IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, 1 3 ( 4 ) , 6 2 3 - 6 4 2 (1991)
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CORRELATION BETWEEN MODIFICATION OF MEMBRANE PHOSPHOLIPIDS AND SOME BIOLOGICAL ACTIVITY OF LYMPHOCYTES, NEUTROPHILS AND MACROPHAGES
Francesco Galdiero*, Caterina Romano Canatelli, Concetta Bentivoglio, Ciro Capasso, Santa Cioffi, Antonio Folgore, Femanda Gorga, Raffaele Ianniello, Silvana Mattera, Immacolata NUZZO, Antonietta Rizzo, Maria Antonietta Tufano. Istituto di Microbiologia I Facolta’ di Medicina e Chirurgia Larghetto S.Aniello a Caponapoli, 2 - Universita di Napoli - 80138 NAPOLI, ITALY.
ABSTRACT Our study considered the possibility of modifying the functional response of human neutrophils, of mouse lymphocytes and macrophages treated with phospholipids having different polar groups, different isomerisms with saturated and unsaturated fatty acids from C12 to C20 carbon atoms. The results are as follows. a) Most of the phospholipids containing fatty acids from C12 to C20 cause inhibition of the blastogenic capacity of the polyclonal activators tested. b) The phospholipids tested cause a decrease in adherence of polymorphonuclear leukocytes with the exception of the phosphatidyl-cholinecontaining saturated and unsaturated fatty acids. c) A decrease in polymorphonuclear leukocytes migrational capacity almost always occurs.
Send Correspondence and Proofs to: Prof.F.Galdiero - Istituto di Microbiologia I Facolta di Medicina e Chirurgia, Larghetto S.Aniello a Caponapoli, 2 - UniversitA degli Studi di Napoli 80138 NAPOLI. ITALY.
1 Abbreviations used in the text: FAs, Microbial fatty acids; PLs, phospholipids; C o d , concanavalin A, LPS,lipopolysaccharide. 623 Copyright 0 1991 by Marcel Dekker, Inc.
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GALDIERO ET A L .
The cells treated with L-phosphatidyl-ethanolaminehaving fatty acids from C14 to C17 show an increase in chemiluminescence; those treated with phosphatidyl-choline and Lphosphatidyl-glycerol show a decrease of the chemiluminescence; L-phosphatidic acid and Lphosphatidyl-ethanolamine having Microbial fatty acids (FAs) at c16 cause a decrease in the formation of phagolisosomes in the macrophages tested. Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/28/14 For personal use only.
d)
INTRODUCTION Numerous research projects have been carried out attempting to manipulate the composition in the fatty acids of prokaryotic and eukaryotic cells. It has been suggested that changes in the physical state of the membrane lipids arising from alterations in fatty acid composition could have a modulating effect on membrane-mediated functions. Microbial fatty acids (FAs) auxotroph have been used to modify the fatty acids composition of biomembrane lipids in order to study the effect of lipid composition on membrane structure, function and assembly (1, 2, 3, 4, 5, 6) and in various other phenomena present in animal cells, such as: a) cell-cell fusion, b) endocytosis, c) antigenic mixing, etc. On the other hand, there is a considerable body of evidence that FAs may exert an influence on immune reactivity both in vivo and in vitro (7, 8 , 9 , 10, 11, 12). In previous research projects (13, 14) we demonstrated that metabolic alterations which determine cellular accumulation of lipids determine a state of lowered specific and nonspecific protection. These alterations vary in degree and in relation to the modifications obtained in the distribution of lipids and proteins within cells. In order to further investigate the effect of lipids supplementation on the biological properties of cells involved in the protective response, we have studied the effects of phospholipids (PLs) with acyl-chains containing FAs from C12 to C20 incorporated by macrophages, polynucleates neutrophils and lymphocytes. The action of each PLs used singly in our experiments allows us to better evaluate the resulting biological effects. At present our research does not foresee a mixture of different PLs, nor the addition of cholesterol.
MATERIALS AND METHODS
Splenic cells were prepared from mice spleens, aseptically removed, minced and washed 3 times in RPMI 1640 (Labtek Laboratories, Eurobio, Paris). Then 0.17M NH4C1
P H O S P H O L I P I D S , L Y M P H O C Y T E S , N E U T R O P H I L S AND M A C R O P H A G E S
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was added to lyse the red blood cells, and after two washings with RPMI 1640 the lymphocytes were isolated on MSL (Milieu de separation des lymphocytes; Eurobio, Paris) at 600 x g for 30 min at room temperature. The isolated lymphocytes were washed and resuspended with RPMI 1640. Cell viability was evaluated with the trypan blue exclusion
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test.
Viable human PMNs were obtained from the heparinized venous blood of normal healthy subjects using mono-ply-resolving medium (MPRM; Flow Laboratories, U.K.) fractionation. Contaminating erythrocytes were lysed by 0.17M NH4CL. The isolated PMNs were washed three times in RPMI 1640 medium (Labtek Laboratories Eurobio, Paris).
Macrophages were obtained by injecting 3 ml of 3% thioglycolate broth in the peritoneal cavity of CD-1 mice. The cells of the peritoneal exudate were washed in RPMI 1640 (Labtek Laboratories, Eurobio, Paris), plated in Falcon flasks and incubated for 2 h at 37°C in a 5 % C 0 2 - air incubator. The adherent cells, harvested and washed in RF'MI 1640 medium, were suspended in RPMI supplemented with 2% fetal calf serum (FCS; Miles) at a final concentration of 2x106 cellslml. The viability was evaluated by trypan blue exclusion test.
The PLs indicated in Tab. 1 were handled as described by Wassef and Alving (15) for the preparation of liposomes. In brief, PLs (Sigma) were mixed in a pear-shaped flask (Wite 9-10 ml) with chloroform, and dried with a rotary evaporator (Heidolph-200) for 1 h under vacuum. The lipids were suspended by adding RPMI 1640 (Setomed) and a small quantity (approx. 70-100 pl for a 10 ml boiling flask) of 0.5 mm glass beads, and then flushed with nitrogen, sealing the flask and vortexing repeatedly and vigorously at top speed. This process caused multilamellar vescicles to form spontaneously. Under the appropriate chemical- physical conditions, many of the PLs used showed the formation of
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GALDIERO ET AL.
multilamellar l i p o m e s when checked on electron microscope (Zeiss EM 109). A cellular suspension with phospholipids added was incubated at 37'C for 4 hrs in 5% C02. The
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cells were then thoroughly washed with RPMI 1640 medium, their viability was checked, and elimination of the phospholipids from the suspension was ascertained. Eventual adherence of phospholipid aggregates to the washed cells was evaluated electron microscope. Phospholipids that unavoidably formed aggregates were not studied. The incorporation and exchange of phospholipids between the suspension medium and the cells has been adequately studied (16, 17, 18, 19). Determination of phospholipid incorporation was carried out by comparing chromatograms of membrane preparations of control cells with those of treated cells (data not shown). Membrane preparation was carried out according to Arvan (20). Chromatography of phospholipids was carried out on a one dimensional thin - layer chromatographysystem (2 1).
The toxicity of PL preparations was assayed on cells studied cultivated in RPMI 1640 (Biochrom KG, Germany) with the addition of 5% fetal calf serum and antibiotics. Scaled quantities of PLs, starting with aliquots at a concentration of 6.25 nmol/lO6 cells, were added to the cultures which were incubated for 2 h at 37'C in 5% C02. The cells were then washed with phosphate buffer saline and the number of dead cells per 500 was evaluated by trypan blue exclusion test. The highest non-toxic quantity of each preparation of PL was used in our experiments.
The treated lymphocytes were resuspended in RPMI 1640 at a concentration of 3.106 cells/ml. Aliquots of 100 p1 placed in multiwell plates with 1,2 pg ConA per well were incubated in 5 % C02 at 37' C for 72 h. Six hours before termination of culture H3thymidine (0.5 yCi; sp. act. 5 Ci/mmol Amersham, Arlington Heights, UK) was added to each culture. Lipopolysaccharide (LPS) extract from Escherichiu coli 0128:B 12 was treated in the same way, and mitogenicity was evaluated on murine lymphocytes incubated with 2 *g LPS/well, by increasing thymidine intake. All cultures were harvested with a multiple automated sample harvester (Skatron cell Harvester-Biotech-Italia) onto glass fiber filters. The filters were dried and placed in vials which were filled with 3 ml of scintillation cocktail (Ready Gel, Beckman) and counted in
PHOSPHOLIPIDS, LYMPHOCYTES, NEUTROPHILS AND MACROPHAGES
a Beckman spectrometer with a standard error E
=
627
0.5%. All determinations were carried
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/28/14 For personal use only.
out in triplicate. Untreated and unstimulated lymphocytes were used as controls.
The adherence of PMNs, treated and untreated with PLs for 2 h at 37" C and then washed, was evaluated according to Krause et al. (23). Briefly, 0.5 ml aliquots of the suspension containing PMNs (5x106 cell/ml) were added to the top of Pasteur pipettes, pressed to a height of 1.5 mm with 40 mg of washed nylon wool. The PMNs in the effluent sample were counted and the percent of PMN adherence was evaluated. PMN adherence for a given specimen is defined as the average value for three columns.
Chemotaxis of PMNs under agarose was determined according to Nelson et al. (23). Briefly, 4 ml of 1% agarose in RPMI 1640 medium were poured into 50 mm diameter Falcon Petri dishes. Three wells (3 mm diameter and spaced 2.4 mm apart) were then cut in these agarose plates; 0.01 ml of PMN suspensions containing 3Ox1O6 cellslml, treated and untreated with phospholipids for 2 h at 37" C and then washed, were added to the central well; 0.01 ml of a pool of human sera activated with Zymosan (5 mg/ml) or 10-4M FMLP (Sigma Chemical Co. Ltd, England) were pipetted into one of the lateral wells, and served as a stimulant for chemotaxis. The other well was filled with 0.01 ml of RPMI.The cells that had migrated were counted using an eye piece grid under x40 magnification and the migration index calculated as described by Nelson (23).
0.2 ml of PMN (1x106 cells/ml) treated and untreated with PLs for 2 h at 37" C and then washed, were incubated at 37' C with 0.4 ml PBS, 0.05 ml of 10-4M luminol (Sigma Chemical Co. Ltd, England), 0.1 ml of pooled normal human serum (opsonizing serum), and 0.1 ml of a 15 mg/ml Zymosan suspension (Sigma Chemical) in PBS (24). CL measurements were taken automatically at 6 sec intervals every 5 min for 60 min, under constant vortexing between the measurements, by a microprocessor-controlled LS 7500 Beckman Scintillation Counter. The temperature was maintained constant at 37'C both
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GALDIERO E T AL.
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during the incubation and the CL measurements. The results are expressed as cpm/lO5 cells. Each experiment was run in triplicate.
Monolayers of lo6 mice peritoneal macrophages in Leighton tubes were covered with 1 ml of RPMI 1640medium containing PLs and then incubated at 37" C for 4 h in 5% C02. At the end of incubation the monolayers were extensively washed with FWMI medium and then stained at 37'C for 10 min with 1 ml acridine orange in RPMI 1640 (1 pg/ml). After staining, the monolayers were washed with RPMI 1640 to remove excess acridine orange, and were coveted with 1 ml of the same medium containing 2x106 Sxerevisiae cells. The monolayem were incubated with the yeast cells at 37" C for different lengths of time (15, 45, 60,min.) and then observed by fluorescence microscopy using a Leitz microscope with a BP filter and an excitation wave length of 450-490 nm.
For fluorescence polarization studies, membranes of splenocytes prepared as described by Arvan et al. (20), and pretreated with PLs, were incubated at 25'C for 30 min. as described in a previous paper (25). The fluorescence measurements were performed on 100 pg of membrane suspension in the presence of 2 pM dispersion of DPH (Diphenyl-hesatriene, Sigma) in 50 pM Tris-HC1 buffer pH 7.5, containing 0.1 M NaCl. The degree of fluorescence polarization (P) was measured using an Hitachi Perkin Elmer LS-3B spectrofluorimeter equipped with excitation and emission polarizers. The fluorescence emission was corrected for light scattering, calculated by measuring the fluorescence intensity of the membrane incubated in the same medium, but without the stain. The P value was calculated according to Luly and Shinitzky (26).
Statistical significance of the difference between each test and the respective control was examined by the Student's t-test in most in vitro assays. Significance was determined at the 5% and 1% levels.
PHOSPHOLIPIDS, LYMPHOCYTES, NEUTROPHILS AND MACROPHAGES
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RESULTS After 4 h incubation the PL preparation was absorbed (data not shown). Thin - layer chromatography studies demonstrated that each PL had bands of different dimensions and density. The tests described below were carried out on cells with qualitative variations, even minimal, in the lipidic phase; a dose-response study for each PL will further clarify the effects observed. PL preparations containing choline showed a higher degree of toxicity compared to preparations containing ethanolamine or glycerol. Among the FAs linked to the PC, those saturated with 16-18 carbon atoms were less toxic than those having double links. Non-toxic concentrations of PLs used in our tests are reported in Tab. 1.
From the results reported in Tab. 2 it is evident that most of the PLs containing acylchains from C12 to C20 cause inhibition of the blastogenic capacity of the two polyclonal activators. All the acyl-chains studied from C12 to C18 linked to PA, acyl-chains from Cg to C20 linked to PC always show a decrease in stimulation of lymphocytes from LPS and ConA. This reduction is especially notable in the case of PC-B-18:2(9~,12c)-y-16:0; similarly blastogenesis is also reduced with lymphocytes treated with acyl-chains linked to PE and to PG.
The external membrane of the PMNs treated with different PLS undergoes changes which are manifested by an altered adherence to nylon wool. Almost all the PLs tested induce modifications in the cytoplasmatic membrane of the PMN which lead to a decrease in adherence, with the exception of those PCs containing both saturated and unsaturated FAs. The PC causes a decrease only when it is esterified with two different radicals: unsaturated C18 and satured C16-acyl-chains. This lack of effect is generally seen with other PLs tested (PA, PE, PG) which contain acyl-chains lower than C16. Results are reported in Tab. 3.
The migrational capacity of PMN is altered with respect to the nature of the phospholipids and the nature of their acyl-groups. PA with acyl-chains from C12 to c16
6 30
TABLE 1
G A L D I E R O E T AL.
-
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Phospholipids used in the tests
Abbreviations
Concentrations (pg/lo%eIl)
L-a-Phosphatidic Acid Dilauroyl L-a-Phosphatidic Acid Myirtoyl DL-a-Phosphatidic Acid Dipalmitoyl L-a-Phosphatidic Acid Dipalmitoyl L-a-Phosphatidic Acid Distearoyl L-a-Phosphatidic Acid Dioleoyl
PA-I2:O PA-14 0 DL-PA-16:O PA-16:O PA-1810 PA-18:l ( 9 ~ )
L-a-Phosphatidylcholine Dicaproyl L-a-Phosphatidylcholine Didecanoyl DL-a-PhosphatidylcholineDilauroyl L-a-Phosphatidylcholine Dimyristoyl D-a-Phosphatidylcholine Dipalmitoyl L-a-Phosphatidylcholine Dipalmitoyl L-a-Phosphatidylcholine Distearoyl L-a-Phosphatidylcholine Dioleoyl L-a-Phosphatidylcholine O-Oleoyly-Palmitoyl L-a-Phosphatidylcholine O-Oleoyly-Stearoyl L-a-Phosphatidylcholine Dilinoleoyl L-a-Phosphatidylcholine O-Linoleoyly-Palmitoyl L-a-Phosphatidylcholine Diarachidoyl L-a-Phosphatidylcholine O-Arachidonoyly-Stearoyl
Pc-6~0 100 PC-1o:o 6.25 DL-PC-12:O 12.5 PC-140 100 D-PC-16:O 100 PC-16:O 100 PC-18:O 100 PC-18:I (9c) 25 100 PC-O-18:I ( 9 ~ y-16:O ) PC-O-18:I (9c) r-18:O 50 PC-18:2 (9c,l2c) 50 PC-O-18:2 ( 9 ~ , 1 2 y-16:O ~) 100 100 PC-20:4 (5~,8c,llc,14C) PC-O-20:4(5~,8c,lI C , ~ ~~-16:0100 C)
L-a-PhosphatidylethanolamineDilauroyl L-a-PhosphatidylethanolamineDimyristoyl DL-a-PhosphatidylethanolamineDipalmitoyl L-a-PhosphatidylethanolamineDiheptadecanoyl L-a-Phosphatidylethanolamine Distearoyl
PE-12:O PE-140 DL-PE-1610 PE-17:O PE-18:O
100 100 100 100 100
L-a-PhosphatidyCN-N-Dimethylethanolamine Dipalmitoyl PDME-16:O
100
12.5
50 50 100 50 100
L-a-Phosphatidyl-DL-GlycerolDimyristoyl DL-a-PhosphatidyCDL-GlycerolDipalmitoyl L-a-Phosphatidyl-DL-GlycerolDipalmitoyl L-a-Phosphatidyl-DL-Glycerol Distearoyl L-a-Phosphatidyl-DL-Glycerol Dioleoyl
PG-14:O DL-PG-16:O PG-16:O PG-18:O PG-18:l ( 9 ~ )
100 100 100 100 100
DL-a-Phosphatidyl-L-SerineDipalmitoyl
DL-PS-16:O
100
631
PHOSPHOLIPIDS, LYMPHOCYTES, NEUTROPHILS AND MACROPHAGES
-
TABLE 2. Percentage of incorporation variation of 3H-thymidine in lymphocytes treated with phospholipids and stimulated in virro with either LPS from Ecoii 0:128:812 or with ConA. Data reported are average of three experiments +SD.
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3H-thymidine Incorporation(%) PLS added
Lymphocytes a
Lymphocytesb +LPS (2PglWelI)
PA-12:O DL-PA-1610 PA-16:O PA-I 8:O PA-18:1(9~)
-59 -90 -74 -29 +6
+8(d) ?9(d) +9(d) +5 +2
-81 -94 -90
PC- 6:O PC-1o:o PC-1410 DL-PC-16:O PC-16:O PC-18:O PC-18:1(9~) PC-0-18:1(9~)~-16:0 PC-18:2(9~,12~) PC-B-I8:2(9~,12~)7-16:0 PC-20:4(5~,8~,11~,14c) I c4C)7-18:0 ,~ PC-0-20:4(5~,8~,1
-88 -17 -14 -13 -39 -40
-53 +7(d)
-36 -47 -93 +I0 -46
?9(d) +4 k4 +4 +6(d) +6(d) +5 +6(d) +7(d) +10(d) +3 +7(d)
+6(d) 24 *6(d) +8(d) *4 -38 +6(d) +2 +I -37 +6(d) -97 +10(d) -32 +6(d) -86 +9(d)
DL-PE-1610 PE-1710 PE-18:O
-39 -17 -66
+6(d) ?4 +8(d)
-14 -57 -65
-47
+7(d)
PDME-16:O
9(d) +10(d) *9(d) +9(d)
LymphocytesC +ConA (1-2PglWell)
-82 -88 -88 -94
9(d) +9(d) +6(d) +9(d) +10(d)
-68 -63 -26 -9 -51 -3 -11 -37 -29 -98 -23 -73
+8(d) +8(d) +5 +3 *7(d) +2 +3 +6(d) k5 +10(d) +5 +8(d)
+4 +8(d) *8(d)
45 -19 -58
+7(d) *4 t8(d)
-60
+8(d)
-75
+9(d)
-77 +9(d)
-50
+7(d)
-69 0 -14 -48
?8(d)
-22
-84 0
?
-35 -13 -36 -66 -12
PG-14:O DL-PG-16:O PG-1610 PG-18:I ( 9 ~ )
-64 +8(d)
-53 +7(d)
-1 -74
-48 -84
DL-PS-16:O
-34 ?6(d)
+1 +9(d)
aControl: lymphocytes (100%) Control: lymphocytes + LPS (100%) Control: lymphocytes + ConA (100%) (d) Significance (p
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CORRELATION BETWEEN MODIFICATION OF MEMBRANE PHOSPHOLIPIDS AND SOME BIOLOGICAL ACTIVITY OF LYMPHOCYTES, NEUTROPHILS AND MACROPHAGES
Francesco Galdiero*, Caterina Romano Canatelli, Concetta Bentivoglio, Ciro Capasso, Santa Cioffi, Antonio Folgore, Femanda Gorga, Raffaele Ianniello, Silvana Mattera, Immacolata NUZZO, Antonietta Rizzo, Maria Antonietta Tufano. Istituto di Microbiologia I Facolta’ di Medicina e Chirurgia Larghetto S.Aniello a Caponapoli, 2 - Universita di Napoli - 80138 NAPOLI, ITALY.
ABSTRACT Our study considered the possibility of modifying the functional response of human neutrophils, of mouse lymphocytes and macrophages treated with phospholipids having different polar groups, different isomerisms with saturated and unsaturated fatty acids from C12 to C20 carbon atoms. The results are as follows. a) Most of the phospholipids containing fatty acids from C12 to C20 cause inhibition of the blastogenic capacity of the polyclonal activators tested. b) The phospholipids tested cause a decrease in adherence of polymorphonuclear leukocytes with the exception of the phosphatidyl-cholinecontaining saturated and unsaturated fatty acids. c) A decrease in polymorphonuclear leukocytes migrational capacity almost always occurs.
Send Correspondence and Proofs to: Prof.F.Galdiero - Istituto di Microbiologia I Facolta di Medicina e Chirurgia, Larghetto S.Aniello a Caponapoli, 2 - UniversitA degli Studi di Napoli 80138 NAPOLI. ITALY.
1 Abbreviations used in the text: FAs, Microbial fatty acids; PLs, phospholipids; C o d , concanavalin A, LPS,lipopolysaccharide. 623 Copyright 0 1991 by Marcel Dekker, Inc.
6 24
GALDIERO ET A L .
The cells treated with L-phosphatidyl-ethanolaminehaving fatty acids from C14 to C17 show an increase in chemiluminescence; those treated with phosphatidyl-choline and Lphosphatidyl-glycerol show a decrease of the chemiluminescence; L-phosphatidic acid and Lphosphatidyl-ethanolamine having Microbial fatty acids (FAs) at c16 cause a decrease in the formation of phagolisosomes in the macrophages tested. Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/28/14 For personal use only.
d)
INTRODUCTION Numerous research projects have been carried out attempting to manipulate the composition in the fatty acids of prokaryotic and eukaryotic cells. It has been suggested that changes in the physical state of the membrane lipids arising from alterations in fatty acid composition could have a modulating effect on membrane-mediated functions. Microbial fatty acids (FAs) auxotroph have been used to modify the fatty acids composition of biomembrane lipids in order to study the effect of lipid composition on membrane structure, function and assembly (1, 2, 3, 4, 5, 6) and in various other phenomena present in animal cells, such as: a) cell-cell fusion, b) endocytosis, c) antigenic mixing, etc. On the other hand, there is a considerable body of evidence that FAs may exert an influence on immune reactivity both in vivo and in vitro (7, 8 , 9 , 10, 11, 12). In previous research projects (13, 14) we demonstrated that metabolic alterations which determine cellular accumulation of lipids determine a state of lowered specific and nonspecific protection. These alterations vary in degree and in relation to the modifications obtained in the distribution of lipids and proteins within cells. In order to further investigate the effect of lipids supplementation on the biological properties of cells involved in the protective response, we have studied the effects of phospholipids (PLs) with acyl-chains containing FAs from C12 to C20 incorporated by macrophages, polynucleates neutrophils and lymphocytes. The action of each PLs used singly in our experiments allows us to better evaluate the resulting biological effects. At present our research does not foresee a mixture of different PLs, nor the addition of cholesterol.
MATERIALS AND METHODS
Splenic cells were prepared from mice spleens, aseptically removed, minced and washed 3 times in RPMI 1640 (Labtek Laboratories, Eurobio, Paris). Then 0.17M NH4C1
P H O S P H O L I P I D S , L Y M P H O C Y T E S , N E U T R O P H I L S AND M A C R O P H A G E S
625
was added to lyse the red blood cells, and after two washings with RPMI 1640 the lymphocytes were isolated on MSL (Milieu de separation des lymphocytes; Eurobio, Paris) at 600 x g for 30 min at room temperature. The isolated lymphocytes were washed and resuspended with RPMI 1640. Cell viability was evaluated with the trypan blue exclusion
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/28/14 For personal use only.
test.
Viable human PMNs were obtained from the heparinized venous blood of normal healthy subjects using mono-ply-resolving medium (MPRM; Flow Laboratories, U.K.) fractionation. Contaminating erythrocytes were lysed by 0.17M NH4CL. The isolated PMNs were washed three times in RPMI 1640 medium (Labtek Laboratories Eurobio, Paris).
Macrophages were obtained by injecting 3 ml of 3% thioglycolate broth in the peritoneal cavity of CD-1 mice. The cells of the peritoneal exudate were washed in RPMI 1640 (Labtek Laboratories, Eurobio, Paris), plated in Falcon flasks and incubated for 2 h at 37°C in a 5 % C 0 2 - air incubator. The adherent cells, harvested and washed in RF'MI 1640 medium, were suspended in RPMI supplemented with 2% fetal calf serum (FCS; Miles) at a final concentration of 2x106 cellslml. The viability was evaluated by trypan blue exclusion test.
The PLs indicated in Tab. 1 were handled as described by Wassef and Alving (15) for the preparation of liposomes. In brief, PLs (Sigma) were mixed in a pear-shaped flask (Wite 9-10 ml) with chloroform, and dried with a rotary evaporator (Heidolph-200) for 1 h under vacuum. The lipids were suspended by adding RPMI 1640 (Setomed) and a small quantity (approx. 70-100 pl for a 10 ml boiling flask) of 0.5 mm glass beads, and then flushed with nitrogen, sealing the flask and vortexing repeatedly and vigorously at top speed. This process caused multilamellar vescicles to form spontaneously. Under the appropriate chemical- physical conditions, many of the PLs used showed the formation of
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GALDIERO ET AL.
multilamellar l i p o m e s when checked on electron microscope (Zeiss EM 109). A cellular suspension with phospholipids added was incubated at 37'C for 4 hrs in 5% C02. The
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cells were then thoroughly washed with RPMI 1640 medium, their viability was checked, and elimination of the phospholipids from the suspension was ascertained. Eventual adherence of phospholipid aggregates to the washed cells was evaluated electron microscope. Phospholipids that unavoidably formed aggregates were not studied. The incorporation and exchange of phospholipids between the suspension medium and the cells has been adequately studied (16, 17, 18, 19). Determination of phospholipid incorporation was carried out by comparing chromatograms of membrane preparations of control cells with those of treated cells (data not shown). Membrane preparation was carried out according to Arvan (20). Chromatography of phospholipids was carried out on a one dimensional thin - layer chromatographysystem (2 1).
The toxicity of PL preparations was assayed on cells studied cultivated in RPMI 1640 (Biochrom KG, Germany) with the addition of 5% fetal calf serum and antibiotics. Scaled quantities of PLs, starting with aliquots at a concentration of 6.25 nmol/lO6 cells, were added to the cultures which were incubated for 2 h at 37'C in 5% C02. The cells were then washed with phosphate buffer saline and the number of dead cells per 500 was evaluated by trypan blue exclusion test. The highest non-toxic quantity of each preparation of PL was used in our experiments.
The treated lymphocytes were resuspended in RPMI 1640 at a concentration of 3.106 cells/ml. Aliquots of 100 p1 placed in multiwell plates with 1,2 pg ConA per well were incubated in 5 % C02 at 37' C for 72 h. Six hours before termination of culture H3thymidine (0.5 yCi; sp. act. 5 Ci/mmol Amersham, Arlington Heights, UK) was added to each culture. Lipopolysaccharide (LPS) extract from Escherichiu coli 0128:B 12 was treated in the same way, and mitogenicity was evaluated on murine lymphocytes incubated with 2 *g LPS/well, by increasing thymidine intake. All cultures were harvested with a multiple automated sample harvester (Skatron cell Harvester-Biotech-Italia) onto glass fiber filters. The filters were dried and placed in vials which were filled with 3 ml of scintillation cocktail (Ready Gel, Beckman) and counted in
PHOSPHOLIPIDS, LYMPHOCYTES, NEUTROPHILS AND MACROPHAGES
a Beckman spectrometer with a standard error E
=
627
0.5%. All determinations were carried
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out in triplicate. Untreated and unstimulated lymphocytes were used as controls.
The adherence of PMNs, treated and untreated with PLs for 2 h at 37" C and then washed, was evaluated according to Krause et al. (23). Briefly, 0.5 ml aliquots of the suspension containing PMNs (5x106 cell/ml) were added to the top of Pasteur pipettes, pressed to a height of 1.5 mm with 40 mg of washed nylon wool. The PMNs in the effluent sample were counted and the percent of PMN adherence was evaluated. PMN adherence for a given specimen is defined as the average value for three columns.
Chemotaxis of PMNs under agarose was determined according to Nelson et al. (23). Briefly, 4 ml of 1% agarose in RPMI 1640 medium were poured into 50 mm diameter Falcon Petri dishes. Three wells (3 mm diameter and spaced 2.4 mm apart) were then cut in these agarose plates; 0.01 ml of PMN suspensions containing 3Ox1O6 cellslml, treated and untreated with phospholipids for 2 h at 37" C and then washed, were added to the central well; 0.01 ml of a pool of human sera activated with Zymosan (5 mg/ml) or 10-4M FMLP (Sigma Chemical Co. Ltd, England) were pipetted into one of the lateral wells, and served as a stimulant for chemotaxis. The other well was filled with 0.01 ml of RPMI.The cells that had migrated were counted using an eye piece grid under x40 magnification and the migration index calculated as described by Nelson (23).
0.2 ml of PMN (1x106 cells/ml) treated and untreated with PLs for 2 h at 37" C and then washed, were incubated at 37' C with 0.4 ml PBS, 0.05 ml of 10-4M luminol (Sigma Chemical Co. Ltd, England), 0.1 ml of pooled normal human serum (opsonizing serum), and 0.1 ml of a 15 mg/ml Zymosan suspension (Sigma Chemical) in PBS (24). CL measurements were taken automatically at 6 sec intervals every 5 min for 60 min, under constant vortexing between the measurements, by a microprocessor-controlled LS 7500 Beckman Scintillation Counter. The temperature was maintained constant at 37'C both
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during the incubation and the CL measurements. The results are expressed as cpm/lO5 cells. Each experiment was run in triplicate.
Monolayers of lo6 mice peritoneal macrophages in Leighton tubes were covered with 1 ml of RPMI 1640medium containing PLs and then incubated at 37" C for 4 h in 5% C02. At the end of incubation the monolayers were extensively washed with FWMI medium and then stained at 37'C for 10 min with 1 ml acridine orange in RPMI 1640 (1 pg/ml). After staining, the monolayers were washed with RPMI 1640 to remove excess acridine orange, and were coveted with 1 ml of the same medium containing 2x106 Sxerevisiae cells. The monolayem were incubated with the yeast cells at 37" C for different lengths of time (15, 45, 60,min.) and then observed by fluorescence microscopy using a Leitz microscope with a BP filter and an excitation wave length of 450-490 nm.
For fluorescence polarization studies, membranes of splenocytes prepared as described by Arvan et al. (20), and pretreated with PLs, were incubated at 25'C for 30 min. as described in a previous paper (25). The fluorescence measurements were performed on 100 pg of membrane suspension in the presence of 2 pM dispersion of DPH (Diphenyl-hesatriene, Sigma) in 50 pM Tris-HC1 buffer pH 7.5, containing 0.1 M NaCl. The degree of fluorescence polarization (P) was measured using an Hitachi Perkin Elmer LS-3B spectrofluorimeter equipped with excitation and emission polarizers. The fluorescence emission was corrected for light scattering, calculated by measuring the fluorescence intensity of the membrane incubated in the same medium, but without the stain. The P value was calculated according to Luly and Shinitzky (26).
Statistical significance of the difference between each test and the respective control was examined by the Student's t-test in most in vitro assays. Significance was determined at the 5% and 1% levels.
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RESULTS After 4 h incubation the PL preparation was absorbed (data not shown). Thin - layer chromatography studies demonstrated that each PL had bands of different dimensions and density. The tests described below were carried out on cells with qualitative variations, even minimal, in the lipidic phase; a dose-response study for each PL will further clarify the effects observed. PL preparations containing choline showed a higher degree of toxicity compared to preparations containing ethanolamine or glycerol. Among the FAs linked to the PC, those saturated with 16-18 carbon atoms were less toxic than those having double links. Non-toxic concentrations of PLs used in our tests are reported in Tab. 1.
From the results reported in Tab. 2 it is evident that most of the PLs containing acylchains from C12 to C20 cause inhibition of the blastogenic capacity of the two polyclonal activators. All the acyl-chains studied from C12 to C18 linked to PA, acyl-chains from Cg to C20 linked to PC always show a decrease in stimulation of lymphocytes from LPS and ConA. This reduction is especially notable in the case of PC-B-18:2(9~,12c)-y-16:0; similarly blastogenesis is also reduced with lymphocytes treated with acyl-chains linked to PE and to PG.
The external membrane of the PMNs treated with different PLS undergoes changes which are manifested by an altered adherence to nylon wool. Almost all the PLs tested induce modifications in the cytoplasmatic membrane of the PMN which lead to a decrease in adherence, with the exception of those PCs containing both saturated and unsaturated FAs. The PC causes a decrease only when it is esterified with two different radicals: unsaturated C18 and satured C16-acyl-chains. This lack of effect is generally seen with other PLs tested (PA, PE, PG) which contain acyl-chains lower than C16. Results are reported in Tab. 3.
The migrational capacity of PMN is altered with respect to the nature of the phospholipids and the nature of their acyl-groups. PA with acyl-chains from C12 to c16
6 30
TABLE 1
G A L D I E R O E T AL.
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Phospholipids used in the tests
Abbreviations
Concentrations (pg/lo%eIl)
L-a-Phosphatidic Acid Dilauroyl L-a-Phosphatidic Acid Myirtoyl DL-a-Phosphatidic Acid Dipalmitoyl L-a-Phosphatidic Acid Dipalmitoyl L-a-Phosphatidic Acid Distearoyl L-a-Phosphatidic Acid Dioleoyl
PA-I2:O PA-14 0 DL-PA-16:O PA-16:O PA-1810 PA-18:l ( 9 ~ )
L-a-Phosphatidylcholine Dicaproyl L-a-Phosphatidylcholine Didecanoyl DL-a-PhosphatidylcholineDilauroyl L-a-Phosphatidylcholine Dimyristoyl D-a-Phosphatidylcholine Dipalmitoyl L-a-Phosphatidylcholine Dipalmitoyl L-a-Phosphatidylcholine Distearoyl L-a-Phosphatidylcholine Dioleoyl L-a-Phosphatidylcholine O-Oleoyly-Palmitoyl L-a-Phosphatidylcholine O-Oleoyly-Stearoyl L-a-Phosphatidylcholine Dilinoleoyl L-a-Phosphatidylcholine O-Linoleoyly-Palmitoyl L-a-Phosphatidylcholine Diarachidoyl L-a-Phosphatidylcholine O-Arachidonoyly-Stearoyl
Pc-6~0 100 PC-1o:o 6.25 DL-PC-12:O 12.5 PC-140 100 D-PC-16:O 100 PC-16:O 100 PC-18:O 100 PC-18:I (9c) 25 100 PC-O-18:I ( 9 ~ y-16:O ) PC-O-18:I (9c) r-18:O 50 PC-18:2 (9c,l2c) 50 PC-O-18:2 ( 9 ~ , 1 2 y-16:O ~) 100 100 PC-20:4 (5~,8c,llc,14C) PC-O-20:4(5~,8c,lI C , ~ ~~-16:0100 C)
L-a-PhosphatidylethanolamineDilauroyl L-a-PhosphatidylethanolamineDimyristoyl DL-a-PhosphatidylethanolamineDipalmitoyl L-a-PhosphatidylethanolamineDiheptadecanoyl L-a-Phosphatidylethanolamine Distearoyl
PE-12:O PE-140 DL-PE-1610 PE-17:O PE-18:O
100 100 100 100 100
L-a-PhosphatidyCN-N-Dimethylethanolamine Dipalmitoyl PDME-16:O
100
12.5
50 50 100 50 100
L-a-Phosphatidyl-DL-GlycerolDimyristoyl DL-a-PhosphatidyCDL-GlycerolDipalmitoyl L-a-Phosphatidyl-DL-GlycerolDipalmitoyl L-a-Phosphatidyl-DL-Glycerol Distearoyl L-a-Phosphatidyl-DL-Glycerol Dioleoyl
PG-14:O DL-PG-16:O PG-16:O PG-18:O PG-18:l ( 9 ~ )
100 100 100 100 100
DL-a-Phosphatidyl-L-SerineDipalmitoyl
DL-PS-16:O
100
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PHOSPHOLIPIDS, LYMPHOCYTES, NEUTROPHILS AND MACROPHAGES
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TABLE 2. Percentage of incorporation variation of 3H-thymidine in lymphocytes treated with phospholipids and stimulated in virro with either LPS from Ecoii 0:128:812 or with ConA. Data reported are average of three experiments +SD.
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3H-thymidine Incorporation(%) PLS added
Lymphocytes a
Lymphocytesb +LPS (2PglWelI)
PA-12:O DL-PA-1610 PA-16:O PA-I 8:O PA-18:1(9~)
-59 -90 -74 -29 +6
+8(d) ?9(d) +9(d) +5 +2
-81 -94 -90
PC- 6:O PC-1o:o PC-1410 DL-PC-16:O PC-16:O PC-18:O PC-18:1(9~) PC-0-18:1(9~)~-16:0 PC-18:2(9~,12~) PC-B-I8:2(9~,12~)7-16:0 PC-20:4(5~,8~,11~,14c) I c4C)7-18:0 ,~ PC-0-20:4(5~,8~,1
-88 -17 -14 -13 -39 -40
-53 +7(d)
-36 -47 -93 +I0 -46
?9(d) +4 k4 +4 +6(d) +6(d) +5 +6(d) +7(d) +10(d) +3 +7(d)
+6(d) 24 *6(d) +8(d) *4 -38 +6(d) +2 +I -37 +6(d) -97 +10(d) -32 +6(d) -86 +9(d)
DL-PE-1610 PE-1710 PE-18:O
-39 -17 -66
+6(d) ?4 +8(d)
-14 -57 -65
-47
+7(d)
PDME-16:O
9(d) +10(d) *9(d) +9(d)
LymphocytesC +ConA (1-2PglWell)
-82 -88 -88 -94
9(d) +9(d) +6(d) +9(d) +10(d)
-68 -63 -26 -9 -51 -3 -11 -37 -29 -98 -23 -73
+8(d) +8(d) +5 +3 *7(d) +2 +3 +6(d) k5 +10(d) +5 +8(d)
+4 +8(d) *8(d)
45 -19 -58
+7(d) *4 t8(d)
-60
+8(d)
-75
+9(d)
-77 +9(d)
-50
+7(d)
-69 0 -14 -48
?8(d)
-22
-84 0
?
-35 -13 -36 -66 -12
PG-14:O DL-PG-16:O PG-1610 PG-18:I ( 9 ~ )
-64 +8(d)
-53 +7(d)
-1 -74
-48 -84
DL-PS-16:O
-34 ?6(d)
+1 +9(d)
aControl: lymphocytes (100%) Control: lymphocytes + LPS (100%) Control: lymphocytes + ConA (100%) (d) Significance (p
