333

Platelet-Activating Factor Modulates Phospholipid Acylation in Human Neutrophils Jen-sie Tou Department of Biochemistry, Tulane UniversitySchool of Medicine, New Orleans, LA 70112

The present study showed that platelet-activating factor {1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, PAF), but not lysoPAF fl-O-hexadecyl-sn-glycero-3-phosphocholine} rapidly (within 15 sec} stimulated the incorporation of both [1-14C]arachidonate and [1-14C]docosahexaenoate into phosphatidylinositol (PI} and phosphatidylcholine (PC} in human neutrophils. Concomitantly, it inhibited the formation of labeled phosphatidic acid from both fatty acids. The magnitude of stimulation {percentage of control} was greater in PI than in PC for the incorporation of arachidonate and vice versa for the incorporation of docosahexaenoate. It reached a maximum at 10-7 M and started to decline at 10-6 M. Extracellular Ca ~+was not essential for the action of PAF on phospholipid acylation. The distribution of labeled arachidonate in the molecular species of PC was not altered by PAF after 1 min incubation, suggesting that the increased formation of arachidonyl-PC during the early stage of neutrophil-PAF interaction was not originated from the added PAF. No measurable changes in the mass of each phospholipid were detected in neutrophils challenged by PAF from 15 sec to 2 min. The data suggest that the increased incorporation of extracellular fatty acids into P | and PC elicited by PAF may be secondary to increased deacylation of these phospholipids, and the magnitude of stimulation reflects the specificity of acyltransferase catalyzing the acylation of lysoPI and lysoPC by fatty acyl-CoA. Lipids 22, 333-337 {1987).

relatively poor substrate for incorporation into PI compared to arachidonate and eicosapentaenoate {14}. It is thus important to examine whether the incorporation of docosahexaenoate into phospholipids in these cells is responsive to PAF. The present study has demonstrated that PAF rapidly increases {within 15 sec} the incorporation of both arachidonate and docosahexaenoate into PI and PC by human neutrophils. MATERIALS AND METHODS

Preparation of human neutrophils. Human blood was ob-

tained from healthy donors who had received no medication in the previous two weeks. Neutrophils were isolated according to the method of Lee et al. (15}. Thirty-ml portions of venous blood 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 were 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 20 C. After hypotonic lysis of contaminating erythrocytes, leukocytes were washed once and resuspended in a modified Krebs-Ringer HEPES {N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid} buffer {118 mM NaC1, 4.74 mM KC1, 1.19 mM MgSO4, 5 mM glucose, 16.3 mM HEPES, pH 7.4) at 20 X 106 cells/ml. Six-ml cell suspensions were layered on 6-ml Ficoll-Hypaque {Pharmacia} cushions and centrifuged at 400 • g for 20 min at 20 C to yield a neutrophil pellet, Platelet-activating factor {1-O-alkyl-2-acetyl-sn-glycerowhich was washed twice and suspended at a concentra3-phosphocholine, PAF) is a bioactive phospholipid with tion of 20 • 106 cells/ml in the same buffer. Cell counts multiple biological effects {1-4}. At nanomolar concentra- were made in a hemocytometer, and cell viability was tions PAF stimulates human neutrophil chemotactic measured by trypan blue exclusion and by lactate demigration, superoxide production, aggregation, cytochal- hydrogenase release into the incubation medium as asin B-dependent exocytosis of azurophilic and specific described previously {16}. Cell preparations contained lysosomal granules {5,6}. It enhances human neutrophil more than 95% neutrophils. Incubation of cells. [1-14C]Arachidonicacid {54.5 Ci/mol, superoxide generation in response to chemotactic peptide N-formyl-methionyl-leucyl-phenylalanineffMet-Leu-Phe} New England Nuclear Corp., Boston, Massachusetts} (7). This bioactive phospholipid also modulates the or [1-'~C]docosahexaenoate {55.0 Ci/mol, New England metabolism of other phospholipids in neutrophils. It Nuclear} was dissolved in 0.9% NaC1 containing fatty enhances the activity of polyphosphoinositide-specific acid-free bovine serum albumin (Miles Laboratories, Elkphospholipase C in rabbit peritoneal neutrophils {8}, the hart, Indiana; 4 mg/ml}. PAF {1-O-hexadecyl-2-acetylrelease of arachidonate from phosphatidylinositol {PI} and sn-glycero-3-phosphocholine, Novabiochem, L~iufelfingen, phosphatidylcholine (PC} by the action of phospholipase Switzerland}, lysoPAF {1-O-hexadecyl-sn-glycero-3-phosA2 and the production of 5-hydroxyeicosatetraenoate phocholine, Novabiochem} or lysoPC {1-acyl-sn-glycero-3(5-HETE} and 5,12-dihydroxyeicosatetraenoate or leuko- phosphocholine, Applied Science Laboratories, State Coltriene B~ (LTB4) in cytochalasin B-treated rabbit peri- lege, Pennsylvania} was dissolved in 0.9% NaC1 containtoneal neutrophils {9). It also promotes arachidonate in- ing fatty acid-free bovine serum albumin {2.5 mg/ml}. In corporation into PI and PC in neutrophils from guinea a final volume of 2 ml, each tube contained 3.4 • l0 s dpm pig peritoneal exudates riO}. Since neutrophils from {1.4 ~M) [1-'4C]arachidonic acid or 3.6 X l0 s dpm {1.5 ~M} human blood differ from elicited neutrophils in a number [1-'~C]docosahexaenoic acid, 1 mM CaC12, 0-10 -6 M PAF of respects (11-13}, it is important to examine whether {or 10 -7 M lysoPAF or lysoPC) and 20 • 106 cells in the increased incorporation of arachidonate into PI and 1.92 ml Krebs-Ringer HEPES buffer. In control tubes, an equivalent volume of 0.9% NaC1 containing bovine PC occurs in human neutrophils in response to PAF. Recently docosahexaenoate was found to be incorpo- serum albumin (2.5 mg/ml} was included. Incubations rated into PC and phosphatidylethanolamine {PE} were initiated by the addition of 20 X 106 cells to each by human neutrophils (14}. This n-3 fatty acid was a tube and were performed at 37 C. LIPIDS, Vol, 22, No. 5 (1987)

334

J.-S. TOU

Lipid extraction and analysis. Incubations were terminated by the addition of 5 ml methanol to each tube. Lipids were extracted according to the method of Bligh and Dyer (17) and dissolved in chloroform]methanol (2:1, v/v) containing 0.01% butylated hydroxytoluene. Individual phospholipids were resolved and analyzed as described previously (13). Since the phosphorus content of phosphatidic acid (PA) cannot be accurately measured, the radioactivity of individual phospholipids is expressed as dpm/6 X 106 cells. The distribution of ['4C]arachidonate in the molecular species of PC was analyzed as described previously (14). RESULTS

Figure 1 shows the time course of the effect of PAF on the incorporation of [1-"C]arachidonate into phospholipids in human neutrophils. PAF stimulated the incorporation of [1-'~C]arachidonate into PI and PC at all time intervals (15 sec to 2 min). Concomitantly, it inhibited the formation of [1-'4C]arachidonoyl-PA. The magnitude of stimulation (percentage of control) varied widely with cells from individual donors, but an increased acylation of labeled arachidonate into PI and PC was consistently demonstrated after 15 sec incubation of cells with PAF. In two separate experiments, PAF at 10 .7 M caused an increase in the radioactivity of PI and PC to 1278% and 287% of control, respectively, at 15 sec; and the radioactivity of PI and PC became 1888% and 1283% of control, respectively, at 1 min. The magnitude of stimulation on the formation of labeled PI and PC induced by PAF started to decline at 2 min incubation. The incorporation of arachidonate into PE was less responsive to the presence of PAF. An increased radioactivity of PE was observed only after 2 min incubation of neutrophils with

12

12c,~o HIO

4 ~

PE

Pl

Conltol 7M PAF

2F

10

%

PAF. Analysis of phospholipid classes by phosphorus determination showed no measurable changes in phospholipid composition during the neutrophil-PAF interaction. At all time intervals (15 sec to 2 min), the lipid phosphorus (~g) from 6 • 106 cells in the presence or absence of PAF (10.7 M) was 0.803 for PC; 0.580 for PE; 0.197 for phosphatidylserine (PS); 0.125 for PI; and 0.295 for sphingomyelin. Figure 2 demonstrates the time course of the effect of PAF on the incorporation of [1-'4C]docosahexaenoate into phospholipids. PAF inhibited the incorporation of [1-'4C]docosahexaenoate into PA, but it enhanced the incorporation of this n-3 fatty acid into PI and PC. In two separate experiments, the average radioactivities of PI and PC were increased to 403% and 880% of control, respectively, after 15 sec incubation and to 432% and 2118% of control, respectively, after 1 min incubation. The magnitude of stimulation by PAF on the formation of labeled PC but not PI started to decline after 2 min incubation. PAF in a range of 10-'~ -6 M elicited significant changes in the incorporation of [1-'4C]arachidonate into phospholipids after 1 re_in incubation (Fig. 3). Maximal incorporation of [1-'~C]arachidonate into PI and PC was observed at 10-7 M, and a decline in the stimulation of the acylation of both phospholipids occurred at 10 -6 M. At the concentrations tested (10-9-10 -6 M), PAF decreased the formation of ['4C]arachidonoyl-PA. PAF has been shown to promote Ca ~§uptake from extracellular medium by rabbit platelets (18) and rabbit peritoneal neutrophils (8). A requirement for extracellular Ca ~§has been demonstrated for the stimulatory effect of PAF on degranulation (8), but not on polyphosphoinositides-specific phospholipase C (8) or superoxide generation (19). To test if extracellular Ca ~§ is required for the action of PAF on phospholipid acylation by polyunsaturated fatty acids, the incorporation of [1-'~C]arachi donate into phospholipids in cells incubated in the presence of 1 mM Ca 2. was compared with that in cells incubated in the presence of 1 mM EGTA [ethyleneglycolbis-(~-aminoethylether)N,N'-tetracetic acid]. Table 1 shows

8 i

2

8 - 0----0

Control

HIO-7M

"o

6

g 6

8 - PC

ii 'E

PAF

6

"o 4

0

1

2

4

o.

~

2

2

2

2

~D

P

1

2

o

1

2

0

I

2

Minutes ol Incubation

FIG. 1. Time course of the effect of P A F on the acylation of [1-'4C]arachidonate [20:4(n-6}] into phospholipids in human neutrophils. Neutrophiis (20 X 106) were incubated at the indicated .period of time (15 see-2 min) with 3.4 X 10 s dpm [1J4C]arachidonate m the presence ( 9 or absence (O) of 10 -7 M PAF. Each point represents the average value of duplicate incubations from two separate neutrophil preparations. LIPIDS, Vol. 22, No. 5 (1987)

0

1

2

0

1

2

0

1

2

Minutes of Incubation

FIG. 2. Time course of the effect of P A F on the acylation of [1-'4C]docosahexaenote [22:6(n-3)] into phospholipids in human neutrophils. Neutrophils (20 X 106) were incubated at the indicated period of time (15 sec-2 rain) with 3.6 X 105 dpm [1-"C]docosahexaenote in the presence ( 9 ) or absence (O) of 10 -7 M PAF. Each point represents the average value of duplicate incubations from two separate neutrophil preparations.

335 PLATELET-ACTIVATING FACTOR AND PHOSPHOLIPID ACYLATION 10

TABLE 2 Effect of Platelet-Activating Factor (PAF) on the Distribution of [1-'4C]Arachidonate in Diacyl-, Alkylacyl- and Alkenylacylphosphatidyicholine a

== 8 pl

o

% of Total [1-'4]arachidonate in phosphatidylcholine d

pc Control PAF (10-7 M)

2 0

10-I0

i0-9

10-8

i0 -7

i0 -6

PAF {M)

FIG. 3. Dose response to P A F for acylation of [1-"C]arachidonate [20:4(n-6)] into phospholipids in human neutrophils. Neutrophils (20 X 106) were incubated for 1 min at 37 C with 3.4 X 10 s dpm [1-"C]arachidonate and 0-10 -6 M PAF. Each point represents the average value of duplicate incubations from two separate neutrophil preparations.

Diacyl

Alkylacyl

Alkenylacyl

84.7 82.9

14.1 15.7

1.20 1.40

aHuman neutrophils (20 • 106 cells) were incubated for 1 min at 37 C with [1-'4C]arachidonic acid (3.4 X 10Sdpm, 1.4 ~M), Ca2§(1.0 mM) and PAF (10.7 M). In control incubations, PAF was replaced by an equivalent volume of bovine serum albumin (2.5 mg/ml in 0.9% NaCI}. Each phosphatidylchoiine preparation was purified from 40 X 106 neutrophils and was treated with phospholipase C as described in Materials and Methods. The amount of radioactivity in each class of 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.

TABLE 3 TABLE 1 Effect of External Ca 2. on [1-~4ClArachldonate Incorporation into Phospholipids in Human Neutrophils in Response to P A F a

Effect of L y s o P A F and LysoPC on the Incorporation of [1-'4CJArachidonate into PI and PC by Human Neutrophils a

'4C dpm (% of control) Radioactivityb ('~C dpm) Lipid

Control

PAF

Mean % of control in the presence of PAF c

With Ca ~§

PA PI PC

2357 496 383

1146 4558 2038

57 • 11 1188 • 377 574 • 122

Without Ca~

PA PI PC

1626 274 296

1184 2676 1283

66 • 10 1207 • 350 533 • 152

PAF, platelet-activating factor; PA, phosphatidic acid; PI, phosphatidylinositol; PC, phosphatidylcholine. aHuman neutrophils [20 • 106 cells) were incubated for 1 min at 37 C with [1-'4C]arachidonic acid (3.4 • 10s dpm, 1.4 ~M), Ca 2~ (1.0 mM) or EGTA (1 mM), and PAF (10-' M). In control incubations, PAF was replaced by an equivalent volume of bovine serum albumin (2.5 mg/ml in 0.9% NaC1). bThe results from a typical experiment. The radioactivity is expressed as dpm from 6 • 10~ cells. CMean values • S.D. from three experiments.

t h a t e x t r a c e l l u l a r Ca 2§ w a s n o t e s s e n t i a l for t h e i n c r e a s e d i n c o r p o r a t i o n of [1-'4C]arachidonate into P I or PC i n d u c e d b y 10 .7 M P A F after 1 m i n i n c u b a t i o n . A l t h o u g h the b a s a l r a d i o a c t i v i t y of p h o s p h o l i p i d s was less in the p r e s e n c e of 1 m M E G T A t h a n t h a t i n t h e p r e s e n c e of 1 m M Ca ~§ P A F e v o k e d a s i m i l a r m a g n i t u d e of s t i m u l a t i o n in b o t h systems. T h e p o s s i b i l i t y t h a t t h e i n c r e a s e d r a d i o a c t i v i t y of PC w a s o r i g i n a t e d f r o m t h e a d d e d P A F was e x a m i n e d . ['~C]Arachidonoyl-PC w a s t r e a t e d w i t h p h o s p h o l i p a s e C,

Control PAF (10-7 M) LysoPAF (10-7 M) LysoPC (10-7 M)

PA

PI

PC

100 50 • 12 100 • 2.7 78 • 6.5

100 1200 • 391 100 • 0.7 99 • 3.2

100 650 • 182 101 • 2.2 140 • 14

apAF, platelet-activating factor; PC, phosphatidylcholine; PI, phosphatidylinositol; PA, phosphatidic acid. Human neutrophils (20 • 106)suspended in Krebs-Ringer HEPES buffer were incubated for I min at 37 C with [1-'4C]arachidonicacid {3.4 X 10s dpm, 1.4 ~M); Ca2. (1.0 mM); and PAF (10.7 M) or lysoPAF (10-' M) or lysoPC (10-7 M). In control incubations, PAF was replaced by an equivalent volume of bovine serum albumin (2.5 mg/ml in 0.9% NaCI). Data represent the average value • S.D. from four separate neutrophil preparations.

a n d the r e s u l t i n g 1-radyl-2-acylglycerols were acetylated. T h e 1-radyl-2-acyl-3-acetylglycerols f o r m e d were t h e n r e s o l v e d i n t o 1-alkenyl-2-acyl-3-acetylglycerol, 1-alkyl-2acyl-3-acetylglycerol a n d 1,2-diacyl-3-acetylglycerol, a n d t h e r a d i o a c t i v i t y i n each f r a c t i o n was m e a s u r e d as d e s c r i b e d p r e v i o u s l y {14). A f t e r 1 m i n i n c u b a t i o n , m o r e t h a n 80% of the t o t a l r a d i o a c t i v i t y a p p e a r e d in t h e 1,2d i a c y l - 3 - a c e t y l g l y c e r o l f r a c t i o n from b o t h c o n t r o l cells a n d cells e x p o s e d to P A F {Table 2), s u g g e s t i n g t h a t t h e i n c r e a s e d ['4C]arachidonoyl-PC was n o t d e r i v e d from t h e a d d e d P A F u n d e r t h e e x p e r i m e n t a l c o n d i t i o n s . Exo g e n o u s l y s o P A F (10 -7 M) h a d n o effect o n t h e f o r m a t i o n of [14C]arachidonoyl-PC after a 1-min i n c u b a t i o n , whereas e x o g e n o u s l y s o P C (10 _7 M) i n c r e a s e d t h e f o r m a t i o n of ['~C]arachidonoyl-PC a n d c o n c o m i t a n t l y d e c r e a s e d t h e f o r m a t i o n of labeled P A {Table 3). O n the o t h e r h a n d , t h e LIPIDS, Vol. 22, No. 5 (1987)

336

J.-S. TOU presence of lysoPC in the incubation medium exhibited no influence on the incorporation of [1-~4C]arachidonate into PI. DISCUSSION

sensitivity of acyltransferase and/or phospholipase A2 from different cells, as the combined activities of these enzymes control the levels of lysoPC. Human neutrophils have a higher content of alkylacyl-PC than guinea pig peritoneal neutrophils {13,25}, and a CaS§ phospholipase A2 specific for alkylacyl-PC is present in human amniotic fluid 126}. It remains to be studied whether a similar Ca~§ phospholipase As is present in human neutrophils besides the Ca~*-dependent phospholipase A~ {27}. PAF in human neutrophils 128}, rabbit peritoneal neutrophils {29}and rabbit platelets {30,31} is metabolized to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine after a deacetylation-reacylation cycle. Although added PAF caused no measurable changes in the distribution of labeled arachidonate in the diacyl-, alkylacyl- and alkenylacyl-PC after 1 min incubation, the acylation of lysoPAF derived from added PAF would contribute to the increased radioactivity of PC after a longer period of incubation. LysoPC but not lysoPAF was shown to be rapidly acylated by [1-14C]arachidonate to form labeled PC. This finding is in accord with the study demonstrating that extracellular arachidonate was incorporated into diacyl-linked PC more rapidly than into the etherlinked class 113,24,32}. An undetectable acylation of exogenous lysoPAF after 1 min incubation could also be due to a slower rate of lysoPAF uptake by neutrophils, as demonstrated in rabbit platelets {31}. An acylation of exogenous lysoPC into cellular PC was also demonstrated in rabbit peritoneal neutrophils {33} and in human mixed leukocytes from patients with chronic myelogenous leukemia {34}. The increased incorporation of fatty acids into PI and PC during the neutrophil-PAF interaction may be secondary to increased deacylation of these phospholipids by the action of phospholipase As, because no measurable changes in the mass of each phospholipid were detected under the experimental conditions. It remains to be determined whether the activity of the acyltransferase{s} was increased during the neutrophil-PAF interaction in view of the rapid onset of increased formation of labeled PI and PC. An increased acylation of lysoPI and lysoPC by exogenous fatty acids in neutrophils in response to PAF may serve to replenish these phospholipids following deacylation. This may also divert exogenous arachidonate from the 5-1ipoxygenase pathway, thereby attenuating the formation of LTB4 and 5-HETE, both of which are mediators of inflammation {35,36}.

Increased incorporation of arachidonate into PI has been shown to occur in neutrophils in response to fMet-LeuPhe in the presence of cytochalasin B t20), calcium ionophore A23187 {20},phorbol myristate acetate {21} and phagocytosis of inert particles 121}. However, the present study demonstrated a rapid onset {within 15 sec} of increased incorporation of both [1-'~C]arachidonate and [1-'~C]docosahexaenoate into PI and PC during human neutrophil-PAF interaction. It also demonstrated that PAF inhibited the formation of labeled PA from both fatty acids. These findings suggest that the increased formation of labeled PI and PC was not brought about by de novo synthesis but by increased acylation of lysoPI and lysoPC. They also suggest that in the presence of PAF, cellular arachidonate and docosahexaenoate were shunted to the acylation of lysoPI and lysoPC from the acylation of lysoPA. It remains to be determined whether the labeled PA participates in the formation of PI and PC in control neutrophils, as an arachidonate- or docosahexaenoate-labeled cytidine diphosphodiacylglycerol has not been identified, and previous studies have shown only a low level of ["C]docosahexaenoyl-l,2-diacylglycerolin intact human neutrophils {14). The formation of docosahexaenoyl-PA does not seem to be unique in intact neutrophils; it has been demonstrated in bovine retinal microsomes {22}. The magnitude of stimulation by PAF on the formation of labeled PI and PC appears to be a reflection of the specificity of the acyltransferase catalyzing the acylation of lysoPI and lysoPC by fatty acyl-CoA. It was greater in PI than in PC for the incorporation of arachidonate and vice versa for the incorporation of docosahexaenoate. In human neutrophils, arachidonate was a better substrate for incorporation into PI than into PC {9,13,23, 24}, whereas docosahexaenoate was a relatively poor substrate for incorporation into PI as compared to arachidonate 114}. A concentration-dependent stimulation by PAF of the incorporation of arachidonate into PI and PC demonstrated in the present study was also observed in guinea pig peritoneal neutrophils I10) and in lysosomal enzyme secretion by human neutrophils {5). The decreased responsiveness at PAF concentrations above 10-6 M does not appear to be caused by damage of cell membranes, as no increase in the release of lactate dehydrogenase was ACKNOWLEDGMENT detected in the incubation medium. It could be caused by The author is the Albert L. Hyman Research Grant recipient, a rapid neutrophil desensitization for limiting the overall American Heart Association, Louisiana, Inc. cellular response. Extracellular Ca ~§was not found to be essential for the increased incorporation of [1-~'C]arachidonate into PI and REFERENCES PC induced by PAF in human neutrophils under the 1. Ohlrogge, J.B. I1982) Trends Biochem. Sci. 7, 235. experimental conditions. It was required, however, for the 2. Snyder, F. {1982}Ann. Rep. Med. Chem. 17, 242-251. 3. Vargaftig, B.B., and Benveniste, J. ~1983}Trends Pharm. Sci. stimulation by PAF of the formation of arachidonoyl4, 341-343. PC but not arachidonoyl-PI in neutrophils from guinea 4. Hanahan, D.J. {1986}Ann. Rev. Biochem. 55, 483-509. pig peritoneal exudates {10}. The discrepancy in the 5. Shaw,J.O., Pinckard, R.N., Ferrigni,K.S.,McManus,L.M.,and sensitivity toward external Ca s§ for the increased formaHanahan, D.J. 11981)J. Immunol. 127, 1250-1255. tion of arachidonoyl-PC induced by PAF in cells from dif6. Ingraham, L.M., Coates, T.D., Allen, J.M., Higgens, C.P., ferent species is not clear. It may reflect different Ca 2§ Baehner, R.L., and Boxer, L.A. {1982}Blood 59, 1259-1266. LIPIDS, Vol 22, No, 5 (1987)

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LIPIDS, Vol. 22, No. 5 (1987)