Biochiraica et Biophysica Acta, 1044 (1990) 323-331

323

Elsevier BBALIP 33420

Fatty acid m tabolism in human lymphocytes. I. Time-course changes in fatty acid composition and membrane fluidity during blastic transformation of peripheral blood lymphocytes Alberto A n d ~' *, Javier N a v a l 2, Blanca GonzAlez 2 J u a n M a r i a Tortes ', Z o h a i r M i s h a l 1, Jos¢~ Uriel i a n d A n d r 6 s Pi~eiro 2 ; lntti.¢~,t de Recherches Scientifiques sur le Cancer, Vtllejuif (France) and 2 Departamento de Btoquimica y Biologia Molecular. Facultad de Ciencias, Unioersidad de Zaragoza. Zaragoza (Spain)

(Received 20 November 1989) (Revised manuscript receivcct 26 February t990~ Key words: Fatty acid; Blastic transformation; Membrane fluidity; (Human T-lymphocyte)

The t i m ~ changes in fatty acid composition o f h u m a n T - i y m p h o c y t e s during blastic transformation were analysed, as well as the variations in m e m b r a n e fluidity d e t e r m i n e d by fluorescence polarization of 1,6-diphenyi-l,3,5h e x a t r i e n e ( D P H ) , using a f l u o r e s c e n e e - a c t i w t ~ ! cell sorter. T h e m o r e i m p o r t a n t changes observed, in activated relative to quiescent cells, started after 24 h and consisted in an increase in the proportion of oleic (18: l ( n - 9)), d o c o s a p e n t a e n o i c (22: 5 ( n -- 3)) and d o c u s a h e x a e n o i c (22: 6(n -- 3)) acids and a decrease in that of linoleic (18: 2 ( n -- 6)) and arachidonic (20: 4 ( n - 6)) acids. "I'lris represented a relative i ~ r e a s e of 26% for 18: I, 56% for 22: 5 and 84% for 2 2 . 6 in peripheral blood ~ u c l e a r cells 0 P B M C ) a n d 35%, 182% and 94%, respectively, in purified T - l y m p h o ~ t e s ~ b o t h activated f o r 72 h. T h e decrease in n - - 6 fatty acids was o f 42% f o r 1 8 : 2 and 14% for 2 0 : 4 in P B M C and 30% and 19%, respectively, in purified T-cells. T h e s e chmtges mainly affected m a j o r ~ i p i d s ( p l m s p ~ t i d y l c h o l i n e and plmsplmtidylethanolamine) r a t h e r t h a n neutral lipids. T h e 1 8 : 1 / 1 8 : 0 ratio increased greatly in major cell phospholipids. T h e i w o l m r t i ~ of 2 0 : 4 , 2 2 : 5 and 2 2 : 6 in ~ t i d y l i n o s i t o l was n o t significantly altered after 72 h of activation. T h e molar ratio c h o l e s t e r o l / p h o s p l m l i p i d s was reduced in 72-h-activated I)~qphocytes (0.29) c o m p a r e d to qniescient ceils (0.5). O n the other hand, t h e stimulation of h u m a n T - l y m p h o c y t e s cattsed a significant decrease in the o r d e r p a r a m e t e r ( S ) o f D P H , according to the observed c h a n g e s in lipid composition. After 72 h in culture, the S value for quiescent a n d stimulated T - l y m p h o c y t e s was 0.530 a n d 0~326, respectively. In conclusion, the bi~astie transformation of h u n m ~ T - l y m p h o c y t e s is associated with c h a n g e s in lipid c o m p o s i t i o n which modify the physical properties of their m e m b r a n e ~ T h e s e modifications could modulate, in turn, the activity of m e m b r a n e proteins imp4icat~d in the process of blastic transformation. Introduction Biastic t r a n s f o r m a t i o n of T - l y m p h o c y t e s is a c o m -

Abbreviations: PHA. phytohemagglutinin; PBMC. peripheral blood monona¢lear cells; DPH, 1,6-cliphenyl-l,3.5-hexatriene; FP, CS. quorescertce-activatc~l cell sorter; CH, cholesterol; PL, phosphofipids; TO, triacylgl.',eerols; PC, phosphatidylcholine; PE, pho~ph~tid3ilethanolamine; Pi, phosphatidylinosttol; PS, phosphatidylserine; CL. cardiofipin; PA, phosphatidic acid. * On leave from Departamento de Bioqulmica y Biologla Molecular y Celular, Facultad de Ciencias, Zaragoza, Spain. Correspondence: J. Naval, D e p ~ a m e n t o de Bioquimic~ y Biologia Molecular, Facultad de Ciencias, Universidad de Zaragoza, 50009, Zaragoza, Spain.

plex process of g r o w t h and differentiation in which cells u n d e r g o p r o n o u n c e d morphological, biochemical and f u n c t i o n a l changes resulting in m a t u r e blast cells which. in turn, divide to generate the effector T-cells. T h e m o r e easily observable m o r p h o l o g i c a l c h a n g e in activating T - l y m p h o c y t e s is the increase in cell size: the cell diameter m o r e t h a n doubles a n d consequently, the cell v o l u m e a u g m e n t s in m o r e than o n e order of magnitude. This increase ira size is a c c o m p a n i e d by de n o v o synthesis of m e m b r a n e p h o s p h o l i p i d s as welt as by remodelling changes in their fatty acid c o m p o s i t i o n , involving deacylation-reacylation reactions [11. Studies o n calf a n d r a b b i t l y m p h o c y t e s have d e m o n s t r a t e d the enrichm e n t o f these cells in p o l y u n s a t u r a t e d fatty acids, in the early steps of activation [2,3]. W i t h i n m i n u t e s following the interaction of p h y t o h e m a g g l u t i n i n ( P H A ) or concanavalin A with the T-cell r e c e p t o r / C D 3 complex, a p h o s p h o l i p a s e A2 a n d a lysolecithin : acyltransferast, a:

0005-2760/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

324 activated [1]. In this way, saturated fatty aclds in phospholipids are substituted by other polyunsaturates and these changes in composition can be already observed after 4 h of contact with the mitogen [2,31. Changes in fatty acid composition observed during activation of pig [4] and mouse iymphocytes [4,5] are not coincident, however, with those reported for labbit or calf. There is little information on ~he changes in fatty acid composition of T-lymphocytes during the entire period of biastic transformation and, in particular, in the case of h u m a n lymphocytes 16]. On the other hand, there are several studies on the variations of l y m p h o c y t e m e m b r a n e fluidity, detected by fluorescence polarization ( P ) of 1,6-diphenyl-l,3,5hexatriene ( D P H ) and other h y d r o p h o b i c probes, during the blastic t r a n s f o r m a t i e n process [1,5,7-11,13-16]. Using lymphocytes from different animal species, conflicting d a t a on these changes have been obtained [1,5,7-11]. Some authors did not obtain a n y change [5,8,11] while others found a n increase [1,7,9,10] in m e m b r a n e fluidity associated with m.itogenic activation. The majority of these studies use short times of activation (30 r a i n - 4 h) and the changes in P values are not related to changes in lip~.d composition (e.g., cho~est e r o l / p h o s p h o l i p i d s ( C H / P L ) molar ratio, unsaturation of phospholipid fatty acids) [12]. Moreover, the available information o n the changes of m e m b r a n e fluidity of h u m a n lymphocytes is scarce. T w o studies [13,14] are limited to the first h o u r of a~tivation mid in the time-course studies o f Cherenkevich et al. [151 a n d Parola et al. [16], no differences in P values between activated and non-activated cells were found. The aim of the present work ~ a s to study: (a) the changes in the fatty acid composition of phospholipids and other lipids occurring during blastic t r a n s f o r m a t i o n of h u m a n PBMC and isolated T-iymphocytes; and (b) the m a n n e r in which these changes affect m e m b r a n e fluidity of lymphocytes, as d e t e r m i n e d by fluorescence polarization of D P H using fluorescence-activated cell sorting. Results i n d i c a t , that there are an e n r i c h m e n t in fatty acids from the n - 9 a n d n - - 3 series and a decrease in those from n - 6 series, as well as a reduction in the C H / P L molar ratio associated with blastic transformado:-,. These changes, and m o r e probably the reduction ~,f *~.e C H / P L ratio, are associated with a signifieax~z r¢.duction in the P value of proliferating cells. The observed variations in m e m b r a n e fluid;.ty could modulate, in turn, the activity :.f receptors, m e m brane-bound enzymes a n d ion channels ~!7] involved in the process of cell proliferation.

Materials and Methods

Isolation and culture of cells Peripheral blood m o n o n u c l e a r cells were isolated from blood of healthy donors (Service de Transfusion

Sanguine, H r p i t a l Kremlin-Bic~tre, France) by gradient centrifugation in Ficoll-Paque (Pharmacia, Sweden). In some experiments, the cells were further f r a c t i o n a t e d by rosetting with sheep red blood cells treated with 2a m i n o e t h y l i s o t h i o u r o n i u m b r o m i d e (Sigma). Rosetting cells were recovered after a second Ficoll-Paque density centrifugation and the sheep red blood cells were lysed in h y p o t o n i c G e y ' s solution [18]. After the isolation, m o n o n u c l e a r cells were cultured in R P M I 1640 supplem e n t e d with 10% fetal calf serum, 2 m M g l u t a m i n e and antibiotics (penicillin and streptomycin) at a cell d e n s i t y of 1 - 1 0 6 cells/rnl. Viability was controlled by T r y p a n blue exclusion and was always greater than 95%. N o n - a c t i v a t e d cells from the same donors, cultured for different times in s e r u m - s u p p l e m e n t e d R P M I , were used as controls. Boih F B M C mad purified T - l y m p h o cytes were activated by a d d i n g 2 # g of P H A - M (Sigma, U.K.) per ml of culture medium. This c o n c e n t r a t i o n proved to be optimal for cell activation, as d e t e r m i n e d in preliminary studies. In these conditions, 2 - 105 P~IMC activated for 72 h and pulse-labeled with 1 p C i of [3Hlthymidine ( A m e r s h a m , U.K.) d u r i n g the last 7 h of culture i n c o r p o r a t e d 1 0 8 0 0 0 + 15000 c p m ( m e a n of four individual d e t e r m i n a t i o n s on cells f r o m five different donors). T h e s a m e n u m b e r of non-activated P B M C m a i n t a i n e d in culture in the same conditions, incorporated 2500 + 200 clam of [3H]thymidine.

Lipid analysis Cell lipids were extracted with c h l o r o f o r m / m e t h a n o l (2 : 1, v / v ) . Aliquots of the lipid extracts were fractionated in the different lipid a n d phospholipid classes by thin-layer c h r o m a t o g r a p h y in 20 x 20 cm Silica-gel G plates (Merck, F.R.G.). For the separation of lipid classes, the plates were eluted with h e x a n e / diethyl e t h e r / a c e t i c acid (70 : 30 : 1, v / v ) . T h e phospholipid fraction was extracted f r o m Silica-gel using chlorof o r m / m e t h a n o ; (1 : 1, v / v ) . F o r separation of the phospholipid classes, the solvent used was c h l o r o f o r m / h e x a n e / m e t h a n o l / acetic acid ( 5 0 : 3 0 : 15 : 10, v / v ) . The plates were revealed b y spraying with 0.2% 2",7'-dichlorofluorescein in a n h y d r o u s methanol. T h e Silica gel spotted areas were scraped off and the fatty acids were transmethylated, u n d e r nitrogen atmosphere, by r e a c tion with 5% H~SO 4 in a n h y d r o u s m e t h a n o l at 8 0 ~ C for 1.5 h. In o t h e r experiments, the lipid extract was used to prepare the methyl esters o f total fatty acids. First, the lipids were h y d r o l y z e d u n d e r nitrogen a t m o s p h e r e in 2% N a O H in a n h y d r o u s m e t h a n o l 5 rain at 100 ° C . Then, the free fatty acids resulting from the hydrolysis were m e t h y l a t v d u n d e r nitrogen a t m o s p h e r e Using 12% BF3 in a n h y d r o u s m e t h a n o l (5 rain at 1 0 0 ° C ) [19]. The methyl esters were stored at - 3 0 ° C u n d e r nitrogen and a small a , ~ o u n t (20 p g / 1 0 6 cells) o f antioxidant (2,6-di-tertbutyl-4-methylphenol, BHT) was added.

325 Fat:y acid methyl esters were analyzed by gas-liquid c h r o m a t o g r a p h y in a column of G P 10% SP-2330 on C h r o m o s o r b W A W 100/120 (Suppelco, U.S.A.) at 200 o C with helium as carrier zas (flow rate: 20 r n l / m i n ) a n d q,aantified by automatic integration using nheptadecanoic acid ( 1 7 : 0 ) as interred standard. Chromatographic peaks were identified by compari~son with the appropriate c o m m e r c i a l standards (Sigma). Total phospholipids were estimated by measuring the a m o u n t o f phosphorus present in the lipid extracts by an adaptation of the m e t h o d o f Bartlett for micro,quantities [20]. Total cholesterol (ester±fled and free) present in the different types of cell used was d e t e r m i n e d by a stand a r d enzymatic m e t h o d o n lipid samples (Cholesterol CII, Wako, F.R.G.).

brane viscosity directly, but rather on the m e m b r a n e packing [22],

P

t,, +1~

(1)

ltl -- l.L

r~-- I~, +21x

(~)

S~ [1-2r~/ro+5(r~/r°):]l: 2r~/ ro

I+r'/r°

(3)

Statistical significance of the differences in S values were calculated using the Student's t-test for non-paired variates. Results

Fluorescence polariza,'ion studies These studies were carried out using 1,6-diphenyl1,3,5-hexatriene ( D P H ) as fluorescent probe. Labeling of cells with D P H was p e r f o r m e d as indicated by i n b a r and Shinitzky [21]. Briefly, 0.1 ml of 2 m M D P H in t e t r a h y d r o f u r a n was injected into 100 ml of vigorously stirred phosphate-buffered saline (PBS, p H 7.4) giving a final c o n c e n t r a t i o n o f 2 # M in D P H , which is clear a n d practically void of fluorescence. Cells ( 1 . 1 0 6 ) ,vere washed three times with PBS a n d i n c u b a t e d d u ~ n g exactly 30 rain with the D P H suspension at 25 o C. T h e n the cell suspensions were analyzed in a FACS-440 (Becton a n d Dickinson, U.S.A.) cell sorter. T h e i n s t r u m e n t analyses the cells in aqueous suspensions as t h e y pass t h r o u g h an argon-ion laser (model 164-05, Spectra Physics) focused at 363 n m and at an o u t p u t of 100 m W b y simultaneous detection o f a p p a r e n t parallel a n d perpendicular fluorescence emission intensities. D e a d cells were excluded by light scatter-gating measurements. F o r cell size analysis, the cell sorter was calibrated with latex beads o f defined size (Coultronics, France). T h e degree of fluorescence polarization ( P ) was calculated using e q u a t i o n (1), where I , a n d ' are the linearly polarized emissions oriented parallel mad perpendicular, respectively, to the polarized excitation light. The ins t r u m e n t was calibrated with the aid of half wave retardation plates (Oriel, U.S.A.). Fluorescence intensities parallel a n d perpendicular to the laser b e a m were measdred after passing through vertical and horizontal polarizers. T h e steady-state fluorescence anisotropy (r~) values were t.~,leulated from P values, as can be ded u c e d frora F_gln. 2. As indicated in [22], for biological m e m b r a n e s , the estimation of the microviscosity from rs m e a s u r e m e n t s is not reliable, but the d e t e r m i n a t i o n o f the order p a r a m e t e r S is feasible. Thus, the values of S were calculated f r o m r~ using Eqn. 3 a n d a theoretical value for the fluorescence anisotropy in the absence o f a n y rotational m o t i o n of the p r o b e ( r o) of 0.4 (experimental values lie between 0.362 and 0.395) [22,23]. These parameters do not give i n f o r m a t i o n a b o u t m e m -

Changes in the fatty acid composition o] quiescent or PHA-activated T-lymphocytes after short periods of culture T h e total fatty acid composition of T-lymphocytes, freshly isolated o r after 24 h of culture, with or without PHA. in R P M I s u p p l e m e n t e d with 10% fetal calf serum, is s h o w n in Table I. Changes in fatty acid composition were already observed after 30 rain of culture, in oarticular the reduction in the proportion of oleic acid (from 19.8% to 15.8% o f total fatty acids, data not shown). At 24 h of culture, an enrichment in arachidonic

TABLE !

Total fatty acid com,,osition of ~vaescem and PHA-activated haman T-iymphocytes aft¢. 3h¢ -t times of culture (0 - 24 h) Data are expressed as percent (by weight) of the total fatty acids and are the m e a n ± S . D , o f two an_alyses on cells from ,hroe different donors. S a t / u n s a t = sat u r at ed / u n sa t ur a t e d fatty acids ratm nd., not detected. D M A , dimethyl acetal from alkenyl side chain~ of plasmalogens.

Fatty acid 14:0 16:0DMA 16:0 16:!(n-7) 18:0 18:1(n-9) 18:2(n-6) 18:3(n -3) 20:1(n-9) 20:2(n-6) 20:3(n--6) 20:4(n--6) 20:5(n--3) 22:4(n--6) 22:5(n--3) 22:64n-3) Sat/unsat

Noncultured

24 h in culture (quiescent)

24-hactivated

d.5 1.2 27_5 1.7 16.3 19.8 11.1 n.d. 0.4 1.5 2,7 10.1 0.4 l.l n.d. 0.9

n_d. 0.9 23.0 1.1 z8.4 16.5 9.9 n.d. 1.2 2.1 2.1 18.0 1.4 2.0 1.1 1.8

n.d 0.8 23_7 1.4 18.4 18.2 9.4 0.1 0.4 0.8 2.5 17.4 0.5 1.2 2.3 2.2

:t:2.0 +0.5 ±t.7 ±0.7 -rl.O +2.0 ±2,0 ±0.1 ±0.2 ±0.3 ±1.5 i0.1 ±0.2 +0.0

0.98 ± 0.08

±0A _+1.7 ±0.4 ±0.9 +1.0 ±18 +0.5 ±0.3 ±0.7 ±I.1 ±0.I +0.5 +0.3 ±0.5

0.73 + 0.06

+0.1 ±0.7 ±0.0 ±0.5 +_20,2 __+0.2 ±0.0 _+0.1 _+0.0 +0.3 +1.2 ±0,0 ±0.2 +0.1 +_0.2

0.75 ± 0.b 7

326 T A B L E II To;al f a t t y a c i d composition o f quiescent h u m a n P B M C a n d T-lymphocytes cultured f o r 2 4 h a s well a s cells activated with P H A f o r longer periods (48 - 144 h)

Data are expressed as percent (by weight) of the total fatty acids an d are the means 4- S.D. of several dete.,~ainatio,~s on cells from differ~mt donors (n -- number o f donors used for each time-point). S a t / u n s a t = s a t u r a t e d / u n s a t u r a t e d fatty acid~ ratio, n.d., not detected D M A , dimethyi acetal from alkenyl side-chains of plasmalogens.

Fatty acid ( ~ ) 16:0 DMA 16:0 16: l ( n - 7 ) 18:0 18: l ( n - 9 ) 18 : 2(n --6) 18 : 3(n -- 3) 20: l ( n - 9 ) 20: 2(n - 6 ) 20: 3(n - 6 ) 20: 4(n - 6) 20 : .$(n ~ 3) 2 2 : 4 ( n - 6) 22: 5 ( n - 3 ) 22:6(n -3) 18 : 1 / 1 8 : 0 Sat/unsat

Quiescent T-lymphocytes (n = 3)

Quiescent PBMC (n = 7)

48-h-act, T-lymphocytes (n = 3)

72-h-act. T-lymphocytes ( n = 3)

48-h-act. PBMC (n ~ 4)

72-h-act. PBMC (n = 3)

-act. PM~,C (n -- 2)

0.9 23.0 1.1 18.4 16,5 9.9 n.d. 1.2 2.1 2.1 18.0 1,4 2.0 1.1 1.8

1.5 19.9 0,8 18.5 16.4 9,4 0.1 2.5 1.2 1.8 19.7 0.8 1.6 2.3 2.5

1.1 +0.5 22.7 ± 0 . 9 1.1 =1=0.3 16.5 ±0,8 19.8 =1=0.8 7.7 4-0.8 n.d 1.4 ±0.5 1.1 +0.2 2,5 4-0.0 17.2:1:0.6 1.1 ± 0 . 0 1.6 +0.5 2.9 ±0"2 2.8 ±0.2

0.9 + 0 . 3 24.2 + 1 . 0 1,4 -1-0.2 16.3 ~ 0 . 0 22.3 4-0.1 5.7 4-0.1 rt.d 1.0 ± 0 . 0 0.8 +0.1 2.5 +0.1 15.4:1:0.3 1.3 + 0 . 0 1.0 4-0.1 3.1 ± 0 . 2 3.5 ± 0 . 3

0.7 22.6 0.5 18.0 20.1 6.8 0.2 1.2 0.8 2.1 18.1 n.d. 1.6 3.4 3.8

1.9 19.3 0.8 17.2 20.6 6.6 0.3 1.7 0.7 2.5 15.1 0.8 2.0 3.6 4.6

0.5 21.7 0.3 18.8 28.7 4.5 n,d. 2.4 0.5 2.0 12.8 0.5 0.7 2.3 3.8

±0.2 +1.7 ±0.4 ±0.9 ±1.2 4-1.8 +0.5 ±0.3 4-0.7 ±1.1 ±0.1 ±0.5 ±0.3 :t=0.5

+0.4 +0.9 ±0.3 ±1.1 ±1.5 ±1.0 +0.0 +0.5 ±0.2 ±0.2 +2.0 +0.0 ±0.2 ±0.4 ±0.5

4-0.0 :t:0.2 =1=0.1 +0.5 4-1.0 ±0.4 4-0.0 =l=0.1 4-0.0 4-0.1 :t:l.1 4-0.1 ±0.2 ±0.6

4-0.5 ±0.5 +0.1 4-0.8 +0.7 +0.1 +0.1 _+_0.1 4-0.1 ±0.3 4-0.7 +0.0 4-0.3 -1-0.2 ±0.5

4-0.1 =1=2.1 ±0.0 4-0.4 4-0.6 4-0.4 +0.1 +0.1 4-0.1 ±0.1 4-0.0 4-0.2 :F0.0 ±0.8

0.89 4- 0.10

0,88+0.07

1.21 ±0.09

1,364-0.01

1.1 ~_±0.08

1.19 =t=0.10

1.524-0.01

0.73 ± 0.06

0.66 ± 0.04

0.67 ± 0.03

0.71 ± 0.03

0.70 4- 0.01

0.66 4- 0.02

0 . 6 9 t- 0 . 0 5

acid ( 2 0 : 4 ( n - 6)) was observed in b o t h quiescent a n d activated cells relative to freshly isolated cells. T h i s e n r i c h m e n t caused a 24% r e d u c t i o n in t h e s a t u r a t e d / u n s a t u r a t e d fatty acids ratio. T h r o u g h o u t this period, the total fatty acid c o m p o s i t i o n o f quiescent or activated cells was roughly similar. These d a t a s h o w e d that, in short-time (30 rain to 6 h) activated cells, m o d i f i c a t i o n s of cell fatty acid c o m p o s i t i o n m o s t p r o b a b l y result f r o m an a d a p t a t i o n to culture c o n d i t i o n s t h a n to m i t o g e n i c activation itself. However, at 24 h of culture, the prop o r t i o n of 1 8 : l ( n - 9) a n d 22 "~ n - 3) was higher in the activated l y m p h o c y t e s than in the quiescent o n e s ( P < 0 . 0 5 for 1 8 : 1 a n d P < 0 . 0 2 5 for 2 2 : 5 ) . T h e s e differences b e c a m e m o r e significant for longer activation times.

induced by P H A - s t i m u l a t i o n (Table If). Results obtained for activated P B M C a n d T-iymphocytes are a n n marized in Table II and, as it m a y be observed, were similar for b o t h cell populations. T h e t i m ~ changes in the proportion o f major unsaturated fatty acids series is s h o w n in Fig. 1. T h e proportion o f oleic acid (18 : l ( n -- 9)) increased with the time o f activation and, in the case o f purified T-lymphocyt~s, it w a s a c c o m p a n i e d b y a decrease in the percentage o f stearic acid ( 1 8 : 0 ) . A n o t h e r important change associated with blastic transformation was the enrichment in the n - 3 polyunsaturated fatty acids 22 : 5 and 22 : 6, as s h o w n in Table 11. The enrichment in 22 : 5(n -- 3) became significant after 48 h and 72 b o f activation and decreased

Changes in fatty acid composition from cell iipids during blastic transformation of T-lyn,phocytes

30:

A s showed in Table I and c o m m e n t e d above, a culture time of 24 h was sufficient to m o d i f y the fatty acid composition o f quiescient cells without affecting their viability (more than 90%). Longer times o f culture of quiescent cells resulted in a nonspecific activation o f lymphocytes by serum antigens, as observed b y light scattering F A C S analysis and their viability decreased. For this reason, non-activated P B M C or T-lvmphocytes maintained for 24 h in culture were selected as suitable controls ~o evaluate changes in fatty acid c o m p o s i t i o n

20i 10 0 0

24

48 72 96 120 T i m o f n ~ l v a t l o n (h)

144

Fig. 1. Changes in the proportions of n - 9, n - 6 and n-3 fatty acids of human P B M C during blastic transformation. D a t a are expressed as percent of the total fatty acids. T i m e 0 corresponds to quiescent cells cultured fat 24 h. Data are recalculated from T a bl e IL Vertical bars indicate S.D.

327

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~

~

328 thereafter (e.g., at 144 h), while the increased level of 2 2 : 6 ( n - 3) remained throughout the entire activation process. The relative a m o u n t o f n - 6 polyunsaturated fatty acids, n a m e l y linoleic (18 : 2(n - 6)) a n d arachidonic (20 : 4(n - - 6 ) ) was also reduced. T h e reductions in these fatty acids, balanced the increases mentioned in n - 9 a n d n - 3 fatty acids, as illustrated in Fig. 1, and the s a t u r a t e d / u n s a t u r a t e d ratio remained practically unchanged t h r o u g h o u t the blastic transformation period (see Table II). To further assess the relevance of these changes, the fatty acid composition o f neutral iipids and phosphoiipids was determined in quiescent and 72 h P H A activated PBMC from the same donors (Table III). T h e increase in the 1 8 : 1 / 1 8 : 0 ratio was only observed in phospholipid fractions: phosphatidylchol/ne (PC), phosphatidylethanolamine (PE), phosl~hatidylinositol (PI) and cardiol;pin + phosphatidic acid (CL + PA). This change was not detected in the triacylglycerol ('l'G) fraction, where a slight decrease was observed. A signifieant increase in the proportion of 2 2 : 5 ( n - 3) was observed in TG, PE, PS and C L + P A fractions, while that of 2 2 : 6 ( n - 3) increased in PC a n d PE fractions. The reduction in the p . o p o r t i o n of 1 8 : 2 ( n - 6) was observed in PC fraction, while the reduction of 2 0 : 4 ( n - 6 ) proportion was observed in TG, PC a n d PE (see Table IlI). The fatty acid composition of diacylglycerol, free fatty acid and cholesteryl cster fractions f r o m quiescent a n d 72 h-activated cells was also d e t e r m i n e d . The total a m o u n t of fatty acids present in these fractions did not represent m o r e t h a n 9% of *.he total fatty acids and their composition did not show a n y significant change during the blastic transformation period

TABLE

IV

Cholesterol /phospholipid ( C H / PL) molar ratios of quiescent (cultured for 24 h) and activated human T-l),mpho(~ tes and P B M C D a t a a r e t h e m e a n ± S . D . o f d u p l i c a t e d e t e r m i n a t i o n s o n cells f r o m three different d o n n r s . Cholest~:rol (nmol/106cells) Quiescent PBMC 72-h-act. PBMC Quiescent T-lymphocytes 48-h-act. T-lymphocytes

Phospholipids (nmol/106cells)

CH/PL molar ratio

2.20 + 0.3 2.50 :t: 0.1 2.50 ± 0.2

4.37 ± 0.2 8.50 ± 0.6 5.30 :t: 0.3

0.50 ± 0.04 0.29 + 0.02 0.47 + 0,03

3.70 ± 0.2

10.20 + 0.9

0.36 ± 0.01

(data not shown). A n increase in the a m o u n t of cell fatty acids f r o m the different lipid classes was also observed (Table III). The greater e n r i c h m e n t corres p o n d e d to the T G fraction and the relative in, ,'eases a m o n g the different phospholipid fractions analysed were similar.

Changes in the molar ratio cholesteroi / phospholipids T h e a m o u n t of cholesterol and phospholipids present in the lipid extracts of quiescent and P H A - a c t i v a t e d h u m a n T - l y m p h o c y t e s or PBMC, as well as the corresponding cholesterol/phospholipids ( C H / P L ) molar ratios are shown in Table IV. Quiescent cells maintained for 24 h in cultures without P H A were used as controls. Relative to control cells, the activation process i n d u c e d a m a r k e d increase (greater t h a n 90%) in the a m o u n t of cell phospholipids in b o t h P M B C a n d T-

TABLE V

Values of the degree of fluorescence polarization (P), steady-state fluorescence anisotropy (rs) and order parameter (S) of quiescer, and activated iymphocytes D a t a are t h e m~'an o f dupli~ • d e t e r m i n a t i o n s o n cells f r o m d i f f e r e n t d o n o r s . V a l u e s in t h e b r a c k e t s indica'.e t h e n u m b e r o f d i f f e r e n t d o n o r s u s e d in e a c h case. S D w a s h e r e : g r e a t e r t h r m 1 5 ~ o f the m e a n . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s b e t w e e n a c t i v a t e d azzd t h e c o r r e s p o n d i n g n o n - a c t i v a t e d cells: * P < 0.0125; * * P < 0.01 ; * * * P < 0.0025. T-lymphocytes

Non-cultured 24 h in c u l t u r e 48 h 72 h 96 h PHA-activated 30 r a i n (1) 3 h (I) 6 h (1) 24 h (3) 48h (3) 72 h (2) 96 h (2)

4- . i (4) (2) (2) (2)

PBMC

P

r$

S

P

rs

S

0.330 0.283 0.278 0.235 -

0.247 0.208 0.205 0.170 -

0.737 0.640 0.629 0.530 -

0.283 0.255 0.249 0.246

0.208 0-186 0.181 0.179

0.640 0.578 0.:>64 0 551

0.330 0.326 0.309 0 221 0.186 0.152 -

0.247 0.244 0.229 0.159 0.132 0.107 -

0.737 0.730 O. -93 0.495 * * * 0.410 * * 0.326 * -

0.244 0.196 0.12"2_ C.!55

0.177 0.140 0.085 0.109

0.551 0.436 * * 0.252 * * * 0.332 * * *

329 lymphocytes. Since the a m o u n t of cholesterol increased in a lesser extent (14% in P B M C and 48% in Tlymphocytes), the resulting C H / P L molar ratios were m a r k e d l y reduced.

Fluc.rcscence polarization studies The data obtained on the degree of fluorescence polarization ( P ) a n d related p a r a m e t e r s on quie.~cent and PHA-activated h u m a n T - l y m p h o c y t e s a n d P B M C are resumed in Table V. Short times o f activation (30 rain, 3 h or 6 h) did not cause great changes in the o r d e r p a r a m e t e r S, indicating that in the early steps of activation, no significant changes o c c u r r e d in the p a c k i n g of the m e m b r a n e constituents. T h e culture of quiescent cells for 24 h p r o d u c e d a m o d e r a t e decrease o f S value ( f r o m 0.737 to 0.640, P < 0.005). T h e reduction in S value observed in T - l y m p h o c y t e s cultured for 24 h in the presence of P H A was greater than that i n d u c e d b y the culture itself (0.640 in qmescent cells vs. 0.495 in activated T-lymphocvtes, P < 0.0025). This difference was, however, n o t significant in the case of 24-hactivated P B M C (Table V, P < 0.075). At longer times of culture, the differences between quiescent a n d activated cells were significant for b o t h P B M C a n d T - l y m p h o c y t e s ( P < 0.0125). T h e time-course of S values for quiesceint P B M C o r for P H A - a c t i v a t e d P B M C a n d T - l y m p h o c y t e s is shown in Fig. 2. The culture of quiescent cells in s e r u m - s u p p l e m e n t e d m e d i u m producexl a m o d e r a t e decline in S value, comparatively less than that observed at a n y time in activated cells. T h e lowest S value t0.252) was observed for P B M C activated for 72 h. This represented 40% a n d 46% of the S values for quiescent cells cultured for 24 h a n d 72 h, respectively ( P < 0.0005 in b o t h cases). Discussion

L y m p h o c y t e activation leading to proliferation is a basic p h e n o m e n o n in the majority o f i[lUTlunolo~,'r~ events. A l t h o u g h the early metabolic changes in response to a specific antigen (or mitogenic lectin) are

0.8'

~

0.6

°1 0.4

0.2 1

0

24 48 72 96 Tim tn culture(h) Fig. 2. Chan~;cs;n S values(order parameter)duringblast~ctransformation of human T-lymphee3~tes ( I ) and P B M C (A). Non-activated PBMC ( e ) were used as controls. D a t a are taken from Table V. Vertical bars indicate S.D.

well d o c u m e n t e d , the underlying mechanism is not yet completely understood. The present and other studies [1-3,6] d e m o n s t r a t e that the fatzy acid composition of m e m b r a n e phospholipids changes during blastic transformation and t h a t these changes m a y affect, in turn, the process itself. However. it is not clear if: (i) these changes occur in early steps of the activation process and play an. impo_rtant role in the interaction "~ . . . . _ 1 the antigen and the T-cell r e c e p t o r / C D 3 complex, ~s well as in the subsequent process 3f signal t r a n s d u c u o n ; or (ii) the changes in the fatty acid composition arise later, as a secondar3' ~went of the main process of stimulation. In particular, the latter would bring a b o u t the activation o f several key enzymes for lipid biosynthesis, namely, those responsible for the d e a c y l a t i o n - r e acylation process into the phosphohpids, the fatty acid d e s a t u r a s e - e l o n g a s e systems and the enzymes which catalyze the de nova synthesis of phospholipids and triacylgiycerols. In the model p r o p o s e d by Reseh et al. [1], and d u e to the physical proximity between lysolecithin:acyltransferase and the T-cell receptor, the enzyme becomes activated in the early steps o f blastic transformation. The activity of this enzyme, that shows a higher affinity for p o l y u n s a t u r a t e d fatty acids, causes a rapid change in the f~,tty acid composition of plasma m e m b r a n e phospholipids, a p p a r e n t after 4 h of activation~ characterized m a i n l y b y an e n r i c h m e n t in arachidonic acid. T h e s e changes are irreversible as this e n z y m e remains active during the entire period of blastic transformation. A c c o r d i n g to this, the main changes in fatty acid composition o f stimulated T-l)mphoc3~es are p r o d u c e d after short activation times a n d favors an easier signal transduction i n t o the cells. However, the above mentioned sthdies are not directly c o m p a r a b l e with our present data because they were perfo.,xned using rabbit or calf thymocytes cultured in serum-free media, O n the o t h e r hand, our data a n d those of Shires [6], d e m o n s t r a t e that the changes in fatty acid c o m p o s i t i o n occurring during blastic transform a t i o n of h u m a n P B M C and T-lymphocytes are quite different from those f o u n d for rabbit or calf thym6cytes. T h e e n r i c h m e n t in arachidonic acid, observed in these thymocytes, does not take place in h u m a n lymphoeytes. Moreover, in the h u m a n cells, the increase of u n s a t u r a t e d fatty acids of n - 9 (oleic) and n - 3 ( d o c o s a p e n t a e n o i c and docosahexaenoic) series is bala n c e d by a decrease of unsaturated fatty acids of n -- 6 series (linoleic a n d arachidonic). The reduction in the p r o p o r t i o n of 2 0 : 4 ( n - 6) associated with the activation of h u m a n P B M C has b e e n also indirectly obser-,ed b y G i b n e y et al. [24]. This decrease could not be d u e to a local p r o d u c t i o n of arachidonic acid-derived eicosanoids, because h u m a n T- or B-lymphoeytes are unable to synthesize t h e m [25]. However, in PBMC ar,,1 even in purified T - l y m p h o c y t e preparations, there are always a

330 small portion of m o n o c y t e s ( a r o u n d 8% and 7%, respectively) [26], active producers of eicosanoids. A n y w a y , the metabolism o f e n d o g e n o u s 2 0 : 4 ( n - 6) in m o n o cytes would not be e n o u g h to explain the total reduction observed in 2 0 : 4 ( n - 6) levels. A n alternative explanation, as p r o p o s e d by G o l d y n e [27], would be t h a t 20 : 4(n - 6) is released from T - l y m p h o c y t e p h o s p h o lipids and transferred to m o n o c y t c s to syn,,hesize eicosanoids. This possibility suggests the existence of a n intercellular cooperation that c o u l d be involved in t h e control of l y m p h o c y t e proliferation. O n the other hand, n o changes in cellular fatty acid c o m p o s i t i o n were observed after short times o f activation (30 rain to 6 h) a n d o n l y slight changes were noticed up to 24 h. C h a n g e s b e c o m e progressively m o r e i m p o r t a n t fc?_'~.~ g longer periods of activation (48, 72 or 144 h). Therefore, our d a t a indicate that the changes in fatty acid c o m p o s i t i o n of activated h u m a n l y m p h o cytes occur in late activation steps, as a c o n s e q u e n c e o f the c o m b i n e d action of several e n z y m e s of lipid biosynthesis. These changes w o u l d be involved in the m a i n t e n a n c e and d e v e l o p m e n t o f the activated state, sustaining the proliferation of blast cells, r a t h e r t h a n being a trigger of the process itself. Nevertheless, a l t h o u g h we have not detected a n y significant difference b e t w e e n t h e total fatty acid c o m p o s i t i o n o f quiescent or activated h u m a n T-cells d u r i n g the first 6 h of culture, we c a n n o t rule out the existence of a significant t u r n o v e r of fatty acids in individual m e m b r a n e p h o s p h o l i p i d s d u r i n g this period, as has b e e n observed in a n i m a l species [1-3]. This type of m o d i f i c a t i o n w o u l d have r e m a i n e d u n n o t i c e d in out e x p e r i m e n t a l c o n d i t i o n s because fetal s e r u m induces i m p o r t a n t c h a n g e s in fatty acid c o m p o s i tion of resting T-lymphocytes, u n r e l a t e d to t h e activation process itself (Table I). A l t h o u g h we have f o u n d , roughly, t h e same qualitative change in l y m p h o c y t e fatty acid c o m p o s i t i o n as previously relx--'ed for l o n g - t e r m s t i m u l a t e d h u m a n P B M C [6], the • . n o u n t o f arachidonie acid esterified in the PE fraction was higher in o u r analysis a n d coincid e n t with d a t a f r o m several species ( b e t w e e n 19% a n d 31%) [2]. This discrepancy could be d u e to differences in the diets of the d o n o r s or in the c o m p o s i t i o n of t h e o~ '~ "0 ~-d fox the cultures. O t h e r d a t a o n fatty acid cor,apo,~ 0~,.m of h u m a n P B M C indicate that the level o f 2 0 : 4 ( n - 6) in h u m a n l y m p h o c y t e s is > 20% of total fatty acids [24,28,29]. W i t h regard to m e m b r a n e ~ u i d i t y studies, o u r d a t a o n the order p a r a m e t e r values ( S ) indicate t h a t activation o f h u m a n T - l y m p h o c y t e s reduces i m p o r t a n t reductions o n the m e m b r a n e p a c k i n g after , ~ g t i m e s o f activation (i.e., > 24 h), being irrelevant m th~ early steps. These reductions shnu!d b e related to t h e changes in the fatty acid c o m p o s i t i o n a n d in the c h o l e s t e r o l / p h o s p h o l i p i d s ( C H / P L ) m o l a r ratio [12]. Since the ratio o f s a t u r a t e 0 / u n s a t u r a t e d fatty acids d i d n o t c h a n g e

significantly d u r i n g blastic transform~,tion, the low value o f the C H / P L ratio observed in activated T - l y m p h o cytes a n d P B M C relative to q u i e s c e n t cells, m u s t b e the m a i n c o n t r i b u t o r to the observed r e d u c t i o n s in m e m b r a n e packing. Moreover, as indicated in ReL 30, the m o d i f i c a t i o n o f the fatty acid c o m p o s i t i o n of several cell types did n o t c h a n g e t h e P values or the C H / P L ratio. In cellular m e m b r a n e s , P values seem to be m o r e related to the C H / P L ratio t h a n to the degree of u n s a t u r a t i o n o f p h o s p h o l i p i d fatty acids. However, the decrease in t h e C H / P L ratio m a y have n o t b e e n the o n l y c o n t r i b u t o r to t h e decrease in fluorescence polarization. T h e possibility that D P H m a y have p a r t i t i o n e d p a r t l y i n t o lipid d r o p l e t s [5] o r r e a c h e d the e n d o p l a s m i c r e t i c u l u m m e m b r a n e s c a n n o t be r u l e d out. Analysis of m e m b r a n e s u b f r a c t i o n s w o u l d be necessary to det e r m i n e their c o n t r i b u t i o n to the c h a n g e s observed. Nevertheless, I n b a r et al. [21] indicated that, in the e x p e r i m e n t a l c o n d i t i o n s used, D P H is m o s t l y incorporated i n t o p l a s m a m e m b r a n e . C h e r e n k e v i c h et al. [15] a n d Parola et al. [16] observed r e d u c t i o n s in P values o f activated h u m a n l y m p h o c y t e s similar to those described by us. C o n t r a r y to o u r results, however, t h e y observed n o differences b e t w e e n q u i e s c e n t a n d activated cells. T h e m a i n methodological difference b e t w e e n o u r s t u d y a n d theirs was t h e use o f a F A C S analyser i n s t e a d o f a fluorimeter. As r e p o r t e d above, the p e r c e n t a g e of d e a d cells in cultures t r e a t e d w i t h t h e m i t o g e n was negligible, b u t in q u i e s c e n t cells this p e r c e n t a g e raised u p to 20% with the t i m e o f culture. D e a d cells p r o d u c e a s h a r p fluorescent p e a k a n d very low values o f P. If the c o n t r i b u t i o n o f this p e a k c a n n o t b e e l i m i n a t e d (case of m e s u r e m e n t s m a d e with c o n v e n t i o n a l fluorimeters), t h e m e a s u r e d value w o u l d be m u c h lower t h a n t h e real value c o r r e s p o n d i n g to living cells only. T h i s c o u l d explain the low P (or S ) values previously o b t a i n e d for quiescent cells in l o n g t e r m c u l t u r e [15,16]. In c o n c l u s i o n , sigJaificant c h a n g e s in fatty acid c o m p o s i t i o n o c c u r late d u r i n g blastic t r a n s f o r m a t i o n of h u m a n T - l y m p h o c y t e s : an increase in t h e c o n t e n t of oleic, d o c o s a p e n t a e n o i c a n d d o c o s a h e x a e n o i c acids a n d a c o n c o m i t a n t decrease in that o f linoleic a n d a r a c h i d o n i c acids. T h e s e c h a n g e s m a y c o n t r i b u t e to the a u g m e n t a t i o n in m e m b r a n e fluidity of T-cells, t h o u g h the m a j o r f a c t o r seems to be t h e decrease in the c h o l e s t e r o l / p h o s p h o l i p i d s m o l a r ratio. T h e m e c h a n i s m s by which s,.;_mulated l y m p h o c y t e s mo,4ify their fatty acid c o m p o s i t i o n (i.e., in situ synthesis or u p t a k e ) are n o t well k n o w n a n d will be further ir, vestigated. Acknowledgements

W e th~.nk P r o f e s s o r F. G r a n d e - C o v i h n for his help i n statis:ical analysis. T h i s w o r k was s u p p o r t e d by g r a n t s from F o n d o d e lnvestigaciones Sanitarias d e la Seguri-

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