Inhibition of the CD3-mediated calcium signal by protein kinase C activators in human T (Jurkat) lymphoblastoid cells

Immunology Letters, 20 (1989) 47-52

Elsevier IML 01150

Inhibition of the CD3-mediated calcium signal by protein kinase C activators in human T (Jurkat) lymphoblastoid cells A. Tordai, B. S a r k a d i , Gy. G 6 r 6 g a n d G. Ggtrdos National Institute of Haematology and Blood Transfusion, Budapest, Hungary

(Received 29 August 1988; accepted 9 October 1988)

1. Summary

2. Introduction

The changes in the cytoplasmic free calcium concentrations (Cai) were investigated in h u m a n T (Jurkat) lymphoblastoid cells, loaded with the calciumsensitive fluorescent dye Indo-1. A rapid increase in Ca i could be evoked by monoclonal antibodies (mAb) directed against the CD3 antigen, as well as by the mitogenic lectin concanavalin A (conA), The protein kinase C (pKC) activators TPA and DiC8 did not increase Cai, but the addition of any of these two compounds prior to m A b eliminated the mAbinduced calcium signal. In contrast, the calcium signal evoked by C o n A was not blocked by TPA or DiC8. These results suggest that the CD3-dependent calcium signal is selectively down-regulated by the activation o f pKC.

The possible intracellular mechanisms of signal transduction involved in T-lymphocyte activation has been extensively investigated (for reviews see [1, 2, 3]). It is now well established that following the binding of the antigen to the T-cell receptor (TCR), phosphoinositide breakdown occurs which results in significantly higher levels of cellular IP 3 and DAG [4]. IP 3 is known to mobilize Ca ++ from internal stores which causes rapid elevation of Ca i [5]. The requirement for the elevated Ca i in T cell activation is strongly supported by the findings that this activation is inhibited in Ca + +-free media [6, 7] and the effect of T cell activators can be, at least partially, mimicked by calcium ionophores, e.g. by A23187 [8, 9]. The CD3 protein complex appears to be closely linked to the antigen recognizing receptor of T lymphocytes (TCR), and involved in the transduction of the calcium signal induced by various antigens [1, 2]. It has been shown that several monoclonal antibodies directed against the T cell receptor or the CD3 antigen are able to induce T lymphocyte activation and produce a rapid increase in cellular calcium levels [10-13]. The other product liberated during cell stimulation from the phosphoinositides, DAG, is known to activate protein kinase C [14]. The functional consequences of pKC activation are not well established as yet but some of these are mediated by the phosphorylation of various membrane proteins, e.g. receptors (see [14, 15]). In the case of T lymphocytes, the pKC dependent phosphorylation of the 7subunit of the CD3 protein has been reported and a consequent down-regulation of the TCR-CD3

Key words: Calcium signal; T cells; Jurkat; Protein kinase C;

Phorbol ester; CD3 antigen Correspondence to: Prof. G. G~irdos,National Institute of Haematology and Blood Transfusion, 1113Budapest, Dar6czi u. 24, Hungary. Abbreviations: aCD3, monoclonal antibody against CD3 anti-

gen; aTCR, monoclonal antibody against the T-cellreceptor; Cai, cytoplasmic free Ca + + concentration; ConA, concanavalin A; DAG, diacyl-glycerol;DiC8, 1,2-dioctanoylglycerol; IL-2, Interleukin 2; IP3, inositol-trisphosphate; iTPA, phorbol 12,13dibutyrate; mAb, monoclonal antibody; pKC, protein kinase C; TCR, T-cell receptor; TPA, phorbol 12-myristate 13-acetate (PMA).

0165-2478 / 89 / $ 3.50 © 1989 Elsevier Science Publishers BN. (Biomedical Division)

47

complex from the cell surface also demonstrated [16, 1, 17]. On the other hand, lymphokine production in Jurkat cells requires the synergistic effects of pKC activation and the induction o f a calcium signal by lectins, mAbs, or calcium ionophores, respectively

[181. In the present work we intended to examine the effects of pKC activation on the CD3- (and TCR-)mediated generation o f the calcium signal in T cells. Therefore we studied the effects o f the PKC activating phorbol ester (TPA) and a synthetic diacyl glycerol (DiCS) in a well established T lymphoblastoid cell line (Jurkat), when the calcium signal was triggered by anti-CD3 mAbs, by mAbs directed against the TCR, or by the mitogenic lectin, ConA. In these experiments we found a direct and selective down-regulation o f the CD3- (and TCR)-mediated calcium signal by the activation of pKC. 3. Materials and methods

3.1. Chemicals and reagents The mitogenic lectin concanavalin A (ConA) and the Indo-1 acetoxymethylester were purchased from Serva, phorbol 12-myristate 13-acetate (TPA), phorbol 12,13-dibutyrate (iTPA) and 1,2-dioctanoyl glycerol (DiC8) were from Sigma. Functionally active monoclonal antibodies against the CD3 antigen and TCR were obtained from OKT-3 hybridoma cells and from the panels o f the Third International Workshop on H u m a n Leukocyte Differentiation Antigens, as used in [19]. 3.2. Cells The h u m a n leukemic T cell line, Jurkat was maintained under standard conditions in R P M I 1640 solution supplemented with 10070 fetal calf serum. 3.3. Cell loading and fluorescence measurements Cells were loaded during incubation with 1 /zM Indo-I acetoxymethylester in R P M I 1640 solution supplemented with 10°70 fetal calf serum for 1 h at 37 °C. The cells were then centrifuged with 1000xg for 10 min and resuspended in R P M I 1640. Before each measurement, 1 × 10 6 cells were sedimented by a rapid centrifugation (10 sec at 10000xg) in an Ep48

pendorf microfuge and the pellet was rinsed three times with H P M I (containing 100 m M NaCI, 5.4 m M KC1, 0.4 m M MgC12, 0.04 mM CaC12, 10 m M Hepes, 20 m M glucose, 24 m M N a H C O 3, and 5 m M Na2HPO4 at p H 7.1). The cells were resuspended in H P M I and the fluorescence measurements were carried out in quartz cuvettes containing 2 ml suspension of 1 z 106 cells at 37 °C, with continuous stirring, in a Hitachi F-4000 fluorescence spectrophotometer. The excitation and emission wavelengths were 331 and 410 nm, respectively. 3.4. Calibration and calculation o f Ca i levels Calibration was carried out after each measurement by using 12.5 m M digitonin as detergent in the presence of 0.5 m M Ca ++ for obtaining the maxim u m fluorescence, and with 1.3 m M EGTA at p H 8 (adjusted with Tris), respectively, for measuring minimum fluorescence. The intracellular Ca ++ concentration values were calculated by the equation [20]: Cai = KD Indo - 1 × [F-Fo/(Fma x -F)], where F = actual fluorescence; F 0 = minimum fluorescence; Fmax = m a x i m u m fluorescence, and KD I n d o - 1 = 250 nM. 4. Results

The resting Ca i values in Jurkat cells as found in our experiments were between 120 and 180 nM and the addition of EGTA to the medium only slightly lowered Ca i. These values proved to be stable within 20 min. Leakage of the hydrolyzed dye I n d o - 1 was negligible within the measurement periods of 20 min. Figure 1 shows the effect of the pKC activator phorbol ester, TPA, on the calcium signal evoked by an aCD3 mAb, OKT-3. In the control experiments (Fig. 1A and B) a CD3 induced a rapid rise in Ca i which peaked within I min. At this peak of the calcium signal, Ca i was about three times greater (reaching 3 0 0 - 5 0 0 nM) than the resting values. The initial peaks were similar, but the curves then were different if Ca + + was present or absent in the incubation media. When 0.5 m M Ca + + was present, Ca i remained on a level significantly greater ( 3 0 0 - 4 0 0 nM) than the starting value (Fig. 1A). This increased calcium

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Fig. l. Effect of TPA on the calcium signal evoked by the anti-CD3 mAb, OKT-3,in Jurkat cells. Fluorescence measurements with Indo - 1 loaded cells were carried out as described in section 3. The reagents indicated on the fig. were added in the following final concentrations: EGTA: 0.5 raM; Ca: 1 mM CaCI2; eth: 0.25% ethanol; TPA: 6.25 riM; aCD3:OKT-3 hybridoma ascites in 4×10 -4 dilution; ConA: 100 #g/ml. The traces are representative of at least five similar experiments with different Jurkat cell preparations.

level proved to be relatively stable within 20 min. In contrast, when Ca + ÷ was absent f r o m the medium, after the m A b - i n d u c e d peak the curve rapidly returned to the basal or even to a lower level (Fig. 1B). The following addition o f C a + ÷, exceeding the E G T A concentration in the m e d i u m by 0.5 m M , caused again a rapid Ca i elevation, reaching about ten times the values o f the starting level. Panels C and D in Fig. 1 show the effect o f T P A u n d e r the same experimental conditions. TPA was a d d e d to the m e d i u m prior to a C D 3 by two minutes and it slightly lowered C%. U n d e r these conditions the O K T 3 m A b could not evoke any calcium signal. As shown in Fig. 1, the absence or presence o f external Ca ÷ ÷ did not cause any difference in the inhibitory effect o f TPA. The effects o f several other aCD3

mAbs, distributed by the Third International Workshop o f H u m a n Leukocyte Differentiation Antigens and functionally characterized as mitogenic activators in [19], were also examined. These m A b s (Nos. 127,472, and 490) p r o d u c e d a similar calcium signal in Jurkat as OKT-3, and these calcium signals were also f o u n d to be eliminated by the addition o f TPA (data not shown here). Nevertheless, in the TPAtreated cells the addition o f the mitogenic lectin, C o n A , induced a calcium signal similar to that observed in the control experiments (the m a g n i t u d e o f this C o n A signal was slightly smaller and the timecourse o f its development somewhat delayed as compared to that seen in the absence o f TPA - not shown). 49

Ca++1 Ai

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Fig. 2. Effect of DiC8 on the calcium signal evoked by the antiCD3 mAb, OKT-3, in Jurkat cells. For the measurements and the concentrations of the reagents added see the legend to Fig. 1. The final concentration of DiC8 was 1.25 /~M.

Fig. 3. Effect of iTPA on the calcium signal evoked by the antiCD3 mAb, OKT-3, in Jurkat cells. For the measurements and the concentrations of the reagents added see the legend to Fig. 1. The final concentration of iTPA was 12.5 nM.

Figure 2 shows a similar experiment to that in Fig. 1C, with another pKC activator, the synthetic diacyl glycerol, DiC8. This compound also proved to be effective in eliminating the aCD3-signal, although in a considerably greater concentration than TPA (corresponding to the concentration requirements for the activation o f pKC - see [21]). DiC8 slightly lowered the magnitude o f the signal evoked by ConA but did not eliminate the lectin-induced signal even in significantly greater concentrations. Figure 3 shows the effect o f phorbol 12,13dibutyrate (iTPA) on the development o f calcium signal in Jurkat cells. This compound in similar concentrations is much less effective in activating pKC than TPA, although its chemical structure is similar to the latter [22, 23]. The curve we obtained using 2 - 20 nM iTPA was nearly identical to that observed in the control experiments (see Fig. 1A). The effect of iTPA was also studied in Ca + ÷-free media but no inhibition o f the calcium signal was observed here either.

fluorescent calcium indicator I n d o - 1, which has no significant cellular calcium buffering effect in the concentrations applied (see [20, 24, 25]), allowed us to follow the dynamics of the changes of the intracellular Ca ++ concentrations in intact Jurkat lymphoblastoid cells. In agreement with previous reports in the literature [11-13, 26, 27], our results also show that a marked and rapid calcium signal can be evoked by several mAbs, including OKT-3, raised against the CD3 antigen of T cells, or by the mitogenic lectin, ConA. The finding that such a signal can be observed also in the absence of external Ca + + shows that the sources of the primary rapid elevation are intracellular calcium pools. The persisting increase in Ca i when the cells are stimulated in the presence of external calcium and the rapid secondary calcium influx, when external calcium concentration is increased after stimulation in a calcium-free medium, indicate the opening of plasma-membrane calcium channels as well [27, 28]. Experiments carried out with a mAb directed against the antigen-recognizing receptor of Tlymphocytes (aTCR mAb, No. 480 of Ref. [19]), showed similar results to those presented in Fig. 1. Thus, we observed a similar calcium signal evoked

5. Discussion

The use o f the recently developed, highly sensitive 50

by aTCR, and this could be inhibited by incubation o f the Jurkat cells with the TPA concentrations used in the experiments in Fig. 1 (data not shown), The results presented above demonstrate that the calcium signal evoked by aCD3 or aTCR mAbs can be rapidly inhibited by the direct activation of pKC by phorbol ester or diacyl glycerol. This is in line with earlier results in the literature, reporting the phosphorylation and a consequent down-regulation of the CD3-TCR complex from the cell surface following the stimulation of T lymphocytes or by the activation o f p K C [16, 17, 29]. This has been suggested to be a feed-back down-regulation mechanism that might prevent the transduction of consecutive activating signals in lymphocytes [15, 29] and may be a general way o f regulating transmembrane signaling [30]. The rapid loss o f the calcium signal observed by us may occur because the phosphorylated CD3 antigen immediately loses its aCD3 binding capacity (this is unlikely in the light of the data of Cantrell et al. - see [16, 29]), or by the inability of the CD3-TCR complex to transfer the activation towards other elements of the signal transduction pathway. Our results are supported by the finding [31] that TPA pretreatment of T lymphocytes suppressed the stimulus-induced phosphatidylinositol response. In our experiments we found that in the TPA- or DiC8-treated cells the addition o f the calcium ionophore ionomycin, similarly to that in the control cells, induced a rapid rise in Ca i even in the absence of external Ca + + (data not shown). Moreover, the calcium signal response to C o n A (which could be selectively blocked by the addition o f a-methyl-Dmannoside) was preserved. Thus m a j o r changes in the calcium stores or alterations of various calcium transport mechanisms, independent o f the TCRCD3 pathway, are unlikely to occur in the TPA or DiC8 treated cells. The finding that the Ca i increase triggered by aCD3 or aTCR mAbs could be blocked by TPA or DiC8, while the ConA-induced calcium signal was relatively insensitive to these agents, deserves some attention. Although C o n A is also supposed to act mainly through the TCR-CD3 complex, the lectin may have several other cell-surface binding sites (see [2, 4]) and some of these may transfer the activation signal insensitively to the action of pKC. A TPA-insensitive development o f the calcium signal in Jurkat cells stimulated by the C305 IgM

m A b (directed against the antigen-receptor complex) was reported by I m b o d e n and Stobo [11]. In contrast to the inhibitory effects of TPA on the surface expression of the TCR-CD3 complex and on the development of the calcium signal (as shown above), it has been suggested that activators of pKC have synergistic actions with the calcium signal generating agents during T cell activation [1, 15, 32, 33]. In Jurkat cells, when added simultaneously, TPA was synergistic with aCD3 or C o n A in inducing interferon and IL-2 production [18]. We observed that in the case of a simultaneous addition o f TPA and aCD3 the magnitude of the calcium signal evoked by aCD3 only slightly decreased (data not shown). This suggests that the effect of pKC on the inhibition of calcium signal requires 1 0 - 3 0 sec to develop at 37 °C. Therefore, in order to further clarify the functional regulatory role of pKC, it would be important to examine the effect of TPA added prior to, simultaneously with, or after aCD3 on the changes in lymphokine production and on the development of the calcium signal. These experiments are currently under way in our laboratory.

Acknowledgements The skilful and precise technical assistance of Ms. M. Sarkadi, M. Feh6r and E. Bitai is gratefully acknowledged. We also wish to thank Dr. M. Benczur for the consultations during the experiments. This work has been supported by a grant of O K K F T Tt. 1.5.1.3. of the Hungarian Academy of Sciences.

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