of PKC inhibitors on ROI production in LPS-primed and OpZ-stimulated cells. MM6 cells (1 x 106/ml) were separately incubated with or without the indicated inhibitor and LPS (100 ng/ml) for 18 h. ROI production (respiratory burst activity) was measured by luminol-enhanced chemiluminescence using OpZ (125 µg/ml) as a stimulant. Controls are MM6 cells incubated in the absence of inhibitors with LPS for 18 h. Inhibitor concentrations are given in MATERIALS AND METHODS.

 
Effect of DPPC on PKC isoform activation. To gain insight into the isoforms involved in DPPC inhibition of ROI production via PKC, an assessment of PKC isoform translocation in Triton-soluble and Triton-insoluble fractions was determined by Western blotting, since activity measurements do not differentiate between the activities of individual PKC isoforms within a class. Western blots were analyzed by densitometry to evaluate the amount of protein in each fraction and to confirm no overall loss of PKC protein in cells treated with DPPC. In control monocytes, the majority of PKC{delta} is present in the cytosol fraction (Fig. 6). Incubation of MM6 cells with PMA or LPS/OpZ results in a significant reduction in PKC{delta} in the cytosol and a concomitant increase in the membrane fractions of PMA- or LPS/OpZ-stimulated cells, respectively (Fig. 6). In MM6 cells incubated with DPPC, the increase in membrane PKC{delta} protein upon stimulation with PMA or LPS/OpZ was less than those cells incubated without DPPC. These data suggest that DPPC may inhibit the PMA- or OpZ-stimulated PKC{delta} membrane translocation. In addition, Western blotting for the phosphorylated form of PKC{delta} (indicative of PKC activation) demonstrated that DPPC attenuates PKC{delta} activation by PMA and LPS/OpZ, thus supporting observations in the PKC translocation experiments (Fig. 7). The effects of DPPC are unlikely to be a consequence of cofactor activity, since DPPC does not inhibit ROI production in a cell-free system (data not shown). Western blotting experiments showed that PKC{alpha}, a classic PKC isoform, is not translocated from the cytosol to the membrane in response to PMA (Fig. 8). This suggests that DPPC may be inhibiting the respiratory burst by selectively inhibiting PKC isoforms such as PKC{delta}.



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Fig. 6. Effect of DPPC on PKC{delta} translocation in PMA- or OpZ-stimulated cells. MM6 cells (1 x 106 cells/ml) were incubated with DPPC (250 µg/ml) for 2 h followed by washing in PBS (3x). Cells were stimulated with PMA (100 ng/ml) for 15 min (A) or cells were primed with LPS (100 ng/ml) for 18 h and stimulated with OpZ (125 µg/ml) (B). After stimulation, membrane and cytosolic fractions were prepared as described in MATERIALS AND METHODS. Top: lysates were subjected to protein quantitation, and equivalent amounts of protein for each sample (see {beta}-actin panel) were analyzed by SDS-8% PAGE and immunoblotting with anti-PKC{delta}. Bottom: relative levels of PKC{delta} were determined by densitometry as a ratio between the signal intensity of the PKC{delta} band in the cytosol or membrane fraction compared with total signal intensity (intensity of membrane + cytosol).

 


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Fig. 7. Immunoblot of the effect of DPPC on phosphorylated (phospho) PKC{delta} in PMA- or LPS + OpZ-stimulated cells. MM6 cells (1 x 106 cells/ml) were incubated with DPPC (250 µg/ml) for 2 h followed by washing in PBS (3x). Cells were stimulated with PMA (100 ng/ml) for 15 min. After stimulation, cell lysates were prepared as described in MATERIALS AND METHODS, and immunoblotting was performed with an antiphospho-PKC{delta} antibody. Data shown are representative of 3 independent experiments.

 


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Fig. 8. Immunoblot of the effect of DPPC on PKC{alpha} translocation in PMA stimulated cells. MM6 cells (1 x 106 cells/ml) were incubated with DPPC (250 µg/ml) for 2 h followed by washing in PBS (3x). Cells were stimulated with PMA (100 ng/ml) for 15 min. After stimulation, membrane and cytosolic fractions were prepared as described in MATERIALS AND METHODS. A: lysates were subjected to protein quantitation, and equivalent amounts of protein for each sample (see {beta}-actin panel) were analyzed by SDS-8% PAGE and immunoblotting with anti-PKC{alpha}. B: relative levels of PKC{alpha} were determined as a ratio between the signal intensity of the PKC{alpha} band in the cytosol or membrane fraction compared with total signal intensity (intensity of membrane + cytosol).

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The results of this study support our previous findings that surfactant phospholipids suppress the production of ROIs by monocytic cells (37, 38). In addition, we provide further evidence of the suppressive effects of commercially available surfactant preparations on the production of ROIs in both OpZ- and PMA-stimulated MM6 cells. Moreover, our results suggest that the mechanism by which the surfactants modulate ROI generation depend, at least in part, on suppression of PKC. Our present data provide new insights into the mechanisms by which the components of surfactant, particularly DPPC, mediate its effect on the respiratory burst and subsequent ROI production.

Using chemiluminescence, the present study indicates that the surfactant preparations Survanta, Curosurf, or Exosurf, all used in the treatment of respiratory distress syndromes, are able to inhibit the release of ROIs from MM6 cells in a dose-dependent manner in response to either particulate (OpZ) or soluble (PMA) stimuli (Fig. 1). Moreover, the active concentrations of the surfactant preparations (100–500 µg/ml) are consistent with reported in vivo concentrations (20). Surfactant has previously been reported to enhance (42), suppress (20, 21), or have no effect on the production of superoxide by alveolar macrophages or monocytes (34). However, the different models and methods used have probably contributed to the conflicting reports. Our current study supports previous findings that Curosurf or Exosurf downregulates the production of other inflammatory mediators, such as cytokines, in LPS-stimulated monocytes (3, 27, 36, 41). Furthermore, the natural surfactants used in this study either exclude SP-A [Survanta and Curosurf (33)] or are devoid of all surfactant-associated proteins (Exosurf) (35), indicating a role for surfactant lipids in the effects seen.

Most investigators have only examined short-term incubation (<24 h) of surfactant on inflammatory functions. However, in vivo, alveolar macrophages constantly reside in pulmonary surfactant. Our results with DPPC, the major constituent of surfactant, show that it has suppressive effects on ROI production in cells for extended periods (>9 days of culture). Furthermore, the ability of DPPC to suppress ROI production is removed once the cells are incubated in medium that does not contain DPPC. However, this effect requires at least 36 h in culture in the absence of lipid for it to be observed. This suggests that DPPC uptake is required for an effect on ROI production to take place and that this effect is only slowly reversible. We have previously demonstrated by HPLC that DPPC is taken up into MM6 cells in a dose-dependent manner (38).

The fact that the surfactants or DPPC did not interfere with the chemiluminescent assay and did not induce cell cytotoxicity at any concentration used suggests that these immunomodulators were acting at specific cellular targets to inhibit the oxidative burst. The inhibitory effect of DPPC on ROI production was also noted in human peripheral blood monocytes. These studies confirm that the downregulation of ROI production in our study is attributable to DPPC alone and is not a feature of MM6 cells or due to assay interference.

Surfactant and/or DPPC might alter NADPH oxidase activity by either inhibiting signal transduction events, which trigger oxidase assembly, or blocking the assembly steps directly. To distinguish between these possible mechanisms of action, we utilized a reconstituted cell preparation, composed of membrane and soluble fractions, that has been shown to allow assembly and activation of NADPH oxidase directly by stimuli such as arachidonic acid or SDS (5). This cell fraction system represents the direct pathway of activation of the oxidase in intact cells, and all the components necessary for activity of the oxidase in intact cells are also required in this "reconstituted" cell system. Activation of the NADPH oxidase results in consumption of O2 and ROI production, and both these can be measured in this cell fraction system. Furthermore, it was shown that the same cell fractions prepared from cells from chronic granulomatous disease patients do not show oxidase activation and do not consume O2 or produce ROI (5).

DPPC did not inhibit the oxygen consumption or the production of ROI by the NADPH oxidase complex in the reconstituted cell system. Therefore, DPPC does not inhibit the production of ROIs by inhibiting the assembly of cytoplasmic and membranous components into a functional enzyme. This suggests that DPPC is acting at a site in the activation pathway proximal to the assembly of the NADPH oxidase, supporting our findings that it modulates PKC activation. In contrast, Geertsma et al. (15) demonstrated that sheep surfactant inhibited the assembly of the NADPH oxidase by an unknown mechanism, a response that may be related to the different components within sheep surfactant including surfactant proteins. Recently, Crowther et al. (10) demonstrated that SP-A inhibited macrophage ROI production through a reduction in NADPH oxidase activity by altering oxidase assembly on phagosomal membranes.

Activation of the respiratory burst can occur in response to both particulate and soluble agents via different signal transduction mechanisms. PMA, an analog of DAG, is believed to induce translocation of oxidase subunits and oxidase activation through both PKC-dependent and -independent pathways that ultimately lead to phosphorylation of multiple serine residues on p47phox (29). Inhibition of ROI production in whole cells stimulated with LPS and OpZ with various kinase inhibitors (Fig. 5) demonstrated the involvement of PKC in the monocyte respiratory burst. Although bis-indolylmaleimide I, a selective inhibitor for all PKC isoforms, inhibited ROI production by ~50%, its potency in whole cell assays is known to be reduced due to competitive kinetics with ATP (39). Almost complete inhibition of ROI was achieved with rottlerin, reported to be a specific and selective PKC{delta} inhibitor (18). However, some recent reports suggest that rottlerin may inhibit other protein kinases, and its purity and specificity is thus subject to controversy (12). Although we acknowledge that PKC inhibitors may have other possible effects, at the concentrations used in our experiments the results suggest that PKC and isoforms such as PKC{delta} are involved in the stimulation of the respiratory burst in MM6 cells.

Recent studies have confirmed a prominent role for PKC in the activation of phagocyte NADPH oxidase (14). Chou et al. (9) have demonstrated an almost complete inhibition of ROI production in mice null deficient for PKC{delta}. In addition, Brown et al. (7) have demonstrated in human neutrophils that depletion of PKC{delta} leads to impaired superoxide production. It has also been demonstrated that inhibition of PKC{delta} activity inhibits ROI production through the perturbation of phosphorylation and translocation of p47phox, a constituent of the NADPH oxidase (4). It is becoming apparent that different PKC isoforms may activate the NADPH oxidase via different stimulatory pathways (2). The results of our Western blotting experiments suggest that DPPC can inhibit the NADPH oxidase via effects on the novel isoform, PKC{delta}, but not the classic isoform, PKC{alpha}. A number of recent studies suggest that conventional PKC isoforms may activate the respiratory burst to PMA, whereas the novel isoforms, including PKC{delta}, activate the responses to particulate stimuli (32). However, in our studies we find that DPPC has similar effects on PKC{delta} in response to both soluble and particulate stimuli. These differences may reflect the different cell types used in these studies and the possibility that isoforms such as PKC{delta} may be regulating the respiratory burst through multiple pathways. Although this study has investigated both a classic and novel PKC isoform expressed in monocytes, we cannot rule out the possibility that other PKC isoforms may regulate the respiratory burst in MM6 cells. However, recent experiments suggest that the novel isoform PKC{epsilon} is not affected by DPPC in these cells (Parton and Jackson, unpublished observations). Despite the fact that DPPC was seen to affect the translocation of cytosolic PKC{delta} to the membrane, there may be other pathways by which DPPC could be regulating NADPH oxidase activation. The inhibitory effects of pulmonary surfactant on ROI production have previously been attributed to inhibition of PKC and stimulation of PKA (16). However, that study did not investigate the effects of surfactant lipids and, in particular, DPPC directly on PKC activity or on particular PKC isoforms. The mechanism by which DPPC inhibits PKC activity is unclear. A recent study has shown that PKC{delta} can be activated directly by H2O2 without translocation to the membrane (25). This raises the possibility that DPPC may be inhibiting PKC{delta} activation via several pathways, some of which may not require membrane translocation. A major proportion of PKC activity is constitutively associated with the plasma membrane in alveolar macrophages. Membrane perturbation by surfactant phospholipid would therefore be an attractive mechanism for the suppression of inflammatory responses to LPS and OpZ. Such a notion is supported by a recent study, demonstrating that surfactant modulates cAMP accumulation in monocytes through a membrane-controlled mechanism (33).

The many different facets of surfactant function in the airways and the alveolus need to be considered in the interpretation of pathological mechanisms of lung diseases. Inflammatory cells such as neutrophils migrate out of the circulation and accumulate in large numbers in the lung during episodes of ARDS. Defects or deficiencies in lung surfactant phospholipids, such as those seen in ARDS (28), may facilitate lung destruction and loss of function since the attenuation of ROI production will have been removed. The observation that lung surfactant, particularly DPPC, can markedly attenuate monocyte respiratory burst activity through inhibition of PKC suggests an additional rationale for the administration of surfactant to patients with ARDS and related inflammatory lung diseases. Understanding the mechanisms by which pulmonary surfactant lipids alter macrophage responses to infectious stimuli will advance our understanding of innate immune responses in the lung and might aid the development of lipid-based agents as therapies for inflammatory lung diseases.

In summary, this study has shown that surfactant preparations and their major constituent DPPC can downregulate oxidative functions in monocytes by a mechanism that may involve PKC regulation. Although observed reductions in release of inflammatory products were not complete, a 50% reduction in the release of ROIs would offer a considerable protection against inflammatory damage in the delicate alveolar region of the lung. Indeed, complete inhibition of such responses would be deleterious to the individual as macrophage-mediated responses are the central immune defense mechanism in this region of the respiratory tract.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported in part by a grant from the Wales Office of Research and Development in Health and Social Sciences.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Tonks, Dept. of Haematology (7th fl.), School of Medicine, Wales College of Medicine, Cardiff Univ., Heath Park, Cardiff CF14 4XN, UK (E-mail: tonksa{at}cf.ac.uk)

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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 GRANTS
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