Jan 26, 1988 - SUMMARY. The aim of this study was to seek in vitro evidence to support our hypothesis that the ... Dose response studies conducted in serum-free medium, have demon- ... phospholipids found in pulmonary surfactant PC, PG, PI, PE ..... The writers thank Mr P. J. Townsend for technical advice, Mr D. J..
Clin. exp. Immunol. (1988) 73, 117-122
Immunoregulatory properties of pulmonary surfactant: influence of variations in the phospholipid profile MARGARET L. WILSHER, D. A. HUGHES & PATRICIA L. HASLAM Cell Biology Unit, Department of Cardiothoracic Surgery, Cardiothoracic Institute, London
(Acceptedfor publication 26 January 1988)
SUMMARY The aim of this study was to seek in vitro evidence to support our hypothesis that the immunosuppressive properties of pulmonary surfactant might be influenced by variations in phospholipid composition. Dose response studies conducted in serum-free medium, have demonstrated that pure phospholipids of the major types found in pulmonary surfactant, namely phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylinositol (PI) exhibit significant dose dependent suppression of lymphocyte response to phytohaemagglutinin (PHA) (PC > PG > PI). By contrast, phosphatidylethanolamine (PE), sphingomyelin (SM), and the neutral lipid cholesterol (CH) augment the lymphoproliferative response in serum-free medium. The degree of suppression obtained using mixtures of various ratios of PC, PG and PI depended on the proportion of the most suppressive phospholipids (PC and PG). Similarly, increasing the proportion of PC in mixtures containing variable proportions of PE or CH resulted in increasing suppression of lymphoproliferation. Comparisons of the lipid fractions purified from pulmonary surfactant of three species (human, pig and rabbit) showed that the levels of suppression they induced reflected their relative content of phospholipids with the greatest immunosuppressive properties. We conclude that variations in phospholipid composition may affect the immunoregulatory properties of pulmonary surfactant.
Keywords pulmonary surfactant phospholipids cholesterol immunosuppression mitogen
INTRODUCTION We have recently reported that pulmonary surfactant obtained
by bronchoalveolar lavage (BAL) from three species (human, pig, rabbit), suppresses the in vitro proliferative responses of human peripheral blood lymphocytes to mitogens and alloantigens (Wilsher, Hughes & Haslam, 1987). The purified lipids suppressed these responses to a greater degree than whole surfactant, suggesting that the lipid fraction of pulmonary surfactant is responsible for its immunosuppressive properties. The majority of lipids in pulmonary surfactant are phospholipids, in particular phosphatidylcholine (PC), but also others including phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Neutral lipids such as cholesterol, triglycerides and free fatty acids make up about 15% of the total lipid composition (Clements & King, 1976). Although considerable interest has focussed on the immunosuppressive properties of serum lipoproteins (Morse, Witte & Goodman 1977; Curtiss & Edgington, 1977), the immunoreguCorrespondence: Dr P. L. Haslam, Head, Cell Biology Unit, Department of Cardiothoracic Surgery, Cardiothoracic Institute, Fulham Road, London SW3 6HP, UK.
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latory properties of lipids in pulmonary surfactant have received little attention. Ansfield and co-workers have shown that pure preparations of PC, PG and PI all suppress lymphocyte proliferation in a dose-dependent manner (Ansfield & Benson, 1980). It is not known however whether other phospholipids found in surfactant, for example, phosphatidylethanolamine (PE) or phosphatidylserine (PS), share this property. In addition, the neutral lipids and sphingomyelin (SM) may also contribute to its immunoregulatory function. Variations in the pulmonary surfactant phospholipid profile have been demonstrated in a number of pulmonary diseases (Jouanel et al., 1981; Hallman et al., 1982; Hughes & Haslam, 1987). Our group has shown that in untreated patients with the chronic inflammatory lung disease cryptogenic fibrosing alveolitis (CFA), proportions of PG are frequently decreased while those of SM are elevated. Moreover, serial increases in proportions of PG in BAL fluid correlated with steroid responsiveness (Hughes & Haslam, 1987). This has led us to postulate that the relative proportions of phospholipids might determine the immunoregulatory properties of pulmonary surfactant. If this hypothesis is correct, any alteration in the normal phospholipid profile might render pulmonary surfactant less immunosuppressive, thus facilitating potentially damaging immune reactions in
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the alveoli. If this were so, treatment aimed at restoring this imbalance might affect the course of certain inflammatory diseases of the lung. To test this hypothesis we examined, in vitro, the immunosuppressive properties of pure preparations offive main types of phospholipids found in pulmonary surfactant PC, PG, PI, PE and PS, and also SM and cholesterol (CH). Various mixtures of phospholipids, with and without cholesterol, have also been examined. Finally we compared these results with observations on the phospholipid profiles of pulmonary surfactant from the normal lungs of three species (human, pig and rabbit); and investigated how this relates to the relative ability of these surfactant preparations and their purified lipid fractions to induce suppression of mitogen stimulated proliferation. MATERIALS AND METHODS Preparation ofphospholipids and lipids Commercially purified phospholipids (PC, PG, PI, PE, PS) (Sigma) were evaporated to dryness under nitrogen and reconstituted in RPMI 1640 containing 25 mm Hepes buffer (Gibco) in known weight for volume, by ultrasonication. Cholesterol and sphingomyelin (Sigma) were obtained in powdered form and reconstituted in medium in a similar manner. The tissue sources and Sigma product numbers for the lipids are as follows: PC bovine brain (P6638), PE egg yolk (P5149), PG egg yolk (P0514), PI soybean (P5766), PS bovine brain (P6641), CH porcine liver (CH-PL), SM bovine brain (S7004), DPPC (dipalmitoylphosphatidylcholine) crystalline synthetic (P6267). Preparation ofperipheral blood lymphocytes Peripheral blood was obtained from healthy adult volunteers by venepuncture, defibrinated and diluted with an equal volume of minimal essential medium containing 25 mm Hepes buffer (MEM, Gibco). The sample was then layered in 30 ml fractions over 15 ml lymphocyte separation medium (Flow Laboratories) in 50 ml polypropylene tubes (Falcon), and centrifuged at 500 g for 30 min. The resultant cell layer at the interface was aspirated and washed twice in MEM, then reconstituted in medium at 0-5 x 106 cells per ml. Lymphocyte cultures In serum containing media. All cultures were performed in 6 ml polypropylene tubes (Falcon 2063), and in triplicate. Each culture contained 0-5 x 106 cells in 1 ml RPMI with 10% fetal calf serum (FCS), absorbed to remove isoagglutinins as we have previously described (Wilsher, Hughes & Haslam, 1987), and penicillin 100 IU/ml and streptomycin 100 yg/ml (Gibco). Cultures were performed using lymphocytes from eight subjects in the presence of an optimal concentration (0-5 pg/ml) of phytohaemagglutinin (PHA-P, Wellcome), and the presence or absence of PC, PG, PE, PI and PS in the following concentrations: 0-025, 005, 0 1, 02 mg/ml. In six subjects similar cultures were performed in the presence or absence of the same concentrations of SM and CH. Tritiated thymidine (New England Nuclear) was added 18 h before harvesting. At 72 h the cell suspension was divided in 200 p1 volumes into 96-well microtitre plates and harvested using an Ilacon harvester. Viability was assessed by trypan blue exclusion. Further cultures were performed in a similar manner in three subjects in the presence of 005 mg/ml of the following
phospholipid mixtures: PC/PG and PC/PI 75%/25%; 50%/50%; 25%/75%. In serum free media. Because FCS is an abundant source of lipids particularly cholesterol, we extended the above experiments by using serum free media. Cultures were performed using RPMI containing 25 mm Hepes buffer, and with the following supplements: insulin, transferrin, selenium, linoleic acid, and bovine serum albumin (CR-ITS, Flow Laboratories). Dose response studies were performed in four subjects using the following concentrations of the phospholipids used in the earlier experiments (PC, PE, PG, PI, PS, SM) and also CH: 0-05, 0 1, 0-2, 0 4, 0-8 mg/ml. In order to determine if unsaturated PC behaved differently to the saturated form (DPPC) we compared the two lipids at doses of 0-1 and 0-4 mg/ml in four subjects. Further studies were performed in six subjects using the following mixtures of CH and PC: 75%/25%; 50%/50%; 25%/ 75%. A similar experiment was performed using mixtures of PC/ PE in four subjects. All cultures were performed using 0-5 x 106 PBL/ml in the presence of penicillin and streptomycin, at an optimal concentration of PHA (0-5 pg/ml).
Pulmonary surfactant Bronchoalveolar lavage (BAL) fluid was obtained from normal pigs (n = 3), rabbits (n = 3) and non-smoking humans (n = 3) as previously described (Wilsher, Hughes & Haslam, 1987; Dhillon et al., 1986). The fluid was lyophilized and the resulting 'whole surfactant' preparation used in lymphocyte proliferation studies as previously reported (Wilsher et al., 1987). Total lipids were extracted from the BAL fluid using the chloroform/methanol procedure described by Bligh & Dyer (1959). For use in the lymphocyte studies, the lipid extracts were evaporated to dryness under nitrogen, then reconstituted in RPMI. To determine the proportions of individual phospholipids in the extracts, these were separated by one-dimensional thin layer chromatography according to the method of Gilfillan et al. (1983) alongside known standards. The silica gel plates were then charred and quantification was performed using a Shimadzu CS920 reflectance densitometer from which the proportions of each phospholipid present were computed. Statistical analysis Results are expressed as the mean of triplicate cultures + s.e.m. Statistical analysis was performed using the Wilcoxon matched pairs signed-ranks test for pairs of related samples. RESULTS Cultures in serum-containing medium Comparison of the dose-dependent suppression of lymphocyte proliferation by phospholipids and cholesterol. Except for PS and SM, the phospholipids significantly suppressed the in vitro lymphoproliferative response to optimal PHA (0-5 pg/ml) at all doses tested (P < 0-02, Wilcoxon). PC, PG and PI exhibited dose-dependent suppression, the effect being most marked with PC, and PE was mildly suppressive (Fig. 1). The neutral lipid CH did not exhibit significant suppression at the concentrations tested. No loss of cell viability was observed. Effect of varying the ratios of phospholipids on in vitro lymphocyte proliferation. In a separate experiment, the effect of
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Variable immunoregulatory effects of phospholipids
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