Eur. J. Immunol. 1990. 20: 605-610

M. Angeles Muiioz-Fernhndez., Felipe X. Pimentel-Muiiios., Miguel A. Alonso., Miguel Campaneroo, Francisco Shnchez-Madrid0, August0 SilvaA, Jose Luis AlonsoV and Manuel Fresno.

Centro de Biologia Molecular., CSIC/Universidad Aut6noma de Madrid, Servicio de Inmunologiao, Hospital de la Princesa, Centro de Investigaciones Bio16gicasA, CSIC and Laboratorios Andr6maco S.A.V, Madrid

Tcell activation by TNF and PKC activators

605

Synergy of tumor necrosis factor with protein kinase C activators on T cell activation* The ability of tumor necrosis factor (TNF)-a to activate T lymphocytes in combination with other stimuli has been studied. TNF was strongly co-mitogenic with low doses of anti-CD3 antibodies or phorbol esters (those which are strong activators of protein kinase C, PKC) but poorly with phytohemagglutinin or concanavalin A. No synergism was seen with the calcium ionophore A23187.TNF was co-mitogenic with several phorbol esters known to activate PKC but was uneffective with inactive phorbol esters such as methyl-phorbol 12-myristate 13-acetate. Furthermore, H-7 a known inhibitor of PKC, inhibited the proliferative response of Tcells induced by esters plusTNF. This effect took place at low doses of TNF and was also observed with purified T lymphocytes indicating that the effect of TNF was not dependent on accessory cells.This proliferativeeffect of TNF was inhibited by an anti-interleukin 2 receptor (IL2R) antibody, MAR 108, which blocks IL 2 binding to its receptor. Although PKC activation induced CD25 (IL2R) expression but very little IL2 synthesis, TNF did not synergize by augmenting the synthesis of this lymphokine in peripheral blood lymphocytes stimulated with phorbol esters. By contrast, TNF strongly increased the membrane level of CD25 and to a lesser extent that of the activation antigen, 4F2, over the levels already induced by phorbol esters on T cells. More interestingly, TNF significantly increased the number of high-affinity IL 2R on purified Tcells in the presence of phorbol 12J3-dibutyrate. Our results indicate that TNF is co-mitogenic with those stimuli which strongly activate PKC and suggest that TNF may play a role onTcell activation increasing the number of effective IL 2/IL 2R interactions when these are limiting.

1 Introduction

[14-161. Resting T lymphocytes do not express TNF receptors (TNF-R). However, activation with a variety of TNF, which was initially described as a cytokine cytotoxic stimuli including anti-CD3 antibodies induces the appeafor certain tumor cells [l], has been recently shown to exert rance of TNF-R on resting T lymphocytes [17]. TNF-a pleiotropic effects on several normal cell lines. Thus, TNF modulates the proliferation of mature Tcells by enhancing was able to stimulate fibroblast growth [2] and to enhance antigen and mitogen-induced human T lymphocyte proliMHC cell surface expression either directly or through feration [17-191 .This effect occurs at submitogenicdoses of augmentation of the IFN-y-inducingactivity [3,4]. Recent- several stimuli and it was thought to be mediated through ly, there has been increasing evidence of the immunoregu- an enhancement of IL2R (Tac) expression on the cell latory role of this cytokine.TNF stimulates the cytotoxicity surface [20,21].TNF-a has also been shown to play a role in of several immune effector cells, such as eosinophils [5] and the development of MLR [22] and T cell cytotoxicity monocytes [6], and enhances the antibody-dependent ~ 3 1 . cytotoxicity [7], superoxide production [8] and degranulation of neutrophils [9]. Furthermore, it activates natural The effect of TNF on T cell activation has been generally cytotoxicity [lo] and stimulates B lymphocyte proliferation difficult to interpret because T cell responses are usually dependent on the participation of accessory cells.We have and differentiation [ll]. examined the ability of TNF to induceTcell proliferation in Recently, it has been shown that TNF shares with IL 1a set combination with several activators of PKC; such a system of activities on T cells. Thus, TNF and IL1 enhance avoids the requirement for accessory cells [24]. We have thymocyte proliferation [12, 131. The binding of TNF to found that TNF synergizes with active phorbol esters and specific cell surface receptors appears to be necessary for other known activators of PKC. This activation is probably mediating the multiple effects of this cytokine. High- mediated through the IL2AL2R pathway since TNF affinity receptors have been identified on a variety of cells increases the number and affinity of IL2R on activated T cells; this may allow a more efficient interaction with the low doses of IL2 produced in the presence of phorbol [I 78701 esters.

*

Supported by grants from DGICYT, Fundaci6n Ram611 Areces and CDTILaboratorios Andr6maco S.A.

Correspondence: Manuel Fresno, Centro de Biologia Molecular, Universidad Aut6noma de Madrid, Canto Blanco, E-28049 Madrid, Spain Abbreviation: TfR: Transfemn receptor 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

2 Materials and methods 2.1 Reagents and mAb mAb were used as hybridoma culture SN. mAb specific for the transferrin receptor (TfR; FG2/12), 4F2 (FG1/8) and 0014-2980/90/0303-0$02.50/0

606

Eur. J. Immunol. 1990. 20: 605-610

M. A, Muiioz-Fernlndez, E X. Pimentel-Muitios, M. A. Alonso et al.

CD4 (HP26) from our laboratory have been reported elsewhere [25, 261. W6/32 antibody recognizes a monomorphic determinant of the HLA class I molecule. The anti-CD3 mAb, Leu-4, was purchased from Becton Dickinson (Mountain View, CA). The anti-IL2R (CD25) mAb (MAR 10) and the D3/9 mAb directed to the human T200 (CD45) have been previously described [27,28]. Purified antibodies were obtained from mouse ascites fluid by affinity chromatography on protein A-Sepharose.

and/or TNF-a or anti-CD3 mAb. After 24 h, cell cultures were harvested and the expression of IL2R (CD25),TfR, CD3 and 4F2 was analyzed by immunofluorescence FCM. This analysis was performed on an Epics-C cytolfuorometer (Coulter Scientific, Harpenden, GB). Cells were incubated with hybridoma culture SN, followed by washing and labeling with FITC-labeled goat anti-mouse Ig. 2.6 Binding assay for 1251-labeledIL2 ([1251]IL2)

PMA, PBu2, methyl-PMA (Me-PMA), 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine(H-7), cycloheximide, actinomycin D, mezerein and the Ca2+ionophore A23187 were purchased from Sigma (St. Louis, MO). PHA was from Difco (Detroit, MI). Recombinant TNF-a was a generous gift of Laboratorios Andr6maco S.A., Madrid, Spain. 2.2 Cells PBL were obtained from heparinized venous blood of normal volunteers by Ficoll Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) centrifugation. T cells were purified from PBMC by removal of adherent cells on plastic petri dishes followed by passage through a nylon wool column. Phenotypic analysis of purified T cells by FCM showed that the Tcell population contained > 98% CD3+; c 1% CDllb+ and < 1% CD20+ cells. 2.3 Proliferation assays PBL or purified T cells (1 x lOVwe11) were activated with phorbol esters (Sigma) at different concentrations as indicated, soluble anti-CD3 mAb, anti-CD3 mAb (1 pg/ml) coupled to Sepharose beads, PHA or Con A at different concentrations, A23187 (1 pM),TNF-a (1-lo00 U/ml), or a combination of several of them. Proliferation assays were camed out in triplicate cultures in 96-well U-bottom microtiter plates containing 2% FC S and 5 x lov5M 2-ME in complete DMEM. The cultures were maintained in humidified atmosphere containing 5% COz for 72 h. Cell proliferation was estimated by [3H]dThd (New England Nuclear, Boston, MA) incorporation during the last 16 h of culture. Cells were harvested and the radioactivity measured in a liquid scintillation counter. 2.4 IL2 production assay PBL (106 cells/ml) in DMEM containing 2% FCS and 5X M 2-ME were cultured in 96-well microtiter plates. The cells were activated with different concentrations of TNF-a or purified anti-CD3 mAb either in the absence or in the presence PBu2 (20 ng/ml). After 24 h, culture SN were assayed for IL 2 activity using the mouse Tcell line CTLL-2 as described [29]. The IL2 concentration of each sample was referred to a standard preparation (NIH, Bethesda, MD) and expressed in U/ml.

The [1251]IL2-bindingassay was based on the method of Robb et al. [30],with the modifications described elsewhere [31]. Aliquots of cells (1 x lo6) were incubated at 4°C in 200 pl of PBS, 1% BSA and serial dilutions of [1251]IL2. After 60 min the cell-bound and free radioactivity were separated by centrifugation by measuring the radioactivity not inhibited by excess unlabeled IL 2. The average number and affinity of IL2R were estimated by Scatchard plot analysis using an EBDA computer program.

3 Results 3.1 Synergism of TNF-awith PKC activators in T cell proliferation The ability of TNF-a to provide inductive signals on Tcell activation was initially tested by submitogenic doses of other known stimuli of human T cell activation. In agreement with previous results low doses of TNF-a synergize with soluble anti-CD3 antibodies [19]. In addition, TNF-a synergizes weakly with Con A and PHA. The strongest co-mitogenic effect was observed with low doses of phorbol esters. No significant synergism was seen with the calcium ionophore (Table 1).The co-mitogenic effect of TNF with immobilized anti-CD3 or PBu2 but not with IL2 or soluble anti-CD3 antibodies was also observed with purified T lymphocytes (Table 2).Thus, it appeared that the effect ofTNF was not dependent on accessory cells. Since TNF-a was co-mitogenic with phorbol ester and immobilized anti-CD3 antibodies, known activators of PKC [24,32] but not with PHA or calcium ionophores, the co-mitogenic effect of TNF-a was analyzed in more detail. TNF-a at 10 U/ml was synergistic with active phorbol esters Table 1. Effect of TNF on Tcell activation

Stimulus

None PBu2 PHA PHA ConA AntLCD3 A23187

Concentration 5 ng/ml 0.5 &ml 2 pg/ml 2 pg/ml 2 pg/ml 1 pM

[3H]dThd incorporateda) without TNF with TNF 1625f 131 8802+ 802 35 473 f 2141 61578+5840 30526f2405 28 526 1208 2841 f 251

+

1843+ 204 43412+2981 50 250 f 3821 54511f3987 49581f4027 58 471 f 4036 3027f 347

2.5 Analysis of cell surface expression PBL were incubated as above in plastic plates (24 wells, Costar, Cambridge, MA) in the presence of PBu2 (5 ng/ml)

a) PBL were stimulated for 72 h with the indicated stimulus in the presence or not of TNF-a (100 Ulml). Results shown are the mean cpm of triplicate cultures SD.

+

Eur. J. Immunol. 1990. 20: 605-610

Tcell activation by TNF and PKC activators

607

Table 2. Effect of TNF on the proliferation of purified T cells

Stimulush)

(3H]dThd incorporated*) without TNF with TNF)

None PBu~ PBuz anti-CD3 PBu~ + IL2 IL 2 Anti-CD3 Anti-CD3 (immobilized)

5 189 f 287 13630f 987 36 263 t 2453 39 892 f 3307 6694 f 407 5434 f 1208 10513 t 982

+

5065 f 389 35436f2407 56 084 f 4.033 44591 t 2831 5561 f 347 6587 f 689 30487 f 2405

a) Proliferation was measured 72 h after the initiation of the cultures. Results shown are the mean cpm f SD of triplicate cultures. b) Purified T cells were stimulated with PBu2 (10 pglml), IL2 (10 Ulml), anti-CD3 (0.2 pg/ml) soluble or immobilized on Sepharose beads, alone or in combination as indicated. c) 10Ulml.

I'

5

1

25

PBu, (ng/ml) Figurel. Effect of different concentrations of PBu2 in the comitogenic activity with TNF-a. PBL were activated with different doses of PBu2 in the absence (W) or in the presence of 100 U/ml TNF-a (A).

able to stimulate PKC such as PBu2 or PMA but not with the inactive analogue methyl-PMA. Merezein, a phorbolrelated ester activator of PKC, was also co-mitogenic with TNF. Furthermore, H-7, a known inhibitor of PKC, inhibited the proliferative response (Table 3).The functional effect of TNF-a was clearly seen at submitogenic doses of PBu2 but not at higher doses which are mitogenic by themselves (Fig. 1).O n the other hand,TNF was able to be co-mitogenic at very low doses (1 U/ml) reaching saturation at 100 U/ml.The addition of I L 2 at 20 U/ml to the phorbol ester-stimulated Tcells induced cell proliferation and under those conditions no co-mitogenic effect of TNF-awas seen (Fig. 2). It is well established that calcium ionophores synergize with phorbol esters in triggeringTcel1 proliferation [33], leading to the concept that an increase in free intracellular Ca2+and activation of PKC regulateTcel1 activation [34]. SinceTNF was co-mitogenic with PKC activation, we tested the effect of TNF on intracellular Ca2+levels. However, we could not detect any increase in intracellular free Ca2+ (not shown). Since TNF-R are known to be induced in T cells after activation [17], we investigated the kinetics of T cell proliferation. No significative differences were observed in the time course of the proliferative response induced with

-

i

1

10

100

1000

r T N F (WmO

Figure 2. Dose-response curve of the effect of TNF-a on T cell activation. PBL were activated with PBu2 5 nglml (W), PMA 2 nglml ( 0 )or PBuz 5 nglml plus 20 Ulml IL 2 (A)in the presence of different concentrations of TNF-a.

Table 3. Co-mitogenic effect of TNF with PKC activators Stimulus added None PBu~ PBu~ PMA Met hyl-PMA P B u ~ H-7 Merezein

+

Concentration (ndml) 5

10 10 20 10 5

[3H]dThd incorporateda) with TNF without TNF

1430+ 84 3913 f 121 5241 f 581 11758 f 1043 2303f 201 1480f 103 5 840 k 387

28635 243 20 2% f 2002 25 633 f 1943 38 255 k 2248 3130f 303 3141f 302 28431 f 1943

a) T cells were stimulated for 72 h with the indicated phorbol esters in the presence or the absence of TNF-a(100 Ulml). PKC inhibitor H-7 was added at 25 p~ and incubated with the cells for 30 min before the addition of PBu2. A t that concentration H-7 was not toxic for the cells. Results shown are the mean cpm of triplicate cultures SD.

*

Eur. J. Immunol. 1990.20:605-610

M. A. Muiioz-FernAndez, F. X.Pimentel-Muiiios, M. A. Alonso et al.

608

CD3

CD25

TfR

4F2

16 -

r

TNF

14-

5 2 12-

peu,

U a L

0

& l0-

PBU,

a L

t

0

TNF

5 8 -

c 0

-c

PBU,

u 6-

n

I

t

T3

I %.

4 FLUORESCENCE IA.U.1

2 -

Figure 4. Cell surface expression of CD3, IL2R (CD25),TfR and 2

3

4

5

4F2 on activated PBL. PBL were stimulated withTNF (100 Ulml), PBuz (5 ng/ml), PBu2 (5 ng/ml) plus TNF (100 Ulml) or PBu2

6

DAYS

Figure 3. Kinetics of TNF-induced T cell proliferation. Purified T cells were stimulated with 5 nglml PMA plus 0.5 pglml anti-CD3 mAb ( O ) , 5 ng/ml PMA plus lo00 U/ml TNF (W), 5 ng/ml PMA (A) or anti-CDJ mAb alone (0).

PMA plus anti-CD3 or plus TNF. Maximum incorporation was observed at day 3 and thereafter quickly declined (Fig. 3).

3.2 Involvement of the ILWIL2R pathway in the proliferative effect of TNF The T cell proliferation triggered by phorbol esters and TNF was inhibited by an anti-IL2R antibody, MAR 108, which blocks IL 2 binding [27].This inhibition was stronger than the one obtained upon proliferation to PHA (Table 4). PBuz is known to induce IL 2R expression rather than IL 2 synthesis, and so far most of the stimuli co-mitogenic with it, such as anti-CD3 antibodies or calcium ionophores, are known to induce IL2 synthesis.Therefore, we thought that TNF might induce IL 2 synthesis. However, TNF failed to significantly increase the amount of IL 2 already produced by PBu2 alone in the SN of PBL after 3 days of activation with phorbol esters, in contrast with the increase observed with anti-cD3 antibodies (Table 5).

(5 nglml) plus anti-CD3 mAb (2 pg/ml). Cell surface expression was studied by immunofluorescence FCM before (broken line) or 24 h (continuous line) after activation.The value of the maximum fluorescence with an irrelevant mAb used as negative control is marked with a dash on the abscissa.

By contrast ,TNF strongly increases the level of Tac (CD25) expression on the surface of Tcells over the levels already induced by PBu2. TNF also augmented the expression of the activation antigen 4F2 and t o a lower extent that of the TfR (Fig. 4).PKC activation down-regulated CD3 expression. Interestingly, addition of TNF was able to downregulate CD3 faster than P B q alone. More interestingly, when purified T lymphocytes were incubated in the presence of PBu2 plus"?, a significant increase over PBu2 alone, in the number of high-affinity IL2R, was detected (Table 6).

4 Discussion TNF-a is one of the most pleiotropic lymphokines described so far [35]. Recently, it has been shown that TNF-a has an overlapping set of activities with I L 1 in T cell activation [12, 31. Thus, it has been shown that TNF-a enhances anti-CD3- [191 or alloantigen-induced T cell proliferation [18].We show here that TNF-a is synergistic with other stimuli of T cell activation, namely PKC activators. Activation of PKC is likely to be required for the

Table 4. Inhibition by anti-IL2R on PBuz plus TNF-induced T cell proliferation

A&tiOnS.)

rH]dThd incorporatedb) Control + anti-II. 2R

None mu2

pBo2 mu2

PBUZ PBUZ pBu2 PHA

5 ndml mnghnl 5@+TNF lOU/ml 5 nglml+ TNF 100 U/ml 2Onghnl+TNF lOU/ml 20 nghnl+ TNF 100 U/ml

2 Pdml

1937f 181 6593 f 543 24218f2303 11113f1085 33222 f3114 61389f4827 46476 & 3251 40424 f4003

1966f 135

1352+ 128 2845f 243 1355+ 148 2%2 f 251 4053f 408 3842f 395 16986 f 1305

a) PBL were stimulated with the indicated stimuli. b) [3H]dThd incorporation was measured in the presence or the absence of 10 pglml purified MAR108 anti-IL2R mAb after 72 h of incubation. Results shown are the mean cpm ? SD of triplicate cultures.

Eur. J. Immunol. 1990. 20: 60-610

Tcell activation by TNF and PKC activators

Table 5. Failure of TNF to significantly increase IL2 production by PBL

None

TNF (10Uld) TNF (loo Uhnl) Anti-CD3 (2 @d)