Immunology 1990 69 110-116
Synergism between AS101 and PMA in lymphokine production B. SREDNI, Y. KALECHMAN, F. SHALIT & M. ALBECK Bar Ilan University, Ramat Gan, Israel
Acceptedfor publication II September 1989
SUMMARY ASIOI [ammonium trichloro (dioxyethylene-o-o') tellurate] has been reported to stimulate normal mouse and human lymphoid cells to proliferate and to produce lymphokines such as interleukin-2 (IL-2) and colony-stimulating factor (CSF), regulators of lymphopoiesis and myelopoiesis. In this study, we demonstrate that the IL-2 secretion and cell proliferation of both human and mouse lymphoctyes, and the production of CSF by mouse spleen cells, was significantly enhanced by the synergistic effect of ASIO1 and phorbol myristate acetate (PMA). ASIOI-induced activation was found to be very sensitive to inhibition by EGTA, the Ca2+ channel blocker, nifedipine, and cyclosporin A (CsA), an agent which selectively suppresses Ca2+-activated steps in this process. Our results suggest that ASIOI may efficiently trigger the Ca2+ signal required to initiate lymphocyte activation, but that the enhancement observed when cells are stimulated with both AS 101 and PMA may be due to the generation of a second signal, probably the activation ofprotein kinase C (PKC). A more thorough understanding of the mechanism of action of the immunomodulator AS101, presently under clinical trials on cancer and AIDS patients, is highly relevant to the assessment of its optimal application.
INTRODUCTION A new synthetic compound, ammonium tricholoro (dioxyethylene-o-o') tellurate (AS 101) has been synthesized in our laboratory and has been shown to have immunomodulating properties and minimal toxicity (Sredni et al., 1987). AS101 was found to induce proliferation and IL-2 production by human lymphocytes in vitro, and to enhance the production of IL-2 and colonystimulating factor (CSF) by mouse spleen cells. AS101 also enhanced the ratio of OKT4 to OKT8 positive cells in cultured mononuclear cells from patients with acquired immune deficiency syndrome (AIDS). In vivo, splenocytes from infected mice exhibited increased IL-2 and CSF production as well as high levels of IL-2 receptors (Sredni et al., 1987). The drug also mediated an anti-tumour effect in mice. Phase I clinical trials, currently in progress on cancer and AIDS patients, show a general enhancement of immunological parameters similar to results described above in vitro and in animal models (Sredni et al., 1988). We proposed earlier that the mechanism by which ASIOI stimulates IL-2 secretion involves transmembrane calcium flux (Sredni et al., 1987). This was based upon the rapid increase in intracellular Ca2+ noted using the quin-2 acetoxy methyl ester Abbreviations: CsA, cyclosporin A; CSF, colony-stimulating factor; DAG, diacylglycerol; IL-2, interleukin-2; InsP3, inositol triphosphate; MNC, mononuclear cells; PKC, protein kinase C; PMA, phorbol myristate acetate. Correspondence: Professor B. Sredni, Dept. of Life Sciences, Bar Ilan University, Ramat Gan 52100, Israel.
assay (Gelfand et al., 1986) when mouse splenocytes were incubated with ASI l. It appears that the interaction of ASI01 with the cell membrane triggers the Ca2+ signal that is believed to play a key role in inducing lymphocyte activation. In order to elucidate further the role of Ca2+ in the activation and IL-2 secretion of cells stimulated with ASIOI, we investigated the effect of EGTA (a chelator of extracellular Ca2+), nifedipine (the Ca 2+ channel blocker), and cyclosporin A (which selectively inhibits Ca2+-activated steps in the lymphocyte activation process) (Manger et al., 1986; Gelfand et al., 1986). We found that these compounds strongly inhibited both processes. Evidence has accumulated that the interaction of a wide variety of biologically active substances with their specific cell surface receptors is accompanied by an immediate breakdown of membrane inositol phospholipids and that this, in turn, is associated with an increase in intracellular Ca2+ (Berridge, 1984). These biochemical events appear to mediate many physiological responses of cells. Two main products of the breakdown of inositol phospholipids are diacylglycerol (DAG) and inositol triphosphate (InsP3) (Nishizuka, 1984). DAG activates the enzyme PKC (Nishizuka, 1984), InsP3 mobilizes intracellular Ca2+ (Berridge, 1984). At present, it is generally accepted that PKC is the cellular target for PMA (Neidel, Kuhn & Vandenbark, 1983). These data lead us to postulate that PMA and ASIOI might exert cumulative, perhaps synergistic, effects on mouse spleen cells or human MNC, and that this could result in overt enhancement of lymphokine secretion and cell proliferation.
Synergism between ASJO] and PMA
In the present study, we investigated, in depth, the T-cell activation induced by AS 101 and PMA by testing the effects of these agents on human and mouse T-cell proliferation and IL-2 release and on mouse CSF production. We found that AS 101, when added to cultures in conjunction with PMA, significantly enhanced IL-2 secretion and cell proliferation. These observations imply that the initiation of lymphocyte activation by AS101 may involve transmembrane Ca2+ flux and that the enhancement of IL-2 secretion and cell proliferation, observed when the cells were stimulated with PMA in conjunction with ASlOI, might be due to the generation of a second signal, probably the activation of PKC.
effect on IL-2 release. The cultures were incubated for 48 hr and 24 hr, respectively. Supernatants were collected and assayed for IL-2 activity. The ability of the supernatant fraction to support the growth of the IL-2-dependent CTLL clone was used to assay IL-2 production. CTLL cells (104 per well) were seeded in triplicate in culture medium, with or without dilutions of the supernatant fractions. After 48 hr, [3H]thymidine uptake was determined in a liquid scintillation counter. One unit of IL-2 activity was defined as the reciprocal log 2 dilution required to give 50% ofthe maximal proliferation of 104 IL-2-dependent murine CTLL cells after 48 hr of culture.
MATERIALS AND METHODS
CSF production Mouse spleen cells (107/ml) were suspended in enriched RPMI 1640 medium with 10% FCS and supplemented with various concentrations of ASlOl, PMA, or both. The cultures were incubated for 24 hr or 48 hr. Supernatants were collected and assayed for CSF activity.
Reagents The following reagents were employed, concanavalin A (Con A; Sigma Chemical Co., St Louis, MO), calcium ionophore (A23187; Calbiochem, La Jolla, CA), phorbol myristate acetate, 12-o-tetradecanoylphorbol 13-acetate (PMA; Sigma Chemical Co.). Nifedipine (Miles Laboratories, Toronto, Ontario, Canada) was prepared as a 10 mm stock solution in DMSO. EGTA (Sigma Chemical Co.) was prepared as a solution of 10 mM with the pH adjusted to 7-2. A23187 and PMA were first dissolved in ethanol and then in serum-free culture medium. The maximal dose of ethanol in A23187 solution was 0-1 % and in PMA 0-001 %. Cyclosporin A (CsA, Sandoz Inc., Hanover, NJ) was dissolved in I mg/ml ethanol and then serially diluted in culture medium to give the desired concentrations. Stock solutions were stored protected from light. RPMI-1640 was obtained from Gibco (Grand Island, NY) and fetal calf serum (FCS) from Sera Lab (Israel). PHA (Difco, St Louis, MO) was dissolved in distilled water; the stock concentration was 10 mg/ml.
Assay for CSF activity CSF was quantified by determining the number of colonies that developed from BM cells cloned in the presence of the CSFcontaining fraction to be tested. The soft-agar technique described by Pluznik & Sachs (1965) was used to clone BM cells. Briefly, supernatants containing CSF were incorporated in 2 ml of hard medium in a 35-mm Petri dish; 105 BM cells in 1 ml of soft agar medium (0-3%) were cloned above the hard agar layer. After 7 days of incubation at 370 in a humidified atmosphere of 8 5% CO2 in air, the number of colonies that had grown in the soft agar layer was scored.
RESULTS Cell preparation Heparinized blood was obtained from healthy adult donors and peripheral blood mononuclear cells (MNC) were isolated following Ficoll-Hypaque gradient centrifugation. The isolated MNC were washed and suspended in enriched RPMI-1640 at a concentration of 106/ml. Spleens were removed from male BALB/c mice (6-8 weeks old). Cells were passed through stainless steel mesh nets, treated with hypotonic solution to lyse erythrocytes and washed three times. Cells were brought to a concentration of either 106/ml or 107/ml enriched RPMI.
Lymphocyte proliferation The various compounds to be tested for their effect on human or mouse cell proliferation were added in the appropriate concentrations to suspensions of MNC or mouse spleen cells at a concentration of 106/ml. The cells were seeded in triplicate 0-2ml cultures in 96-well tissue culture trays. The cultures were incubated for 72 hr and pulsed with 1 yCi of [3H]thymidine per well during the final 14 hr. The cells were harvested and samples counted in a liquid scintillation counter. IL-2 studies Human MNC (106/ml) or mouse spleen cells (107/ml) were suspended in enriched RPMI 1640 medium with 10% FCS and various concentrations of the compounds to be tested for their
Effect of CsA on AS101-induced mouse IL-2 secretion In an earlier investigation, we postulated that activation of T cells by ASIOI involves transmembrane calcium flux (Sredni et al., 1987). In order to confirm this, we analysed the effect of CsA on IL-2 secretion. It was reported that the production of IL-2 requires an increase in Ca2+ concentration and does not take place in the absence of extracellular free Ca2+; CsA is known to be associated with inhibition of mitogen-induced changes in Ca2+ concentration (Mills et al., 1985a). Increasing concentrations of CsA were added to suspensions of mouse spleen cells containing 0 5 Mg/ml AS101. Supernatants were collected and tested for IL-2 bioactivity. Table 1 shows that the addition of CsA is accompanied by a dose-dependent inhibition of IL-2 secretion. The addition of 0 05 yg/ml CsA to a spleen cell suspension containing AS101 resulted in approximately 50% inhibition of IL-2 secretion. Concentrations of 0- 1 Mg/ml or more completely abrogated the production of IL-2. As the supernatants tested for IL-2 bioactivity contained increasing concentrations of CsA, we wished to ascertain that it was indeed the inhibition of IL-2 secretion that we were measuring and not suppression of the proliferation of CTLL cells by CsA. We therefore determined the effect of the appropriate CsA concentrations on the proliferation of the
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Table 1. Effect of CsA on AS101-induced mouse IL-2 secretion. Increasing concentrations of CsA were added to mouse spleen cell suspensions containing 0 5 pg/ml ASI01. The controls were supplemented either with AS101 or with CsA. Supernatants were collected after 24 hr and tested for IL-2 content
CsA (pg/ml) 5 1
0.1 0-05 0-01
05 0-5 05 0-5 0-5 0-5
1-4±0-49 1-4+0-49 15+0-35 4-2+0-35 7-5+1-62 7-8 +2-35
Table 2. Inhibition of mouse IL-2 secretion by nifedipine and EGTA. Spleen cells (107/ml) were incubated with increasing concentrations of nifedipine or 2 mm EGTA for 15 min in serum-free medium. AS10 was added and the suspension incubated at 37°. The cells were then washed three times and cultured in enriched RPMI supplemented with 10% FCS for 24 hr in the presence (right column) or absence (left column) of AS10. Supernatants were collected and tested for IL-2
bioactivity IL-2 (units/ml)
15' Concentration of the drug Medium Medium EGTA 2 mM EGTA 2 mm Nifedipine 10 PM 30 pM 50 pM 100p M 200 yM
No AS101 readded
Medium AS10 (1 pg/ml) Medium AS10 (1 pg/ml)
1 28 1 6
27 25 21 22
ASIOI (1 pg/ml)
30 14 12
27 26 27 22 20
AS101 (1 pg/ml) AS10 (1 pg/ml) ASIOI (I pg/ml) ASIOI (I pg/ml)
IL-2-addicted CTLL line. We found that at the various dilutions of IL-2 tested, CsA had no effect on the proliferation of the cell line (data not shown). Inhibition of mouse IL-2 secretion by nifedipine and EGTA In order to further elucidate the role of Ca2+ in IL-2 secretion of cells stimulated with ASlOl, we tested the effect of nifedipine and EGTA on this process. Table 2 shows that there was dose-dependent inhibition of IL-2 secretion, determined from the [3H]thymidine incorporation of the IL-2-dependent CTLL cells, in cultures incubated with nifedipine. EGTA (2 mM) suppressed IL-2 secretion by
Table 3. Inhibition of human IL-2 secretion by nifedipine and EGTA. Human MNC (106/ml) were incubated with 50 pM nifedipine or 2 mM EGTA for 15 min. Various concentrations of AS101 were added and cultured in enriched RPMI for 48 hr in the presence of ASIOl. Supernatants were tested for IL-2 content IL-2 secretion (units/ml)
Concentration of ASI01 (pg/ml)
o 0-1 0-5
0-1 0-5 I 0.1 0-5 1
Nifedipine50pM Nifedipine 50pM EGTA 2 mM EGTA 2 mM EGTA 2 mM
No AS101 readded
2+0-4 1+0-l 2+0-3 1+0-2
4+0-7 3+0-4 9+0i9 4+0 1
79%. The trypan blue dye exclusion test for cell viability revealed no cell death at the drug concentrations employed. In our search for potentially toxic effects of the drugs, we reintroduced AS1Ol to the cultures after the drugs had been washed out. It was noted that at concentrations of 100 pM or greater, nifedipine had some minor irreversible inhibitory effects on AS101-induced IL-2 secretion. Nifedipine (50 gM), and 2 mm EGTA inhibited the AS 101-induced IL-2 secretion by 58% and 79%, respectively; the effects were almost entirely reversible (Table 2). The addition of AS101, after the initial incubation with AS 101, and either one of the drugs, restores the ability of the cells to secrete IL-2, indicating that the 1-hr incubation with EGTA or nifedipine and AS1O1 did not damage the cells irreversibly. Cells that had been incubated with either of the drugs and with ASlO for 1 hr and then washed, did not proliferate. Apparently, the washing steps had removed sufficient AS 101 to prevent inhibition of the activation steps leading to IL-2 secretion after the cells had been placed in a culture medium containing Ca2+ but devoid of AS 101. Inhibition of human IL-2 secretion by nifedipine and EGTA In order to determine whether nifedipine and EGTA exert the same effect on human IL-2 secretion as they do on mouse spleen cells, human MNC were incubated with 50 pM nifedipine or 2 mM EGTA for 15 min. AS101 was added in increasing concentrations and the cultures incubated for I hr. They were then washed and incubated in culture medium for 48 hr. Supernatants were assayed for IL-2 content. The potentially toxic effects of the drug on human MNC were assessed just as on mouse spleen cells. Table 3 shows that both nifedipine and EGTA strongly inhibit the secretion of IL-2 by human MNC. The presence of EGTA during the 1-hr incubation with AS1Ol completely abrogated IL-2 production. Nifedipine (at 50 pM) inhibited IL-2 secretion by 60%. The production of IL-2 could be restored in these cells if AS1O1 was reintroduced into the cultures washed free of EGTA and of nifedipine. These data confirm that the secretion of IL-2 in response to ASlO is dependent on Ca2+ uptake.
Synergism between ASJOJ and PMA 50
-a- AS-IOI+PMA .40- AS-101
0.4 0.8 0.6 AS-101 (pLg/ml)
0.6 0.8 0.4 AS-101 (/Lg/ml)
Figure 1. Synergism between ASIO1 and PMA in stimulating human cell proliferation and IL-2 production. (a) Proliferation of human MNC incubated with ASlOl in vitro in the presence (2 5 ng/ml) or absence of PMA. (b) IL-2 production by human MNC stimulated with ASIOI with (2 5 ng/ml) or without PMA.
Figure 2. Synergism between ASIO1 and PMA in stimulating mouse cell proliferation and IL-2 production. (a) Proliferation of mouse spleen cells incubated with AS101 in vitro in the presence of (20 ng/ml) or absence of PMA. (b) IL-2 production by mouse spleen cells stimulated with ASIOI with (20 ng/ml) or without PMA.
Synergism between ASlOl and PMA in stimulating human and mouse cell proliferation and IL-2 production In order to test the separate and combined effects of AS O1 and PMA on the proliferation and IL-2 production of mouse spleen cells or human MNC, we first stimulated human or mouse cells with increasing concentrations of ASIOl alone, and then with AS 101 and an equal concentration of PMA. Figure I shows that significant enhancement of IL-2 secretion and cell proliferation of both human and mouse lymphocytes were obtained by the synergistic activation of ASIO1 and PMA. Concentrations of 0.1 and 0-5 pg/ml were found to be optimal for inducing MNC proliferation and IL-2 production (Fig. la and lb). ASO1l at 0-05-025 Mg/ml induced optimal IL-2 production by mouse spleen cells while optimal cell proliferation was attained with 0-05 Mg/ml (Fig. 2a and 2b). Negligible amounts of IL-2 were produced when mouse and human cells were stimulated with 20 and 5 ng/ml PMA, respectively. At these concentrations of PMA, cell proliferation was minimal. It is noteworthy that ASIO 1 in the absence of PMA induced both human and mouse IL-2 secretion; it also stimulated human but not mouse cell
needed for maximal effect. As seen in Fig. 3b, PMA by itself, even at high concentrations, did not induce human cells to release IL-2, although it was able to stimulate a remarkable increase in proliferation (Fig. 3a). Spleen cells were not affected similarly (data not shown). The optimal concentration was defined as the lowest dose of PMA which exerts maximal synergistic effect with AS 101, but which, when added by itself to the cultures, does not stimulate the cells to proliferate or secrete IL-2. This concentration was found to be 15 ng/ml for human cell proliferation, 5-10 ng/ml for human IL-2 secretion (Fig. 3) and 10 ng/ml for mouse cell proliferation and IL-2 secretion (data not shown).
proliferation. After having established that the two compounds act synergistically, we determined the optimal PMA concentration
Synergistic effect of AS101 or A23187 and PMA on IL-2 production In order to compare the effects of ASI01 and of the calcium ionophore, A23187 (two structurally distinct compounds), on IL-2 production, optimal concentrations of each compound were added to human or mouse cells, either alone or with PMA. As can be seen in Fig. 4, cells incubated with either PMA or A23187 stimulated the release of negligible amounts of IL-2. ASIOI induced a significant amount of IL-2. An overt synergis-
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a... 0 - 60
PMA( 5) PMA( IO)
cE 30 %-
0.1 0.5 ASIOI (A.g/ml)
Figure 3. Determination ofoptimal concentrations of PMA for inducing maximal synergistic effect with ASlO for human MNC proliferation and IL-2 secretion. Increasing concentrations of ASI 01 and PMA (0 510 ng/ml) were added to MNC cell suspensions. Cultures were assayed for cell proliferation (a) or IL-2 secretion (b).
60 r *
N Mouse U Human
C 30 cm
ASIOI ASIOI+PMA A23187+PMA
Figure 5. Induction of human MNC or mouse spleen cell proliferation by ASlOI or A23187 and PMA. Human or mouse spleen cells were cultured with the reagents for 72 hr. Proliferation was assessed by [3H]thymidine uptake. The concentrations used were: ASIOI, 0 1 Ig/ml; A23187, 1 uM; PMA, 20 ng/ml for spleen cells and 1 5 ng/ml for MNC.
tic effect was evident when mouse spleen cells or human MNC were stimulated with either A23187 or ASIOl in conjunction with PMA: the IL-2 levels were much higher than those obtained with either agent alone.
Synergistic effect of AS101 or A23187 and PMA on cell proliferation ASO1l and the calcium ionophore A23187 had a similar effect when added to cultures either by themselves or in conjunction with PMA (Fig. 5). After 72 hr in culture, control human MNC or mouse spleen cells exhibited minimal incorporation of [3H]thymidine; A23187 and AS101 both induced an extensive response of MNC to either compound alone. A23 187 or ASIO1 together with PMA lead to remarkable levels of cell proliferation (Fig. 4).
Synergism between AS101 and PMA in the induction of CSF secretion by mouse spleen cells It is interesting to know whether the synergistic effect of AS IOl and PMA is confined to IL-2 secretion and cell proliferation or whether this phenomenon is more general and exists in processes that lead to the secretion of other lymphokines. We therefore tested the effect of AS101 and PMA on the induction of CSF secretion. Increasing concentrations of AS 101 were mixed with PMA and incubated with mouse spleen cells (107) for 24 hr and 48 hr, respectively. Control cultures were supplemented only with AS1O1. When the supernatants were assayed for CSF activity, it was found that ASlO can stimulate mouse spleen cells to secrete CSF. However, secretions were maximal when both ASlO and PMA were added to the cultures.
Figure 4. The synergistic effect of AS101 (0 5 pg/ml for human MNC; 0-l yg/ml for mouse spleen cells), or A23187 1 ,M, and PMA (5 ng/ml for human MNC; 20 ng/ml for mouse spleen cells), on IL-2 release. Human MNC or mouse spleen cells were cultured for 48 hr and 24 hr, respectively. The supernatant fractions were analysed for IL-2 bioactivity using their ability to stimulate the growth of the IL-2-dependent Tcell clone, CTLL.
DISCUSSION The results reported here suggest that action of the immunomodulator AS1Ol in inducing IL-2 and CSF production is one of inducing changes in [Ca2+]. Calcium has been implicated as being pivotal as a transmembrane messenger in the activation signal for T lymphocytes (Greene, Parker & Parker, 1976). The
Synergism between ASJOJ and PMA prevention of the change in [Ca2+], inhibits the proliferative response of T cells and the secretion of IL-2 (Mills etal., 1985a; Gelfand etal., 1986). The effect of preventing changes in [Ca2+]J might be confined to IL-2-secreting cells, as IL-2-receptor expression and IL-2-induced T-cell proliferation take place in the absence of [Ca2+] changes (Mills et al., 1985a; Mills et al., 1985b). We show in this study that prevention of changes in [Ca2+] by CsA, Ca2+ chelators or Ca2+ channel blockers is accompanied by the inability of cells stimulated with ASlOl to trigger IL-2 release. Table 1 shows CsA inhibition of IL-2 secretion as dose-dependent. CsA has been reported to inhibit selectively reaction pathways that require an increase in [Ca2+]J (Manger et al., 1986; Gelfand et al., 1987). The fact that CsA-induced inhibition could not be eliminated with Ca2+ ionophores leads us to conclude that CsA probably acted on pathways distal from those that require an increase in [Ca2+]i and not on the initial plasma membrane-associated events in the activation process. Many intracellular Ca2+-dependent events are regulated by the Ca2+-binding protein calmodulin. The role of calmodulin in CsA action is controversial. Colombani, Rubb & Hess (1985) demonstrated that CsA binds to calmodulin and inhibits calmodulin-dependent phosphodiesterase activity in vitro. Although inhibition of a specific Ca2+-calmodulin-regulated event in T-cell activation signalling is an attractive explanation for the inhibitory effects of CsA, other proteins also bind CsA, like cyclophilin, a cytosolic protein whose function is unknown (Handschemacher et al., 1984). To further document the role of extracellular Ca2+, and particularly Ca2+ uptake, in the initiation of T-cell activation by ASIOI leading to IL-2 secretion, we used the drugs EGTA and nifedipine. Both EGTA and nifedipine caused a significant inhibition of both human and mouse IL-2 secretion which was reversible if the drugs were washed out and the cells were cultured in the presence of ASI01 (Table 2 and 3). The behaviour of Ca2+-channel blockers, such as nifedipine, supports our contention that Ca2+ uptake is not just associated with, but is critical for initiating AS 101-induced IL-2 secretion. It directly confirms the results observed with EGTA (Kay, 1971). Nifedipine can block the entry of Ca2+ into cells (Birx, Berger & Fleischer, 1984); in our study the drug was found to inhibit AS10-induced IL-2 secretion (Tables 2 and 3). The ability of PMA to synergize with Ca2+ influx inducers in stimulating the production and secretion of IL-2 and other lymphokines by T cells is well known (Rosenstreich & Meizel, 1974; Koretzky, Daniels & Nowell, 1982). We were therefore intersted in analysing the effects of PMA and AS 101 alone and when combined in order to assess the relative role of PKC activation and the Ca2+ signal obtained by ASlOl in various aspects of T-cell activation. Figures 1 and 2 show a significant elevation of human and mouse IL-2 secretion and cell proliferation after stimulation with AS101 in conjunction with PMA. PMA alone induced proliferation of human MNC at high concentrations (Fig. 3), while the same concentration of PMA did not affect IL-2 secretion (Fig. 3). On the other hand, AS1O1 induced both IL-2 secretion and cell proliferation although its effect could be impressively elevated by co-operation with PMA. The ability of PMA to stimulate human MNC to proliferate is known to be a function ofits concentration (Isakov et al., 1985b; Moolenaar, Tertoolen & Delaat, 1984). This suggests that T-cell proliferation is related to PKC activation. Interestingly, when
human T cells are induced to proliferate by PMA, no transcription of the IL-2 gene or secretion of IL-2 is detectable (Isakov et al., 1985b). Moreover, this proliferation cannot be blocked by a neutralizing anti-IL-2 antibody (Isakov et al., 1985a). These findings indicate that in addition to the conventional IL-2dependent pathways of T-cell proliferation, another IL-2independent pathway operates, probably via the activation of PKC. ASIOI and A23187 were both able to stimulate cell proliferation although substantially less than that obtained in conjunction with PMA (Fig. 4). The Ca2+ ionophore can mimic the physiological signal of InsP3 and increase [Ca2+1i, and ASIOI has been shown to affect the Ca2+ influx. However, as PKC is a Ca2+-dependent enzyme that can be activated irreversibly by a Ca2+-dependent protease, a sufficient increase in [Ca2+] may directly activate PKC in the absence of phosphatrdyl inositol diphosphate (PtdInsP2) hydrolysis and DAG generation. This may be the reason why ASlOI was able to induce cellular proliferation. In human T cells, either PMA or A23187 provides a sufficient stimulus to trigger IL-2 receptor expression and proliferation (Isakov etal., 1985a). This proliferative response is probably IL-2-independent. However, mouse spleen cells do not proliferate in response to either compound alone, but only to a combination of both, although they are induced to develop IL-2 receptors. The proliferation of mouse spleen cells is probably IL-2 dependent. The basis for this difference may reflect quantitatively or qualitatively different requirements for activation signals by human and murine T cells. ASIOI and the calcium ionophore (CI) seem to have the same effect on mouse and human T cell proliferation (Fig. 5). The signal provided by PMA or CI seems sufficient for the induction of IL-2 receptors in mouse or human T cells, yet neither signal alone can induce IL-2 production (or IL-2 mRNA). Only a combination of the two signals trigger the process leading to IL-2 secretion. AS101 may deliver an additional signal that CI may not, which demonstrates that the two compounds differ not only structurally (AS101 is a tellurate derivative and the CI is a polyether carboxylic acid) but also functionally. Studies are now in progress to determine whether IL-2 secretion induced by AS101 is caused by elevated Ca2+ alone or also by another cellular response to AS10 not directly related to the Ca2+ signal. As shown in Table 4, 0 05 Mg/ml AS101 induced spleen cells to secrete CSF. The same concentration of AS101 in conjunction with PMA induced the secretion of a very large quantity of CSF (132 colonies relative to 34). The ability of murine spleen or BM cells to secrete CSF following induction by PMA and LPS has been reported by Pluznik and colleagues (Pluznik, 1983; Baum, Pluznik & Rozenszajn, 1985). To summarize, we demonstrated that significant enhancement of IL-2 secretion and cell proliferation of both human and mouse cells can be obtained by the synergistic effect of AS101 and the tumour promotor PMA (Figs 1 and 2). Co-operation between the two compounds also resulted in enhanced CSF production by mouse spleen cells (Table 4). These results led us to the conclusion that the synergistic action of AS101 and PMA could be explained by the action of AS 101 on [Ca2+], influx and the effect of PMA on PKC activation. Preliminary laboratory results have shown that bryostatins, that are also PKC inducers, also synergize in vitro with AS 101 to induce lymphokine production. As ASlO may be injected in
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Table 4. Synergism between ASIOI and PMA in the induction of CSF secretion by mouse spleen cells. Increasing concentrations of ASlOl, with or without 20 ng/ml PMA, were added to 107/ml spleen cells. Supernatants were collected after 24 hr and 48 hr and tested for CSF content, as reflected by the number of colonies developed from 10 nucleated BM cells No. of colonies/105 BM cells
Incubation time (hr) ASIOI
PMA (20 ng/ml)
+ 0 01 005 0-1 0-5 1 0-01 0-05 0-1 05 1
+ + + + +
1+0-8 2+0-2 0 3+04 2+0-1 2+0-2 1+0 1 12+0-8 10+0 9 4+0 3 0 2+0-2
4+0 3 36+2-1 15+2-0 34+3-2
3+07 2+0-1 2+0 1 48+26 132+8-4 21 +2-1 20+1-7 19+ 1-6
vivo, unlike CI, to induce lymphokine production, and as bryostatins were found to have anti-neoplastic activity (Pettit et al., 1984), it would be interesting to test whether ASIOI as a Ca2+ influx-inducer and bryostatin as a PKC-inducer would synergize in vivo in lymphokine production and anti-tumour activity. We believe that a better understanding of the mechanism ofaction of the immunomodulator ASIO1 will enable us to optimize its application both in vitro and in clinical trials.
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