Diacylglycerol production in Jurkat T-cells: Differences between CD3, CD2 and PHA activation pathways

Cellular Signalling Vol. 3, No. I, pp. 35--40, 1991 Printed in Great Britain.

0898-6568/91 $3.00+.00 © 1991PergamonPress pie

D I A C Y L G L Y C E R O L P R O D U C T I O N IN J U R K A T T-CELLS: D I F F E R E N C E S BETWEEN CD3, CD2 A N D PHA ACTIVATION PATHWAYS CLAUDETTE PELASSY,DmmR MARY and CLAUDE AUSSEL* Unit6 de Recherches en Immunologie Cellulaire et Mol~culaire, U210 INSERM, Facult6 de Medecine (Pasteur), 06034 Nice C~dex, Franc. (Received 3 July 1990; and accepted 1 October 1990)

Abstraet--Diacylglycerol (DAG) production induced after stimulation with either CD3 mAb, a pair of CD2 mAbs or phytohaemagglutinin has been monitored in Jurkat T-cells prelabelled to isotopic equilibrium with seven [3H]- or ["C]fatty acids. It was found that CD3 induced a high production of arachidonic acid-labelled DAG and a modest production of oleic acid-DAG. The reverse was observed when using CD2 as activator. Phytohaemagglutinin induced a high production of these two DAG subspecies and in addition induced the production of linolenic acid-labelled DAG. Whatever the activator used no changes were observed in DAG production when cellular phospholipids were prelabelled with either myristic, palmitic, stearic or linoleic acids. All together our results strongly suggest that the three activation pathways previously described in Tlymphocytes might differ either at the level of the transduction mechanism or the phospholipid pools solicited during the activation process. Key words: Diacylglycerol, T cell activation

INTRODUCTION

lizes Ca 2+ from intracellular stores. The combination of Ca 2+ and D A G activates protein kinase C (PKC), a protein that appears to play a key role in lymphocyte activation [8, 9]. In order to study in more detail the fatty acid composition of DAGs produced during lymphocyte activation, we have monitored D A G production in Jurkat cells prelabelled to isotopic equilibrium with seven different fatty acids, and then activated them with either CD3, CD2 mAbs or PHA. The results described demonstrate for the first time differences in the DAGs induced by the different activation pathways.

T-CELL activation and proliferation are initiated by the interaction between an antigen linked to a membrane-bound major histocompatibility complex (MHC) molecule, and a corresponding T-cell receptor complex (TCR). Experimentally, T-cell activation can be mimicked by mitogenic lectins or mAbs directed against the TCR complex (CD3 mAbs) or the sheep erythrocyte receptor (CD2 mAbs) [1-4]. Signals generated by these cell surface interactions are believed to be transduced via a GTP-binding protein coupled to a phosphodiesterase (phospholipase C) that cleaves phosphatidylinositol bisphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG) [5-7]. IP3 in turn mobi-

MATERIALS A N D METHODS Ce//s The human T-cell leukemic line Jurkat was kindly supplied by Dr A.M. Schmitt-Verhulst (Centre d'Immunologie, Marseille-Luminy, France). Cells were cloned by limiting dilution, clone D was selected on the basis of its high interleukin-2 produc-

* A u t h o r to w h o m correspondence should be addressed. Abbreviations: P E - - p h o s p h a t i d y l e t h a n o l a m i n e ; P C - - p h o s phatidylcholine; PI---phosphatidylinositol; PIP/PIP2--phosphatidylinositol m o n o - a n d bisphosphate; P S - - p h o s p h a t i dylserine; DAG--1,2-diacylglycerol; N L - - n e u t r a l lipids. C~LLS 3:1-C

35

36

C. PELASSYet al. .fill

.

.

.

.

.

.

.

.

+

I 41

.

.

.

.

.

.

.

.

.

.

.

.

.

~

I l!I

.

.

.

.

.

.

.

.

.

.

.

A

yQ .

.

.

.

.

.

.

.

.

~1[

'

.+

o

i

o

I

I

*

I

i

~

i

I

i

I 16

.

~

'

I

i

£ot~3 .

.

.

.

.

.

.

g

.

Iml:

.

L O

i

o

l

l

1

1

4

1

1

1

1

1

,

1

1

1

1

.

1

1

1

l

8

FIG. I. Separation of [3H]oleic acid labelled lipids (A) and phospholipids (B) by thin layer chromatography on LK6D silica gel plates as described in Materials and Methods. Radioactivity on chromatograms was determined by using an automatic linear analyser equipped with an 8 mm window and an evaluation time of 5 rain. The different lipids were identified by using lipids standards revealed with iodine vapors. Abbreviations: PL--phospholipids; DAG--diacylglycerol; TG---triglycefides; CE---cholesterol esters; PIP/PIP~--phosphatidylinositol mono- and bisphusphate; PC--pbosphatidylcholine; PI/PS--phosphatidylinositol and phosphatidylserine; PE--phosphatidylethanolamine; PA--phosphatidic acid; NL--neutral lipids.

tion [22] and used throughout this study. Cells were cultured in RPMI-1640 (Seromed, Lille, France) supplemented with 5% foetal calf serum, 50 units/ml penicillin, 50 #g/ml streptomycin, 2 mM L.glutamine, I mM pyruvate and 0.1 #M/~-mercaptoethanol. Monoclonal antibodies

The CD3 monoclonal antibody, secreted by the hybridoma X35, was kindly supplied by Dr D. Bourrel (CRTS, Rennes, France). The CD2 mAb (IOT 11) was purchased from Immunotoch (Marseille, France), the other CD2 mAb (GT2) was kindly supplied by Dr A. Bernard (IGR, Villejuif, France).

Chemicals

[5,6,8,9,11,12,14,15-3H]Arachidonic acid (C20 : 4) (l.85-2.2Glkl/mmol); [9,10(n)-3H]palmitic acid (C16:0) (1.5-2.2 TBq/mmol); [1-'4C]stearic acid (C18:0) (1.85-2.2 Glkl/mmol); [l-~L-']olvic acid (C18:1) (1.85-2.2 Glkl/mmoi); [l-I~-']linoleic acid (1=18 : 2) 0.85-2.2 Glkl]mmoi); [1-i~-llinolenic acid (C18 : 3) (1.85-2.2 GBq/mmol); and [9,I0(n)3H]myristic acid (C14:0) (1.5-2.2 TBq/mmol) were purchased from Amersham, France. DAG production

Cells were prelabelled for 24-48 h in complete medium with 1-5/zCi of either [3HI- or [~+C]-labelled

Diacylglycerol production in T-cells

37

[3H]- or ['~C]-labels were used, all the results are given as a % relative to a control.

fatty acids. After washing the cells were incubated at 37°C for different periods of time, between 30 s and 30 min, in the absence (controls) or presence of either PHA (50/~g/ml), CD3 (2 #g/ml) or a pair of CD2 mAbs (2/~g/mi each). The concentration of the effectors used was chosen to be equal to the concentration able to induce maximal interleukin-2 synthesis (in the presence of phorbolesters) in Jurkat cells. After lipid extraction by the method of Bligh and Dyer [10], the organic phase was collected and further analysed by thin layer chromatography (TLC) on silica gel plates LK6D (Whatman) with the use of a solvent system composed of n-hexane/diethylether/fomfic acid (80 : 20 : 3). This solvent system separated phospholipids from diacylglycerol, trigiycerides and cholesterol esters (Fig. 1A). For phospholipid analysis, the organic phase was separated on the same TLC plates as above and developed in a solvent system composed of chloroform/methanol/acetic acid/water (75 : 45 : 12 : 3). This solvent system allowed the separation of six peaks corresponding, respectively, to phosphatidylinositol mono- and bisphosphate (PIP+PIP2), phosphatidylcholine (PC), phosphatidylinositol + phosphatidylserine (PS + PI), phosphatidylethanolamine (PE), phosphatidic acid (PA) and neutral lipids (NL) (Fig. 1B). The lipid migration was monitored by using unlabelled lipid standards revealed with iodine vapours. Radioactivity was determined with the use of an automatic linear thin layer radiochromatography scanner (Berthold). In order to compare data from experiments in which

RESULTS

Incorporation of labelled fatty acid in Jurkat T-cells Incorporation o f the different [3H]- or [14C]labelled fatty acids into Jurkat lipids was measured after either 24 or 48 h of incubation in complete culture medium at 37°C in a humidified atmosphere containing 5% CO2/ 95% air. N o significant changes were observed between 24 and 48 h o f incubation, indicating that isotopic equilibrium was reached after a 24 h incubation period. This preincubation time was thus chosen for subsequent studies. The incorporation of the different labels is shown in Table 1. Palmitic and oleic acids were found preferentially incorporated into phosphatidylcholine (PC). The majority of the highly unsaturated fatty acid, arachidonic acid, was incorporated into both phosphatidylethanolamine (PE) and the phosphatidylinositol-phosphatidylserine group (PI/PS). The other fatty

TABLE 1. DISTRIBUTIONOF FATTYACreS IN JURKATCELLPHOSPHOLIPIDS Fatty acid Myristic (C14:0) Palmitic (C16:0) Stearic (C18:0) Oleic (ClS: 1) Linoleic (C18:2) Linolenic (C18:3) Arachidonic (C20:4)

PIP/PIP2

PC

PS/PI

PE

NL

2.77 __ 1.23 8.79 _ 1.36 2.03 ___0.28 4.87 -+ 1.59 9.37 -+ 1.60 4.58 _+1.17 8.25 _+1.71

26.54 -+ 3.60 41.95 _+4.75 25.83 _+5.98 43.31 -+6.33 29.84 _+2.93 25.32 _+3.85 16.04 _+1.12

0.91 _+0.05 10.00 -+ 1.62 23.67 _+1.01 12.71 _+2.23 14.51 -+ 1.61 24.38 _+8.46 26.23 _+5.60

31.34 _ 4.35 14.99 +2.38 9.85 _+0.56 18.73 _+1.01 20.77 _+6.23 14.95 _+3.79 34.75 _+4.03

23.2 -+3.34 5.81 -+0.71 21.13 _+4.72 9.01 _+0.42 4.41 _+1.29 4.87 _+1.79 5.56 _+1.52

Cells were incubated for 24 h in the presence of the different fatty acids, washed, and then phospholipids were extracted with chlorform/methanol. After thin layer chromatography, radioactivity in the different fractions was determined using a linear radiochromatography analyser. Results are expressed as %_+ S.D. (n = 5) of total radioactivity incorporated by cells.

38

C. PELA~Y

200"

OLBC ACID C18:1

180"

i

140

120

100' 80

o

10

20

30

Tree (rain) 200 ¸ ARACtilDONIC ACIO

180' i

DISCUSSION

100' 8O 0

I0

TIME (rain)

200"

20

30

UNOLENIC ACID C18:3

180 160 140 '

pattern was observvd in cells labelled with arachidonic, linolenic and oleic acid. By contrast, in myfistic, palmitic, stearic and linoleic acid labelled cells, no significant change in DAG level has been observed. Comparison of the DAG-subspecies induced by CD3, CD2 or PHA (Table 2) indicated that the three activators, especially CD3 and PHA, generate [C20 : 4]-DAG. Only CD2 and PHA generated a substantial amount of [C18:I]-DAG, and PHA appeared to be the sole activator to generate a high amount of [C18 : 3]-DAG.

160' 140' 120'

,~ 0

et al.

120 Pt~

100' 80

w

10 TIME (rain)

0

20

30

FIG. 2. Kinetics of diacylglycerol production in Jurkat T-cells labelled with either [~%-'~]-or [3H]fatty acids then activated with either CD3 mAb, CD2 mAbs or phytohaemagglutinin. Results are expressed as a % relative to unactivated control cells.

acids studied were equally incorporated by PC, PE and PI/PS, with the exception of myristic acid that appeared to be almost unable to label PUPS.

DAG production In cells preincubated for 24 h in the presence of either [3H]- or [14C]fatty acids, activation with CD3 mAbs, a couple of CD2 m_Abs or PHA resulted in a production of DAG that increased with time, peaked at 5 m in then returned to basal values by 30 min (Fig. 2). This

It has been shown that T-cell activation through either the CD2 or the CD3-TCR pathways results in the stimulation of a phosphodiesterase that cleaves specifically PIP2 into two intracellular second messengers, D A G and IP3, the latter being responsible for an increase in intracellular Ca 2+ concentration [5-7]. Accordingly, T-cell activation results in the production of DAG, which was demonstrated by using cells in which the phospholipid pools have been preiabelled with [3H]glycerol [18, 19]. In addition, we have recently demonstrated that during PHA, CD2 and CD3 activation of T-cells, a second source of DAG, independent of the phosphatidylinositol cycle, occurred. This second source of DAG appeared to be generated through the hydrolysis of a pool of phosphatidylcholine arising from the transmethylation of phosphatidylethanolamine [20]. These results strongly suggest that both the antigen dependent CD3-TCR pathway and the alternative, the antigen independent CD2pathway, share the same transduction mechanism. This was supported by the fact that T-cell activation through the CD2 molecules necessitates the presence, on the cell surface, of the CD3-TCR complex, leading to the concept that the CD2 pathway is functionally linked to the CD3-TCR complex in a large subset of T-cells [11, 21, 13]. However, other evidence suggests that the CD2 utilizes a separate pathway. During thymic ontogeny, CD2 expression precedes CD3-TCR expression, and is fully

Diacylglycerolproductionin T-cells

39

TABLE2. DAG PRODUCTIONBYJUKKATT-C~LLS Fatty acid Myristic (C14:0) Palmitic (C16:0) Stearic (C18:0) Oleic (C18:1) Linoleic (C18:2) Linolenic (C18:3) Arachidonic (C20:4)

CD3

CD2

99+5 96+6 97_+13 127-+8" 121_+24 101-+23 198_+22"

98+8 110+27 82-1-15 185-+24" 131+27 96-+5 134_+10~"

PHA 101-1-6 108+17 109-+11 170-+18" 111+25 147_+13~" 172_+37t"

Cells were incubated for 24 h in the presence of the different fatty acids, then washed and activated for 5 min with either PHA, CD3 or a couple of CD2 mAbs. Results are expressed as a % DAG relative to unactivated control cells (taken as 100%). Standard deviation was calculated from three to five independent experiments done in duplicate. *Indicates significantly different from control at P < 0.01 and t at P < 0.05.

functional, since triggering with CD2 mAbs caused Ca e+ mobilization, IL2-receptor expression and T-cell proliferation [14]. In addition, it has been shown that many T-cells and T-cell clones express a functional CD2 molecule in the absence of CD3-TCR expression [14, 15]. Finally, Jurkat CD3-TCR negative mutants could be stimulated through the CD2 molecules [16]. Thus, in certain cases, CD2 functions appear to be independent of the CD3-TCR. To explain these conflicting results it has been proposed that CD2 molecules and the CD3TCR complex are linked in some T-cells, while in others CD2 mediates a CD3-TCR independent pathway. These concepts could be reevaluated in view of the present work. DAG generated by triggering the CD2 pathway appears to be composed of different subspecies than DAG generated through the CD3 pathway. Although [C 18 : 1]-DAG and [C20 : 4]-DAG were common to both the CD2 and the CD3-TCR pathways it appeared that [C18 : 1]-DAG is produced in greater amounts by CD2 mAb than by CD3 mAb. On the contrary, CD3 mAb generated higher amounts of [C20 : 4]-DAG than CD2 mAb. Accordingly, CD2 appears to be able to generate different amounts of DAG subspecies when compared to CD3.

Our data differs from those previously published by Rosoff e t a l . [17]. Indeed, these authors have shown that treatment with CD3 mAb of a different clone of Jurkat cells induces the production of both [C18: I]-DAG and [CIS:0]-DAG, while our results show that CD3 did not induce a significant production of [C 18 : 0]-DAG. The basis of the differences seen with the use of different subclones of Jurkat is not, however, known. Another difference between this and the previous work is the lack of [C18 : I]-DAG and [C18 : 0]-DAG observed in cells treated with CD2 mAbs, whilst in this study we demonstrate that CD2 produced [C18: 1]-DAG. It is possible that the difference may be due to the fact that Rosoff et al. [17] used a single nonactivating CD2 mAb while we used an activating couple of CD2 (GT2+IOTll). On the other hand, our work confirms the work of Pantaleo et al. [7], demonstrating that PHA, CD3 and CD2 mAbs induce [C20 : 4]-DAG in Jurkat T-cells, and agree with that of Hasegawa-Sasaki and Sasaki [21] demonstrating that lectin mitogens induce [C20 : 4]-DAG in rat lymphocytes. It appears from our experiments that PHA is able to activate together both the CD2 and CD3-TCR pathways, since the lectin induces high production of both [CIS:I]-DAG and

40

C. l~J~r~ et al.

[C20: 4]-DAG. In addition, P H A generated [C18 : 3]-DAG. Whether this particular D A G is responsible for IL-2 production through the activation of a particular P K C subspecies is not known but is suggested by the observation that the lectin alone, in contrast to CD3 or CD2 mAbs, is able to induce IL2 synthesis in the absence o f phorbolesters [22]. REFERENCES I. Isakov N., Mally M. I., Scholz W. and Altman A. (1987) Immunol. Rev. 95, 89-111. 2. Linch D. C., Wallace D. L. and O'Flynn K. (1987) lmmunol. Rev. 95, 138-159. 3. Alcover A., Ramarly D., Richardson N. E., Chang H. C. and Reinherz E. L. (1987) lmmunol. Rev. 95, 5-36. 4. Clevers H., Alarcon B., Wileman T. and Terhorst C. (1988) A. Rev. Immunol. 6, 629-662. 5. Imboden J., Weiss A. and Stobo J. (1985) J. lmmunol. 134, 663. 6. Weiss A., Imboden J., Schoback D. and Stobo J. (1984) Proc. natn. Aead. Sci. U.S.A. 81, 4168-4173. 7. Pantaleo G., Olive D., Poggi A., Kosumbo W. J., Moretta L. and Moretta A. (1987) Fur. J. lmmunoi. 1, 55-60. 8. Manger B., Weiss A., Imboden J., Laing T. and Stobo, J. (1987) J. lmmunol. 139, 2755-2760. 9. Berry N., Ase K., Kikkawa U., Kishimoto A. and Nishizuka Y. (1989) J. Immunol. 143, 1407-1413.

10. Bligh E. G. and Dyer W. J. (1959) Can. J. Bioehem. 37, 911-919. 11. Alcover A., Alberini C., Acuto O., Clayton L. K., Transy C., Spagnoli G., Moingeon P., Lopez P. and Reinherz E. L. (1973) E M B O J. 7, 11-18. 12. Bockenstedt L. K., Goldsmith M. A., Dustin M., Olive D., Springer T. A. and Weiss A. (1988) J. ImmunoL 141, 1904-1910. 13. Moingeon P., Chang H. C., Sayre P. H., Clayton L. K., Alcover A., Gardner P. and Reinherz E. L. (1989)ImmunoL Rev. 111, 111-126. 14. Fox D. A., Hussey R. E., Fitzgerald K. A., Bensussan A., Daley J. F., Schlossman S. F. and Reinherz E. L. (1985) J. lmmunoL 134, 330-336. 15. Schmidt R,, Michon J., Woronicz J., Schlossman S., Reinhertz E. L. and Ritz J. (1987) J. clin. Invest. 79, 305-311. 16. Moretta A., Poggi A., Olive D., Bottino C., Fortis C., Pantaleo G. and Moretta L. (1987) Proc. natn. Acad. Sci. U.S.A. 84, 1654-1658. 17. Rosoff P. M., Savage N. and Dinarello C. A. (1988) Cell 54, 73-80. 18. Bismuth G., Theodorou I., Gouy H., LeGouvello S., Bernard A. and Debre P. (1988) Fur. J. ImmunoL 18, 1351-1357. 19. Hasegawa-Sasaki H. and Sasaki T. (1983) Biochim. biophys. Acta 754, 305-312. 20. Aussel C., Pelassy C. and Rossi B. (1990) J. Lipid Mediators 2, 103-116. 21. Hasegawa-Sasaki H. and Sasaki T. (1982) 3.. Bioehem. 91, 463-467. 22. Aussel C., Mary D., Peyron J. F., Pelassy C., Ferrua B. and Fehlmann M. (1988) J. ImmunoL 140, 215-220.