324

Acylation of Docosahexaenoic Acid into Phospholipids by Intact Human Neutrophils Jen-sie Tou Department of Biochemistry, Tulane University School of Medicine, New Orleans, LA 7Ol12

Docosahexaenoic acid was not only acylated into phospholipids but also into triacylglycerols by intact human neutrophils. The distribution of radiolabeled docosahexaenoic acid among individual phospholipids was dependent on the incubation time. [1-14C]Docosahexaenoic acid at all concentrations (1 to 8 pM) was acylated mainly into phosphatidic acid after 1-2 min incubation, and the radioactivity of phosphatidic acid started to decline after a longer period of incubation, suggesting the participation of docosahexaenoyl-phosphatidic acid in the synthesis of other glycerolipids. It was acylated primarily into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) after a 2-hr incubation. The labeled phosphatidic acid may be rapidly deacylated and the 22:6(n-3) moiety is then reacylated into other lysophospholipids. The low levels of [~4C]22:6(n-3) in 1,2-diacylglycerol suggest that the deacylation-reacylation cycle may be a major pathway in the formation of ['4C]22:6(n-3)-PC and -PE in intact neutrophils. This n-3 fatty acid was a relatively poor substrate for acylation into phosphatidylinositol as compared to arachidonic acid and eicosapentaenoic acid. However, the patterns of distribution of all three polyunsaturated fatty acids among the diacyl- and ether-linked class compositions of PC and PE were similar. These data suggest the potential of increasing the content of docosahexaenoic acid of membrane lipids in neutrophils by dietary supplement of this fatty acid. Lipids 21, 324-327 (1986). Polyunsaturated fatty acids appear to participate in the regulation of neutrophil functions. In response to physiological or chemical stimuli, leukotriene B, (LTB~; 5,12-dihydroxyeicosatetraenoic acid) and 5-hydroxyeicosatetraenoic acid (5-HETE) are formed by the action of 5-1ipoxygenase from either exogenous arachidonic acid (20:4[n-6]} (1,2) or endogenous 20:4(n-6) released from neutrophil phospholipids by activation of phospholipase A2 (3}. LTB4 and 5-HETE are not only chemotactic stimuli toward neutrophils (4,5) but also modulate the formation of another potent chemotactic agent, platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine), by enhancing the expression of phospholipase A2 (6). Eicosapentaenoic acid (20:5[n-3]), a major polyunsaturated acid in fish oils, is anti-inflammatory by inhibiting LTB~ formation in human neutrophils (7,8). This fatty acid is metabolized to leukotriene B5 (LTBs; 5,12-dihydroxyeicosapentaenoic acid) which is a much weaker chemotactic agent for human neutrophils {9). In vitro studies have demonstrated that the patterns of the incorporation of 20:4(n-6} and 20:5(n-3} into individual phospholipids by neutrophils are similar (10}, suggesting the potential of modification of fatty acid composition in neutrophil phospholipids by dietary supplements of 20:5(n-3). Docosahexaenoic acid (22:6[n~ is also a major polyunsaturated fatty acid in fish oils. It is a poor substrate for LIPIDS, Vol, 21, No, 5 (1986)

the leukotriene-synthesizing system (11}. In response to ionophore A23187, human neutrophils metabolize exogenous 22:6(n-3) to 7-hydroxydocosahexaenoic acid (8,12), which is not chemotactic for neutrophils (8). The present study was undertaken to examine whether human neutrophils can acylate 22:6(n-3) into cellular lipids. The results show that resting human neutrophils can acylate [1-1'C]22:6(n-3) into phospholipids and triacylglycerols. MATERIALS AND METHODS

Preparation of human neutrophils. Human neutrophils were prepared according to the method of Lee et al. (13). Thirty-ml portions of venous blood from normal donors were each mixed with 4 ml of 0.15 M sodium citrate, pH 5.2, and 5 ml of 5% dextran T500 (Pharmacia Fine Chemicals, Piscataway, New Jersey) in 0.15 M NaC1 and allowed to sediment at room temperature for 30 min. The supernatants containing leukocyte-rich plasma were removed and centrifuged at 250 • g for 10 min at 25 C. After hypotonic lysis of contaminating erythrocytes, leukocytes were washed once and resuspended in KrebsRinger phosphate buffer modified to contain 1.3 mM CaC12 and 5 mM glucose at 1-2 • 108 cells/ml. Three-ml cell suspensions were layered on 3 ml Ficoll-Hypaque (Pharmacia) cushions and centrifuged at 400 • g for 20 rain at 25 C to yield a neutrophil pellet which was washed twice and suspended at a concentration of 20 • 106 cells/ml in Krebs-Ringer phosphate buffer. Cell counts were made in a hemocytometer, and cell viability was measured by trypan blue exclusion. Cell preparations contained more than 95% neutrophils.

Incubation of cells with [1-14C]docosahexaenoic acid. [1-14C]Docosahexaenoic acid {55.0 Ci/mol, New England Nuclear Corp., Boston, Massachusetts) was dissolved in dimethylsulfoxide (DMSO) and mixed with fatty acid-free bovine serum albumin {Miles Laboratories, Elkhart, Indiana, 4 mg/ml 0.9% NaCl}. In a final volume of 2 ml, each tube contained 2.44 • 105 dpm (1 ~M} to 1.95 X 106 dpm (8 ~M) [1-"C]22:6(n-3} and 20 • 106 neutrophils. The system was incubated at 37 C for various periods of time (1 to 120 min). The highest concentration of DMSO in the incubation medium was 0.1% and had no adverse effect on cell viability. Incubations were terminated by the addition of 10 ml methanol to each tube. Lipid extraction and analysis. Neutrophil lipids were extracted (14) and dissolved in chloroform/methanol (2:1, v/v) containing 0.01% butylated hydroxytoluene. Triacylglycerols, 1,2-diacylglycerols and total phospholipids were separated by one-dimensional thin layer chromatography (TLC) on silica gel H {Analtech, Newark, Delaware) developed with petroleum ether/diethyl ether/acetic acid (80:20:1, v/v/v) (15}. The radioactivity in each fraction was counted by liquid scintillation and expressed as a percentage of the total radioactivity in each incubation system. Individual phospholipids were resolved by twodimensional TLC on silica gel H and analyzed as described

325

22:6(n-3) IN NEUTROPHIL PHOSPHOLIPIDS previously (16). The c h r o m a t o g r a m was developed with chioroform/methanol/28% aqueous a m m o n i a (65:25:5, v/v/v) for the first dimension and with chioroform/methanol/acetic acid/water (95:10:30:3, v/v/v/v) for the second dimension. PC and PE were each scraped and e x t r a c t e d from the silica gel with 10 ml chloroform/methanol/water (2:2:0.5, v/v/v) b y vigorous shaking. The e x t r a c t was washed once with 3 ml distilled w a t e r and centrifuged at 1,000 X g for 10 min. The lower chloroform layer was e v a p o r a t e d to dryness and redissolved in diethyl ether for subsequent analyses. T r e a t m e n t of the purified PC and PE with phospholipase C and acetylation of the resulting 1-radyl-2-acylglycerols were performed according to the m e t h o d of W a k u et al. (17). E a c h incubation t u b e contained 0.5 ml of 0.1 M Tris-HC1 buffer, p H 7.4, containing 10 mM CaCI:, 10 t4 (16.7 units, 25 t~g protein) B a c i l l u s c e r e u s phospholipase C (Sigma, St. Louis, Missouri) and 2 ml diethyl ether containing 2 to 2.5 ~g phosphorus of PC or PE. The m i x t u r e was incubated for 16 hr at r o o m t e m p e r a t u r e with c o n s t a n t stirring. The ether layer containing 1-radyl-2-acylglycerols was s e p a r a t e d from the aqueous layer after a brief centrifugation, and the aqueous layer was e x t r a c t e d once more with 2 ml diethyl ether. After e v a p o r a t i o n of the combined ether solutions to dryness, 0.2 ml of acetic anhydride/pyridine (10:1, v/v) was added to each tube. Acetylation was performed at r o o m ternp e r a t u r e for 16 hr, and it was t e r m i n a t e d b y the addition of 1 ml distilled w a t e r to each tube. The mixture was extracted three times each with 2 ml hexane. After evaporation of the combined hexane solutions to dryness, the r e s i d u e was redissolved in chloroform/methanol (2:1, v/v) containing 0.01% b u t y l a t e d hydroxytoluene. The resulting 1-radyl-2-acyl-3-acetylglycerols were resolved into 1-alkenyl-2-acyl-3-acetylglycerol, 1-alkyl-2-acyl-3-acetylglycerol and 1,2-diacyl-3-acetylglycerol b y TLC as described b y Rendonen and L u u k k o n e n (18) on silica gel H. The radioactivity of each lipid class was m e a s u r e d b y liquid scintillation, and the a m o u n t of radioactivity in each class of the resulting diglyceride acetates was expressed as a percent of the total radioactivity recovered f r o m the thin layer plate.

TABLE 1 Incorporation of [1-14C]22:6(n-3) into Neutrophil Triacylglycerols and Phospholipids a

Time (min)

Triacylglycerols

1

5.09 14.6 41.0 66.2 68.9 69.1 69.5 69.2

2

5 10 2o 30 6o 120

• • • • • • • •

0.8 1.2 4.4 6.8 7.3 6.7 7.5 8.4

1,2 Diacylglycerols (% of total radioactivity) 0.281 0.587 0.832 0.448 0.398 0.323 0.215 0.212

• • • • • • • •

0.02 0.03 0.03 0.02 0.01 0.01 0.01 0.02

2.67 4.50 9.54 9.70 10.3 10.3 10.4 10.7

• • • • • • • •

0.22 0.17 0.51 0.44 0.72 0.81 0.95 1.0

aNeutrophils (20 X 106 cells)were incubated with [1-'4C]22:6(n-3)(2.44 )4 10s dpm, 1 ~M} at 37 C at indicated time. Lipids were extracted and resolved as described in Materials and Methods. Each value is mean • SD from three separate experiments and is expressed as a percentage of the total radioactivity in each incubation system.

_~

x ~"

351

"*PC

3.0 i

2,5 k /

~

PE

x 2.0

-0 _~i~ '.L 1.5 ~, .~. 1.0 c~r ~-~

"PI

9

0.5

"--" --o

RESULTS AND DISCUSSION

The results in Table 1 show t h a t [1-'~C]22:6(n-3) was acylated not only into phospholipids but also into triacylglycerols b y intact h u m a n neutrophils. The rate of acylation of the f a t t y acid was a rapid process and reached m a x i m u m acylation into triacylglycerols and t o t a l phospholipids in 20 min under the experimental conditions in which the concentration of bovine serum albumin in the incubation m e d i u m was 0.1 mg/ml. This process was slower in the presence of a higher ratio of albumin to [1-'~C]22:6(n-3) in the incubation medium (data not shown). After a 2-hr incubation of 20 • 106 cells with 1 ~M [1-"C]22:6(n-3), 69.2% labeled f a t t y acid was acylated into triacylglycerols and 10.7% into phospholipids. The radioactivity of 1,2-diacylglycerol (1,2-DG) at all time intervals was less t h a n 1% of the total radioact i v i t y in the incubation s y s t e m and reached a m a x i m u m after a 5-min incubation. I t started to decline after longer periods of incubation, s u g g e s t i n g its participation in the formation of other lipids.

Phospholipids

0

20

60 0 Minutes of Incubation

100

8 PS PA

120

FIG. 1. Time-dependent acylation of [1-'4C]22:6(n-3) into individual neutrophil phospholipids. Human neutrophils (20 X 106 cells) were incubated at 37 C at the indicated time with 1 ~M [1-~4C]22:6(n-3) (2.44 X 105 dpm) as described in Materials and Methods. Each point represents the average value from three separate experiments.

In h u m a n neutrophils the average molar phospholipid composition measured in the present s t u d y from five s e p a r a t e cell p r e p a r a t i o n s was PC, 39.0%; PE, 27.9%; phosphatidylserine (PS), 10.9%; phosphatidylinositol (PI), 6.8%; and sphingomyelin, 15.4%. There were 1030 nmol of lipid phosphorus/108 cells. These values are in general a g r e e m e n t with published values (19). The p h o s p h o r u s content of phosphatidic acid (PA) cannot be accurately measured; thus the distribution of radioactivity a m o n g individual phospholipids is expressed as dpm/6 • 106 cells. As shown in Figure 1, [1-14C]22:6(n-3) was acylated LIPIDS, VoL 21, No. 5 (1986)

326

J.-S. TOU mainly into PA after 1-2 min of incubation, and the radioactivity of PA started to decline after a 5-min incubation. The labeled PA may be rapidly deacylated and the 22:6(n-3) moiety is then reacylated into other lysophospholipids. The low levels of [14C]22:6(n-3) in 1,2-DG suggest that the deacylation-reacylation cycle may be a major pathway in the formation of [14C]22:6(n-3)PC and -PE in intact neutrophils. Also shown in Figure 1 is that 22:6(n-3) was a relatively poor substrate for acylation into PI as compared to 20:4(n-6) and 20:5(n-3) (10). However, the time course of the acylation of [1-'4C]22:6(n-3) into PC and PE resembled those of [1-'4C]20:4(n-6) and [1-'~C]20:5(n-3) (10). The radioactivity of PC reached a maximum after a 20-min incubation with 1 ~M [1-'~C]22:6(n-3) and began to decline thereafter with increasing incubation time, whereas that of PE continued to increase. These data suggest a transfer of the [1-'~C]22:6(n-3) from PC to PE in neutrophils. There is evidence for the presence of a CoA-mediated, ATPindependent acyltransferase catalyzing the transfer of 20:4(n-6) from PC to lysoPE in mouse thymocytes (20), and a CoA-independent transacylase catalyzing the transfer of 20:4(n-6) from diacyl-PC to 1-alkenyl-lysoPE in human platelets (21) and to 1-acyl-lysoPE in dog heart membranes (22). PS contained the lowest radioactivity among the phospholipids, and sphingomyelin was not labeled by [1-'~C]22:6(n-3). Figure 2 illustrates a time-dependent incorporation of [1-~'C]22:6(n-3) into individual phospholipids by neutrophils in the presence of various concentrations of [1-'4C]22:6(n-3) in the incubation medium. After a 2-min incubation, 6 ~M of [1-~'C]22:6(n-3) was approximately substrate-saturating for acylation of this fatty acid into all phospholipids, and PA exhibited the highest radioactivity at all concentrations of [1-1'C]22:6(n-3) tested. After a 20-min incubation of cells with 1 to 8 ~M [1-'4C]22:6(n-3), the radioactivity of PC became the highest among the phospholipids and it was markedly increased with increasing [1-~'C]22:6(n-3) concentrations in the incubation medium. These data suggest that in intact neutrophils lysoPA serves as a better acceptor than lysoPC for 22:6(n-3)-CoA under the experimental conditions. These data also indicate that [1-'4C]22:6(n-3) was acylated into PE at a slower rate than into PC and PI after a shorter period of incubation. Human neutrophils have a high content of alkylacylPC and alkenylacyl-PE, and these ether-linked species are rich in 20:4(n-6) (23). Previous studies showed that exogenous 20:4(n-6) was acylated more rapidly into the diacyl-linked PC and PE than into the corresponding ether-linked class after a shorter period of incubation (1-20 min); however, more 20:4(n-6) appeared in alkylacylPC and in alkenylacyl-PE after a longer period of incubation (2 hr) (10). These studies were compatible with the distribution of 20:4(n-6) in vivo (23). Similarly, as shown in Table 2, the rate of acylation of [1-'4C]22:6(n-3) into the diacyl-linked and ether-linked PC and PE differed. The acylation of [1-'4C]22:6(n-3) into diacyl-linked PC and PE appears to precede that into the corresponding etherlinked class. With increasing incubation time a decrease in the radioactivity in the diacyl-linked class was accompanied by an increase in the radioactivity in alkylacylPC and alkenylacyl-PE, suggesting that part of the 22:6(n-3) moiety in alkylacyl-PC and alkenylacyl-PE was LIPIDS, Vol. 21, No. 5 (1986)

Incubation

time: 2 rain

PA

4

3

2

u ,O O X ,O r I

PC o

PI PE PS

X

E

Q. -O

2

u~ "nO Q.

4

6

8

Docosahexaenoic Acid (/zM)

20

~" !

Incubation time: 20 rain

PC

,o

15 u 9

!

10

5

PI Ps PA T

2

4

Docosahexaenoic

6

PS

8

Acid (/zM)

FIG. 2. Substrate-dependent acylation of [1-~4C]22:6(n-3) into individual neutrophilphospholipids.Humanneutrophils(20 X 106cells) were incubated at 37 C at the indicated time with 1 to 8 ~M [1-'4C]22:6(n-3)(2.44X 105to 1.95 X 106dpm)as described in Materials and Methods. Each point represents the average value from three separate experiments.

derived from the respective diacyl-linked class. While an enzymatic transfer of 22:6(n-3) or 20:4(n-6) from diacylPE to alkenyl-lysoPE has not been determined, a coenzyme A-independent transacylase catalyzing the transfer of 20:4(n-6) moiety from the sn-2-position of diacyl-linked PC to 1-alkyl-2-1ysoPC has been demonstrated in human platelets (24) and in rabbit macrophages (25,26). It seems likely that a similar transacylase is present in human neutrophils. This enzyme probably exhibits a preference for 20:4(n-6) over 22:6(n-3), if one compares the data in Table 2 with that in previous studies which were performed under identical incubation conditions (10). After a 2-hr incubation about 43% of total labeled 20:4(n-6) in PC fraction was recovered in alkylacyl-PC (10), whereas

327

22:6(n-3) IN NEUTROPHIL PHOSPHOLIPIDS TABLE 2 Distribution of [1-~4C122:6(n-3)in Diacyl-, Alkylacyl- and Alkenylacylphosphatidylcholine and -phosphatidylethanolamine a

Phosphatidylcholine

Phosphatidylethanolamine

Time (min)

Diacyl

Alkylacyl (%)

Alkenylacyl

Diacyl

Alkylacyl (%)

Alkenylacyl

10 20 30 60 120

88.7 85.6 83.3 75.3 72.8

10.1 12.7 15.0 21.7 23.8

1.20 1.70 1.70 3.07 3.43

70.5 62.4 58.2 43.0 38.2

20.0 24.8 22.1 24.0 23.3

9.5 12.8 19.7 33.0 38.5

aNeutrophils (20 X 106 cells) were incubated with [1-~4C]22:6(n-3) (2.44 • l0 s dpm, I t~M)at 37 C at indicated time. PC and PE were purified and treated with phospholipase C as described in Materials and Methods. The amount of radioactivity in each class of the resulting diglyceride acetates is the average value from two separate experiments and is expressed as a percentage of the total radioactivity recovered from the thin layer plate.

a b o u t 24% of t o t a l l a b e l e d 22:6(n-3) in P C f r a c t i o n app e a r e d in a l k y l a c y l - P C (Table 2). The present study demonstrates that human n e u t r o p h i l s c a n a c y l a t e e x o g e n o u s 22:6(n-3) i n t o PC a n d P E , s u g g e s t i n g t h e p o t e n t i a l of i n c r e a s i n g t h e c o n t e n t of 22:6(n-3) of m e m b r a n e lipids in n e u t r o p h i l s b y d i e t a r y supp l e m e n t s of t h i s f a t t y acid. W h e n n e u t r o p h i l s r e s p o n d t o p h y s i o l o g i c a l or c h e m i c a l s t i m u l i , free 22:6(n-3) d e r i v e d e i t h e r f r o m p h o s p h o l i p i d s or f r o m e x t r a c e l l u l a r m e d i u m c o u l d b e o x y g e n a t e d t o f o r m 7-hydroxy-22:6(n-3) w h i c h h a s no c h e m o t a c t i c a c t i v i t y for n e u t r o p h i l s ; i t c o u l d also a t t e n u a t e t h e p r o d u c t i o n of P A F , t h e r e b y m o d i f y i n g t h e p r o c e s s of i n f l a m m a t i o n .

ACKNOWLEDGMENTS The author is the Albert Hyman Research Grant recipient, American Heart Association, Louisiana, Inc.

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R.A., and Austen, K.F. (1984) J. Clin. Invest. 74, 1922-1933. 9. Goldman, D.W., Pickett, W.C., and Goetzl, E.J. (1983)Biochem. Biophys. Res. Comm. 117, 282-288. 10. Tou, J.-S. 11984) Lipids 19, 573-577. 11. Corey, E.J., Shih, C., and Cashman, J.R. (1983)Proc. Natl. Acad. Sci. USA 80, 3581-3584. 12. Fischer, S., Schacky, C.V., Siess, W., Strasser, Th., and Weber, P.C. (1984) Biochem. Biophys. Res. Comm. 120, 907-918. 13. Lee, C.W., Lewis, R.A., Corey, E.J., and Austen, K.F. (1983) Immunology 48, 27-35. 14. Bligh, E.G., and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917. 15. Tou, J.-S. (1979) Biochim. Biophys. Acta 572, 307-313. 16. Tou, J.-S. (1978) Biochim. Biophys. Acta 531, 167-178. 17. Waku, K., Ito, H., Bito, T., and Nakazawa, Y. (1974) J. Biochem. 75, 1307-1312. 18. Rendonen, 0., and Luukkonen, A. (1976) in L i p i d Chromatographic Analysis, Vol. 1, 2nd Edition (Marinetti, G.V., ed.) p. 37, Marcel Dekker, New York. 19. Cockcroft, S., and Allan, D. (1984) Biochem. J. 222, 557-559. 20. Trotter, J., Flesch, I., Schmidt, B., and Ferber, E. (1982) J. Biol. Chem. 257, 1816-1823. 21. Kramer, R.M., and Deykin, D. (1983} J. Biol. Chem. 258, 13806-13811. 22. Reddy, P.V., and Schmid, H.H.O. (1985) Biochem. Biophys. Res. Comm. 129, 381-388. 23. MueUer, H.W., O'Flaherty, J.T., Greene, D.G., Samuel, M.P., and Wykle, R.L. (1984) J. Lipid Res. 25, 383-388. 24. Kramer, R.M., Patton, G.M., Pritzker, C.R., and Deykin, D. (1984) J. Biol. Chem. 259, 13316-13320. 25. Sugiura, T., and Waku, K, (1985) Biochem. Biophys. Res. Comm. 127, 384-390. 26. Robinson, M., Blank, M.L., and Snyder, F. {1985)J. Biol. Chem. 260, 7889-7895. [ R e c e i v e d N o v e m b e r 7, 1985]

LIPIDS, Vol. 21, No. 5 (1986)