Composition and metabolism of phospholipids of human leukocytes

Chemistry and Physics of Lipids, 31 (1982) 169-177 Elsevier Scientific Publishers Ireland Ltd.

169

C O M P O S I T I O N AND M E T A B O L I S M O F P H O S P H O L I P I D S O F H U M A N LEUKOCYTES*

G.V. MARINETTI and K. CATTIEU

Department of Biochemistry, University of Rochester Medical Center, Rochester, N Y 14642 (U.S.A.) Received October 5th, 1981 accepted May 21st, 1982

revision received May 19th, 1982

Human mononuclear (MN) and polymorphonuclear (PMN) leukocytes were analyzed for their phospholipid, triglyceride, cholesterol and fatty acid content. The phospholipid/cholesterol ratio was 1.24 for both cells. MN cells contain more phosphatidylcholine (PC), but less phosphatidylserine (PS), phosphatidylethanolamine 0aE) and sphingomyelin (SPH) than PMN cells when expressed as percent of total phospholipid. When expressed on the basis of lipid content per cell, MN cells contain less PS, PE and SPH but more triglyceride than PMN ceils. PMN cells incorporate palmitic, stearic, linoleic and linolenic acids into their phospholipids, triglycerides or cholesterol esters. The incorporation into triglycerides was highest for all fatty acids. Of the phospholipids, the incorporation was highest into PC. Labeled fatty acids also were found in proteins which had been delipidized by exhaustive extraction with organic solvents. These represent tightly or covalently bound fatty acids. The incorporation of [3H]palmitic acid into this protein fraction is stimulated by insulin.

Keywords: phospholipids; fatty acids; ieukocytes

T h e p h o s p h o l i p i d c o m p o s i t i o n of h u m a n l y m p h o c y t e s a n d P M N leukocytes has b e e n s t u d i e d by G o t t f r i e d [1-3]. L y m p h o c y t e s a n d P M N leukocytes c o n t a i n m u c h m o r e lipid p e r cell t h a n do erythrocytes. P M N cells c o n t a i n m o r e lipid t h a n l y m p h o c y t e s . T h e m a j o r lipids in these l e u k o c y t e s are phospholipids, triglycerides, cholesterol [1-3] a n d glycolipids [4]. B l o m s t r a n d [5] s t u d i e d the fatty acids of isolated h u m a n l y m p h o c y t e s a n d f o u n d that b o t h the p h o s p h o l i p i d a n d triglycerides c o n t a i n e d high a m o u n t s of palmitic, oleic a n d stearic acids. T h e p h o s p h o l i p i d s also h a d a n appreci-

*This work was supported in part by funds from Biomedical Research Support Grant RR05403, Grant IP 50A1-15372 from the National Institutes of Health and Grant PCM-8021564 from the National Science Foundation. 0009-3084/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishers Ireland Ltd.

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able amount of arachidonic acid. Stossel et al. [6] have reported the fatty acid content of the phospholipids of human monocytes, granulocytes and lymphocytes. The phospholipid content and metabolism of leukocytes have been studied in normal and disease states [3, 7] and during active phagocytosis [8--12]. Changes in leukocyte lipid patterns in leukemia and in tissue culture have also been reported [3]. The present study provides data on the phospholipids and fatty acids of normal human MN and PMN cells, the incorporation of labeled palmitic, stearic, linoleic and linolenic acids into specific phospholipids, triglycerides and cholesterol esters of PMN cells, and the effect of insulin on the incorporation of these fatty acids into PMN proteins. These studied extend the earlier studies of Burns et al. [13, 14] and Elsbach [15].

Materials and Methods

Human MN cells (85% lymphocytes, 15% monocytes) and PMN leukocytes (90% granulocytes) were prepared by the method of Boyum [16]. These cells contained negligible amounts of red cells. Cells were washed in Krebs-Ringer-phosphate (KRP) buffer (pH 7.4) and extracted by the method of Folch et al. [17] to obtain the total lipids. The phospholipids were separated by two-dimensional thin-layer chromatography on Merck-Darmstadt silica gel 60 coated glass plates using chloroform/acetone/methanol/acetic acid/water, 5 : 2: 1 : 1 : 0.5, by vol. (CAMAW) as the first solvent and chloroform/propanol/propionic acid/water, 2 : 3 : 2 : 1, by vol. (CPPW) as the second solvent. The chromatogams were dried and exposed to iodine vapors to detect the phospholipid spots. The ninhydrin reagent was used to detect amino-phospholipids. Standard phospholipids PC, PE, PS, SPH and lysophosphatidylcholine (LPC) were obtained from Supelco. Lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), phosphatidylglycerol (PG), phosphatidic acid (PA) and cardiolipin (diphosphatidylglycerol (DPG)) were obtained from Avanti. Saturated and unsaturated fatty acid standards including 14:0 through 24:0, 13:0 through 21:0, 16:1, 18:1, 18:2, 18:3, 20:4, 20:5, 24:1 and 22:6 were obtained from Supelco. The fatty acid methyl esters were prepared by refluxing the different lipids in methanol-HC1 (Supelco, Inc.) as described elsewhere [18]. The analysis of the fatty acid methyl esters was carried out on a Hewlett Packard HP5830 gas chromatograph equipped with a flame detector and microprocessor. The separation was carried out on a 1.8-m glass column (6.3 mm O.D.) packed with 10% diethyleneglycol succinate on Supelcoport (Supelco, Inc.) [18]. The temperature was 190°C and the nitrogen carrier gas flow was 35--40 ml/min. The phospholipid spots were scraped off the chromatograms and eluted

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three times with 3-ml aliquots of methanol-HCl 99/1 at 50°C. The methanol-HC1 extracts were combined and evaporated to dryness and the lipid residue digested with perchloric acid to measure lipid phosphate [19]. The fatty acids of PC, PE and triglycerides were prepared by eluting these components from chromato.grams and refluxing with methanol-HCl. Lipid extractions and evaporation of solvents were done under N2 to minimize lipid peroxidation. The chloroform, acetone, and methanol used for lipid extraction were distilled from glass containers before use. Triglycerides, cholesterol and cholesterol esters were separated by TLC using n-heptane/ethyl ether/acetic acid, 70:20:4 (by vol.) as solvent. The lipid spots were eluted with chloroform/methanol (1:1). Triglycerides were analyzed by the ester group method [20, 21]. Cholesterol was analyzed by standard procedures [21]. Protein was analyzed by the method of Lowry et

al. [221. Incorporation of labeled fatty acids into phospholipids, triglycerides and cholesterol esters was done by incubating PMN cells with the labeled fatty acids, washing the cells, extracting the lipids by the method of Folch et al. [17] and separating the lipids by TLC as described above. The lipid spots were detected by short exposure to iodine vapor. The excess iodine was blown off with nitrogen and the spots were cut off, placed into 10 ml of Amersham ACS cocktail and counted on a Searle Delta 300 Liquid Scintillation Counter after incubating overnight at room temperature. The delipidized pellets were further extracted with chloroform/methanol (1:2) and then with methanol, dissolved in 0.1 N NaOH and counted. The following labeled fatty acids were obtained from Amersham: [1-14C]palmitic acid, 63.3 mCi/mmol; [1-14C]stearic acid, 56.5 mCi/mmol; [1-14C]linoleic acid, 52mCi/mmol; [1-14C]linolenic acid, 56.2mCi/mmol; [9,10(n)-3H]palmitic acid, 500 mCi/mmol.

Results

The phospholipid analyses of leukocyte preparations from seven normal individuals are given in Table I. MN cells contained more PC and LPE than PMN cells. PMN cells contained more PS and possibly more PE than MN cells. The amount of each lipid per 107 cells is given in Table II. Expressed this way, PMN cells had more PS and PE than MN cells. MN cells, however, had more cholesterol esters and triglycerides than PMN cells but the phospholipid/cholesterol ratio was the same for both cells. PMN cells may contain more total phospholipid than MN cells. The fatty acid composition of the leukoeytes is given in Table III. PMN cells contained more 18: 0 dimethylacetal (DMA), 18: 1, and 22: 4 fatty acids

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TABLE I PHOSPHOLIPID COMPOSITION MONONUCLEAR LEUKOCYTES a Phospholipid

PC PE SPH L P C + PI PS PA LPE DPG

OF

HUMAN

POLYMORPHONUCLEAR

AND

% of total lipid phospholipid MN cells

PMN ceils

46-+8 29-+6 9-+4 7-+2 5-+4 1.4 - 0.5 3 -+ 0.1 Trace

32-+ 12 34-+2 13-+2 8-+5 11-+3 1.6 -+ 1 Trace Trace

aThe results are the mean -+S.D. of seven separate cell preparations. The lipids were extracted by the Folch et al, procedure [17] and separated by two-dimensional thin-layer chromatography using C A M A W as the first solvent and CPPW as the second solvent; 2 × los cells were used in each sample.

but less 16: 0, 18: 0 and 20 : 4 fatty acids than M N cells. The D M A originates from plasmalogens. Since our fatty acid analyses were done on the total lipids we cannot compare our results to those of Stossel [15] who analyzed the fatty acids of the total phospholipids. In order to learn how added fatty acids are metabolized by PMN cells we T A B L E II THE NEUTRAL LIPID AND PHOSPHOLIPID COMPOSITION M O N O N U C L E A R A N D P O L Y M O R P H O N U C L E A R CELLS a

OF

HUMAN

nmol/107 cells

PC PE PS SPH Other PL Triglyceride Cholesterol Cholesterol ester PL/cholesterol molar ratio

MN cells

PMN cells

18-+4 11.5-+2 2.8-+0.2 6.5 -+ 0.6 2.2 3.4 -+ 0.7 33 ± 16 7.8 -+ 3.4

16-+2 15-+2 6-+2 8 -+ 2 1.0 2.3 -+ 0.5 37 -+ 5 3 -+ 2

1.24

1.24

aResults are the mean -+S.D. of seven different cell preparations (see Table I); 2 x l0 s cells were used in each sample.

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T A B L E III F A T I ' Y A C I D A N A L Y S I S O F T O T A L LIPIDS O F H U M A N M O N O N U C I ~ A R POLYMORPHONUCLEAR LEUKOCYTES a

AND

A r e a (%)

16:0 18:0 D M A 18:0 18:1 18:2 18:3 20:4 20:5 22:4 22:5 22:6 24 : X (unidentified)

P M N cells

M N cells

13.8---0.8 3.9-+0.4 13.8-+0.7 21.8-+ 1.2 9.4-+ 1.7 2.0-+0.2 11.6_+ 1.8 0.1 -+ 0.01 7.5 ___0.4 1.0-+0.4 0.6-+0.3 1.8 -+ 0.6

16.8-+ 1.1 2.5-+0.7 16.8-+ 1.2 14.7-+0.5 8.3-+0.3 1.9---0.4 19.7-+0.8 1.2-+0.9 4.4-+0.6 1.5---0.1 1.9-+0.5 1.4 - 0.3

"Results are t h e m e a n +-S.D. of four separate cell preparations; 2 x 10s cells were used in each sample. Fatty acids were prepared and analyzed as described previously [18].

studied the incorporation of four labeled fatty acids into phospholipids, triglycerides and cholesterol esters. The results in Table IV show that all four fatty acids are incorporated into various phospholipids. The greatest incorporation occurred in PC with all fatty acids. There were differences in the relative incorporation of the fatty acids into the different phospholipids. Thus, stearic acid was incorporated into PS at a higher level than palmitic, linoleic or linolenic acid. Palmitic acid showed the greatest incorporation into PC. With all fatty acids except stearic acid, LPE was more heavily labeled than PE. However, stearic acid showed a greater incorporation into PS than did the other fatty acids. The incorporation of the four fatty acids into triglycerides and cholesterol esters of PMN cells is shown in Table V. By far the greatest incorporation occurred in triglycerides. This was also observed by Burns et al. [13]. As with phospholipids, palmitic acid was incorporated into triglycerides more than stearic, linoleic and linolenic acids. The saturated fatty acids were incorporated to a greater extent into triglycerides and cholesterol esters than were the unsaturated fatty acids. We also examined the incorporation of fatty acids into the delipidized proteins of PMN cells. The PMN cells were extracted exhaustively with organic solvents to remove loosely bound fatty acids. The protein residues were then measured for radioactivity. The results in Table VI show that all

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TABLE IV INCORPORATION OF LABELED FATI'Y ACIDS INTO PHOSPHOLIPIDS OF H U M A N POLYMORPHONUCLEAR LEUKOCYTES 1.1 x 107 PMN cells were incubated with 2 # Ci of either [14C]linolenic, [14C]linoleic, [14C]stearic or [14C]palmitic acid in 1 ml of KRP buffer (pH 7.4) containing 5 mM glucose and 1% BSA. Samples were run in triplicate and incubated for 1 h, 2 h and 3 h at 37~C. The cells were washed twice with buffer and extracted by the Folch et al. method [17]. The delipidized protein pellets were used to measure tightly bound lipids (see Table VI). The lipid extracts were separated into various phospholipid classes by two-dimensional chromatography on silica gel coated glass plates using C A M A W as the first solvent and CPPW as the second solvent. The lipid spots were detected by iodine vapor test, scraped off and counted in 10 ml of ACS cocktail. The 2/zCi of ~4C-labeled fatty acids contained the following nmol of fatty acid: iinolenic, 33.3; linoleic, 39.2; stearic, 34.5; palmitic, 35.1. pmol incorporated/107 cells Linolenic acid

PC PE PS SPH PA LPE

Linoleic acid

lh

2h

3h

lh

2h

3h

96 15 3.6 0.6 9.2 25

209 24 6.8 0.8 9.1 54

320 35 10 1.3 10.4 63

137 19 7.5 0.8 10.8 34

260 31 14 1.6 11.7 96

428 44 24 2.7 13 90

Palmitic acid

PC PE PS SPH PA LPE

Stearic acid

lh

2h

3h

lh

2h

3h

202 16 13 15 21 17

527 34 23 38 20 42

814 48 32 63 42 56

110 30 20 3.6 11.6 -

220 67 48 9.9 29 -

372 99 78 19 39 -

four fatty acids were incorporated into proteins but palmitic acid had the greatest incorporation. Hydrolysis of the proteins with HCl and extraction of the hydrolysis products with hexane showed that the fatty acids were incorporated as intact molecules. The bound fatty acids are not removed by prolonged dialysis of sodium dodecylsulfate (SDS)-solubilized proteins or by column fractionation on Sephadex G-25. We believe that these fatty acids a r e c o v a l e n t l y b o u n d t o t h e p r o t e i n s [23].

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TABLE V INCORPORATION OF LABELED F A T I N ACIDS INTO TRIGLYCERIDES AND CHOLESTEROL ESTERS OF H U M A N POLYMORPHONUCLEAR LEUKOCYTES The neutral lipids (triglycerides and cholesterol esters) in the lipid extracts given in Table IV were separated by chromatography on silica gel coated glass plates using n-beptane/etherlacetic acid (70:20:4) as solvent. The triglyceride and cholesterol ester spots were detected by the iodine vapor test, scraped out and counted in ACS cocktail. Fatty acid

[14C]Linolenic

[14C]Linoleic

[14C]Stearic

[14C]Palmitic

Time 0a)

1 2 3 1 2 3 1 2 3 1 2 3

pmol incorporated/107 cells Triglyeeride

Cholesterol ester

581 1188 1474 1217 2122 3899 1460 3370 4968 1764 4481 6652

8 17 26 8 12 16 38 43 54 25 39 52

TABLE VI INCORPORATION OF LABELED FATI'Y ACIDS INTO DELIPIDIZED PROTEINS OF H ~ POLYMORPHONUCLEAR LEUKOCYTES The delipidized protein pellets given in Table IV were further extracted, twice with 3 ml of chloroform/methanol (1 : 2) then twice with 3 ml of methanol. The protein pellets were dissolved in 0.1 N NaOH. Aliquots were taken for protein analysis by the Lowry et al. method [22] and other aliquots were counted in 10 ml of ACS cocktail. Fatty acid

[14C]Linolenic [14C]Linoleic [14C]Stearic [14C]Palmitic

pmol/mg protein lh

2h

3h

203 223 209 589

227 240 195 306

274 255 254 419

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G. v. Marinetti and K. Cattieu, Phospholipids of human leukocytes

Insulin (50 nM) gave a two-fold increase in the incorporation of [3H]palmitic acid into PMN proteins (data not shown). The effect appears largest at 5 min.

Discussion

We have shown that PMN and MN leukocytes have a different phospholipid and fatty acid composition. In comparing our results with those of Gottfried [1], the data for PC, P E and SPH for MN cells are in agreement. Gottfried did not separate PS from phosphatidylinositol or P A from DPG. We find that PS and P A occur in both cells types, but D P G occurs only in trace amounts. Our data on PMN cells are close to those of Gottfried except for PC. His value for PC is 38.6% of the total lipid phospholipid whereas our value for PC is 32%. PMN cells readily incorporate labeled fatty acids into their phospholipids, triglycerides and cholesterol esters. The greatest incorporation occurred in triglycerides with all fatty acids. It is of interest that saturated fatty acids are incorporated to a larger extent into both triglycerides and cholesterol esters than are the unsaturated fatty acids. With the phospholipids, the greatest incorporation occurred in PC. There were relative differences in the incorporation of each fatty acid into the various phospholipids. Our findings are in general agreement with those of Burns et al. [13] except for the absolute uptake values. Their experimental conditions differed from ours. Elsbach [15] used rabbit leukocytes in his studies, thus we cannot directly compare our results with his. As we reported earlier for human red cells [23] we find that palmitic, stearic, linoleic and linolenic acids are incorporated into delipidized PMN proteins. Palmitic acid was incorporated to the greatest extent. Insulin (50 nM) enhanced the incorporation of [3H]palmitic acid into the delipidized PMN proteins. We suggest that these proteins contain covalently bound fatty acids and that these fatty acids may play a role in the insertion and anchoring of proteins into cell membranes. The recent work of Schlessinger et al. [24] supports this view. Since cycloheximide but not actinomycin D inhibits the incorporation of palmitic acid into protein [23], this suggests that the fatty acid is attached to the protein as a post-translational event.

References

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