Dipalmitoylphosphatidylcholine modulates inflammatory functions of monocytic cells independently of mitogen activated protein kinases

Materials

L-α-phosphatidylcholine (TPC; type XVI-E from egg yolk), l-α-phosphatidylethanolamine, l-α-phosphatidylcholine dipalmitoyl (DPPC), l-α-phosphatidylcholine β-arachidonoyl-γ-palmitoyl (PAPC), sphingomeylin, lyso-phosphatidylcholine, lipopolysaccharide (Escherichia coli O111:B4), phenylmethylsulphonyl fluoride, sodium orthovanadate, deoxycholate and tergitol NP-40 were purchased from Sigma Chemical Co. (Dorset, UK). chloroform, methanol and ammonium hydroxide were purchased from BDH (Dorset, UK). silica gel column was obtained from Jones Chromatography (Hengoed, UK)

Cell culture

The human monocytic cell line MonoMac-6 (MM6) was obtained from the German collection of microorganisms and cell cultures (DSM; Braunschweig, Germany). MM6 cells were maintained in RPMI 1640 medium without l-glutamine (Sigma, UK). RPMI was supplemented with 1% bovine insulin, 10% heat inactivated foetal bovine serum (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. Cells were subcultured every 3 days at a density of 0·4 × 10⁶ cells/ml. Unless otherwise stated, prior to experimentation MM6 were ‘weaned’ onto a serum free medium, Ultraculture (supplemented as for RPMI 1640 media without FBS) as indicated by the manufacturer BioWhittaker UK Ltd, Wokingham, UK.

Isolation of human monocytes from peripheral blood was performed by density centrifugation over Ficoll Paque® and negative selection of monocytes using midiMACS (Miltenyi Biotec, Camberley, U.K.). Briefly, EDTA treated whole blood from healthy donors was diluted with 2–4 volumes of PBS. The diluted cell suspension was layered over Ficoll Paque® (1·077 density) and centrifuged at 400 × g for 30–40 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 labelled 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 labelled T-cells, NK cells, B cells, dendritic cells and basophils were retained on a MACS column in the magnetic field of the midiMACS. The isolated monocytes were resuspended at 1 × 10⁶ cells/ml in supplemented RPMI medium and used immediately.

Viability of cells was assessed by trypan blue dye exclusion and the CellTiter AQ_ueous-one solution proliferation assay (Promega, Southampton, UK). The viability of MM6 cells exposed to LPS and/or lipid was greater than 90% in all experiments.

Preparation of lipid media

The physiological concentrations of PC within the lung have been previously estimated to be between 100 and 350 μg/ml [2,22]. The concentrations of phospholipids utilized were approximately those encountered by alveolar macrophages within the lung. The desired amounts of lipids dissolved in chloroform (CHCl₃) were dried as a thin film under nitrogen on ice in acid washed bijoux tubes treated with RepelCote (BDH, Dorset, UK) 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.

Preparation of stimulants

An opsonized zymosan (OpZ) suspension was prepared according to a method as previously reported by Allen, 1986 [23]. OpZ suspensions (2·5 mg/ml) were prepared in advance and stored frozen at −70°C. Phorbol 12-myristate 13-acetate (PMA; Sigma, UK) was dissolved in 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

MM6 cells prepared at a density of 1 × 10⁶/ml were preincubated with tPC, DPPC or PAPC at concentrations of 500, 250, 125, 63 and 2 μg/ml for 2 h at 37°C in 5% CO₂ atmosphere. Cells were washed in PBS (×3) and resuspended in ultraculture, primed for 18 h with LPS (100 ng/ml). Luminol enhanced chemiluminescence (LCL) according to the method of Allen, 1986 [23] with minor modifications was used to quantify the release of ROIs. LCL was detected at room temperature using a standard luminometer. Experiments were performed in quadruplicate and each experiment was repeated three times. Briefly, following treatment, the cells were washed twice with phosphate buffered saline (PBS) and resuspended in standard buffer (4·58 mm KH₂PO₄, 8·03 mm Na₂HPO₄, 0·76% NaCl, 0·033% KCl 0·033, 0·1% glucose, 0·1% endotoxin free albumin 0·1, 0·5 mm MgCl₂, and 0·45 mm CaCl₂, pH 7·3) at 2·0 × 10⁶ cells/ml. To every ml of cell suspension 100 ml of a 3·5-mm solution of luminol (Sigma, UK) was added. Following gentle mixing and a 10 minute adaptation period in the dark, 150 μl cell/luminol suspension were transferred to a 96 well plate (FluoroNunc-PolySorb, Gibco, UK). LCL was initiated by the addition of OpZ (125 μg/ml) or PMA (100 ng/ml). Temporal traces of evolving chemiluminescent reactions were recorded every 5 min for 60 min or until it demonstrated a definite decline. There was no appreciable light emission in this system in the absence of cells or stimulants. LCL results are expressed as relative light units (RLUs) or:

Time course study of PC effects on LCL

In order to determine the effects of a period of incubation on productions of ROIs, preincubation of cells with lipids was performed using the optimal dose of lipid as determined by the previous experiment. MM6 cells were preincubated with the PC species for 0–18 h at 37°C in a 5% CO₂ atmosphere.

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 using FACS analysis as basal levels of isolated monocytes were elevated using the luminol chemiluminescent technique. Briefly, isolated human peripheral blood monocytes (1 ×10⁶/ml) were preincubated with DPPC (125 μg/ml) for 2 h at 37°C in 5% CO₂ atmosphere. Cells were washed in PBS (×3) and resuspended in supplemented RPMI media at (2 × 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 were 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 analysed per tube and data was acquired and analysed using Cellquest (Becton Dickinson, U.K.). The fluorescence distribution was analysed and displayed as a single histogram. Controls were monocytes incubated without lipids and/or DCFH-DA or PMA.

Control experiments

Two control experiments were carried out. The first to determine whether any observed oxidative response was independent of priming with LPS (100 ng/ml). MM6 cells prepared at a density of 1 × 10⁶/ml were primed as above followed by washing in PBS (×3) and incubation with DPPC (125 μg/ml) for 2 h at 37°C in 5% CO₂ atmosphere. Cells were washed and resuspended in standard buffer and chemiluminescence assays performed. The second experiment was to determine whether phospholipids interfered directly with the chemiluminescence assay. MM6 cells prepared at a density of 1 × 10⁶/ml were primed as described above, followed by washing in PBS (×3). Cells were resuspended in standard buffer containing appropriate lipids and chemiluminescent assays were performed immediately.

Measurement of TNF-α release from MM6

MM6 cells prepared at a concentration of 1 × 10⁶/ml, were preincubated with the same concentrations of PC subspecies and at the optimal preincubation time previously used. Following lipid preincubation, MM6 cells were stimulated with LPS (200 ng/ml) and PMA (100 ng/ml) for 4 h at 37°C in 5% CO₂ atmosphere. Previous experiments have shown that PMA alone at this concentration does not stimulate TNF-α production from MM6 cells [24]. TNF-α in MM6 culture supernatants were quantified by a commercially available ELISA kit (R & D, Minneapolis, USA) according to the manufacturer's instruction.

Quantification of uptake of phospholipids into MM6 membrane by HPLC

MM6 cells prepared at a density of 1 × 10⁶/ml were pretreated with 250 μg/ml DPPC for 2 h at 37°C in a 5% CO₂ atmosphere. Cells were washed in PBS (×3) and membrane phospholipids were extracted using a modification to the method of Bligh and Dyer, 1959 [25]. The separation and quantification of membrane phospholipids was determined by HPLC on a silica gel column with a mobile phase consisting of chloroform, methanol and ammonium hydroxide. The uptake of DPPC into MM6 membranes was detected by a light scattering evaporating detector as described previously [26]. A standard phospholipid mixture containing DPPC, PC, sphingomyelin and phosphatidylethanolamine over a range of 5–200 μg/ml were used to obtain a standard curve. This was calculated by regressing the peak areas to obtain the best-fit quadratic line. The quantity of DPPC in MM6 cells after treatment was compared to the mean obtained from untreated cells and reported as a mean percentage increase by weight.

Measurement of membrane fluidity by EPR spectroscopy

MM6 cells at a density of 1 × 10⁶/ml were preincubated with 125 or 250 μg/ml of DPPC for 2 h. After washing (×3) in PBS, the cells were resuspended in PBS at 5 × 10⁶ cells/ml to which 10 μl of 20 mg/ml doxyl stearate in ethanol was added. The concentration of ethanol in the final cell suspension was less than 1%. The cells were incubated for 15 min at room temperature followed by washing in PBS (×3) to remove free spin label. Cells were re-suspended in 100 ml of PBS and membrane fluidity was measured by electron paramagnetic resonance (EPR) spectroscopy as previously described by Darmani, 1993 [27]. The order parameter (S) was calculated from the relationship, S = (A// −A⊥)/25G where A// and A⊥ are the maximal and minimal coupling constants, respectively, obtained from the EPR spectra of the spin labelled cells [27].

Western blot analysis of p44/p42 or p38 MAP kinase

The phosphorylated forms of the P44 (ERK1)/P42 (ERK 2) or P38 MAPK proteins were analysed by a commercially available kit (phosphoplus P44/P42 MAPK or phospho P38 Antibody kit; New England Biolabs UK Ltd, Hertfordshire, UK). Analysis of these MAPKS was performed according to the manufacturer's instructions. MM6 (1 × 10⁶ cells/ml) were preincubated with or without 500, 100 or 10 μg/ml of DPPC for 2 h. Cells were washed in PBS (×3) and primed with or without LPS (100 ng/ml) for 18 h. Cells were stimulated with 100 ng/ml of PMA for 10 min or OpZ for 60 min. These times were determined as a result of experiments to ascertain the optimal conditions for MAPK expression (data not shown). Following stimulation with PMA or OpZ 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% (w/v) deoxycholate, 1% (v/v) NP40, 10 mm MgCl₂, 1 mm phenylsulphonyl fluoride and 100 mm sodium orthovanadate) for 15 min on ice, followed by sonication (Soniprep, Sanyo, Watford, UK) for 10s on ice to reduce sample viscosity. Cells were centrifuged at 13 500 ×g. For 5 min at 4°C and the lysate supernatant fraction was stored at −70°C. Protein concentration of the soluble cytosolic extract was estimated using a modified lowry method (Bio-rad, California, USA). Protein concentration of sample lysate supernatants were adjusted to 5 mg/ml with LDS sample buffer and heated for 10 min at 70°C. Samples were then loaded onto precast 10% SDS PAGE gels (Novex, Groningen, Netherlands) and electrophoresed at 200 V for 50 min. Gels were electroblotted on nitrocellulose membranes (Amersham, UK) and washed in washing buffer (Tris-buffered saline with 0·1% Tween-20). To prevent non-specific protein binding, the membrane was incubated in blocking buffer (Tris-buffered saline, 5% w/v non-fat dry milk and 0·1% Tween-20) for 1 h at room temperature. The appropriate phosphorylated anti-MAPK antibody was incubated with the membrane overnight at 4°C. The membrane was then rinsed with wash buffer (3 × 5 min). Detection of MAPK was achieved by incubating the membrane with horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:2000 dilution) for one hour at room temperature. The membrane was rinsed (3 × 5 min) in wash buffer. Visualization of antibody complex was achieved by enhanced chemiluminescence (ECL) LumiGLO® (1 minute, room temperature) and subsequent exposure on hyperfilm (Amersham, UK). MAPKS were identified by comparison with pre-stained molecular weight markers and MAPK control proteins.

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 (HSD) for pairwise differences for within group comparisons. Differences in medians were analysed by Mann–Whitney. Minitab software version 12·0 (Minitab Inc.) was used for all analyses.

Effect of phosphatidylcholine tPC, DPPC and PAPC on luminol enhanced chemiluminescence (LCL)

Control experiments showed that lipid preparations did not interfere with the chemiluminescent assay under these conditions (data not shown). LCL generation peaked at approximately 55 min after stimulating cells with OpZ. PC exerted no significant effect on the time to peak responses when compared to the primed MM6 control. However, the luminescence generated from MM6 pretreated in this manner with tPC, DPPC and PAPC exhibited significantly (P < 0·0001) different LCL than untreated MM6 primed with LPS. The modulation of peak light emitted was found to be dose dependent in MM6 preincubated with tPC, DPPC and PAPC (Fig. 1). Further analyses of this data using Tukey's pairwise comparison, demonstrated a significant difference (P < 0·001) amongst the means as the concentration of the phospholipid increased to 63, 125 and 250 μg/ml. There were significant differences between tPC and PAPC when compared to DPPC (P < 0·01) again when using Tukey's pairwise comparisons. Using PMA as a stimulant, preincubation of each PC subspecies at 125 μg/ml for 2 h gave a significant reduction in the release of ROIs as determined by LCL (P < 0·0001 using anova) (Fig. 2). Tukey's indicated that DPPC was responsible for the significant decrease in LCL in this part of the study.

Time course of phosphatidylcholine effects on LCL

Using opsonized zymosan as stimulus (125 μg/ml), peak light emission of MM6 cells treated with tPC and DPPC for 2, 6 and 18 h was significantly inhibited (P < 0·01 and P < 0·001, respectively) from that of untreated MM6 (Fig. 3) when analysed by Mann–Whitney. However, 125 μg/ml PAPC significantly enhanced LCL (P < 0·001) at 30 min, 2, 6 and 18 h again when comparing the medians against control.

Control experiments

Experiments were performed to ascertain if the DPPC was affecting the LPS priming process or the stimulation of the cells with PMA or OpZ. Incubation of MM6 cells with DPPC (125 μg/ml) after they were primed with LPS gave significant inhibition of LCL (P < 0·005). This suggests that the DPPC was not affecting the priming process. In addition incorporation of phospholipids into the chemiluminescence buffer had no direct effect on the subsequent LCL from MM6 cells stimulated with PMA or OpZ. This showed that DPPC does not inhibit or quench the LCL once it has been produced.

Effect of DPPC on ROI production in isolated human peripheral blood monocytes

DCF-DA treated human peripheral blood monocytes were stimulated with PMA to induce ROI production and analysed by FACS for the fluorescent oxidized DCF product. Treatment of these cells with DPPC (125 μg/ml) inhibited the ROI response by approximately 30% (P < 0·05 analysed by Mann–Whitney) (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.

Secretion of TNF-α in MM6 cells precultured with PC and its subspecies

Total PC and DPPC significantly reduced TNF-α release by MM6 when compared to controls (P < 0·05). A significant reduction of TNF-α release was seen after preincubation with DPPC and tPC at a concentration of 125 and 250 μg/ml (Table 1). No significant reduction was seen in PAPC treated cells at any of the concentrations tested.

Uptake of DPPC by MM6 cells and effects on membrane fluidity

HPLC analysis of MM6 cell extracts demonstrated that total membrane DPPC was increased by 30 ± 2·8% by weight (n = 3) after 2 h incubation with 250 μg/ml of DPPC (results not shown). No measurable increase in any other quantified phospholipids was seen. EPR spectroscopy showed that the order parameter (S) increased from 0·655 in untreated cells to 0·783 and 0·787 in cells preincubated for 2 h with 125 and 250 μg/ml DPPC, respectively (n = 3). This represents a significant decrease in membrane fluidity (results not shown).

The effect of DPPC on p44 (ERK 1)/p42 (ERK 2) or p38 MAPK activity

The preincubation times shown to affect LCL and TNF-α release were used to study MAPK signalling in DPPC treated cells. There was no observable effect of 10, 100 or 500 μg/ml of DPPC on p44 (ERK 1)/p42 (ERK 2) phosphorylation (Fig. 4) when primed with LPS and stimulated with PMA or OpZ. No observable effect of DPPC on p38 phosphorylation was seen using similar experimental conditions (Fig. 5). Similar results were obtained in cells that were not primed with LPS. Cells not stimulated with PMA or OpZ had little or no activated MAPK.