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Augmentation of cytotoxic responses by prostaglandin E2
CELLULAR
91, 43-5 1 (1985)
IMMUNOLOGY
Augmentation
of Cytotoxic
Responses
BARBEL HACKER-SHAHIN
by Prostaglandin
E2
AND WULF DROGE
Institut fir Immunologie und Genetik, DeutschesKrebsforschungszentrum, D-6900 Heidelberg, West Germany Received August 6, 1984; accepted October 4, I984 The cytotoxic-T-lymphocyte activity in the spleen cells after in vivo immunization of C3H mice with allogeneic spleen cells ip was consistently very weak. Substantial cytotoxic responses were obtained, however, when prostaglandins (PGE,, PGE, , or PG12)were injected ip together with or prior to the immunization. An augmentation of cytotoxic responsesagainst allogeneic stimulator cells was also observed in mixed lymphocyte cultures which were provided with an interleukin 2-containing helper factor. This augmentation was observed when PGEz was added at the start of the culture but not if added 1 day later. Indomethacin was found to be suppressive in these cultures. 0 1985 Academic PBS, Inc.
INTRODUCTION Prostaglandin E2 (PGE2)’ has been reported to mediate positive (l-3) and negative (4-14) regulatory effects on the immune system. In particular, PGE2 has been shown to inhibit the activation of cytotoxic T lymphocytes (CTL) (4, 5). Prostaglandins have been shown to inhibit the proliferative response of lymphocytes including IL-2-dependent cytolytic-T-cell lines [(6- 10); reviewed in ( 11, 12)]. Indomethacin and other inhibitors of prostaglandin synthesis have been shown to enhance the induction of cytotoxic responses against allogeneic cells (8); and finally, PGE2 has been reported to inhibit the production of IL-2 in human lymphocyte preparations (10, 13, 14). Immune-stimulator-y effects of PGEp have been mainly observed in vivo. AntiPGE antibodies have been reported to inhibit the in vivo development of several types of T-cell-mediated immune reactions (1). A PGE2 derivative (diM-PGE*) was found to increase T-cell-mediated immune responses in B16 melanoma-bearing C57BL/6 mice and delayed the development of the tumor, whereas indomethacin was found to enhance tumor development (2). PGE2 has also been implicated as a possible mediator of the biological activity of thymic factor (3). In vitro, PGEz was also shown to augment the immunoglobulin production by primed spleen cell populations (15). We now report an immune stimulatory effect of PGE2 on the cytotoxic response against allogeneic cells in vivo and in vitro. The augmenting ’ Abbreviations used: AC, adherent cells; Con A-spl, concanavalin A-activated spleen cells; CTL, cytotoxic T lymphocytes; IL-2, interleukin 2; PGE,, prostaglandin Es; PG12,prostacyclin; TNP, trinitrophenyl; Hepes, 4-(2-hydroxyethyl> 1-piperazineethanesulfonic acid; BSS, balanced saline solution; PMA, phorbol myristic acetate. 43 0008-8749185$3.00 Copyright 0 1985 by Academic Ras, Inc. All rights of reproduction in any form reserved.
44
HiiCKER-SHAHIN
AND
DRijGE
effect of PGE2 in vitro was observed in cultures with additional helper factor(s). The suppressive effect of PGE2 was usually observed in cultures without an external source of helper factor(s) (4). The demonstration of the augmenting effect of PGE2 in cultures excludes certain interpretations, such as altered migration pattern of lymphocytes in vivo, and it may be useful to analyze the mechanism of the immune stimulatory effects of PGEz in detail. MATERIAL AND METHODS Animals. The mice were obtained from Bomholtgard, Ry, Denmark. Most of the experiments were performed with 8- to lo-week-old male animals. Activation of cytotoxic T lymphocytes in vivo. C3H mice were immunized by injection of 5 X 10’ irradiated (1500 t-ad) allogeneic spleen cells ip. The mice were sacrificed 5 days later, and their spleen cells were tested for cytotoxic activity against “0-labeled target cells at the indicated attacker:target cell ratios. Activation of cytotoxic T lymphocytes in vitro. Twenty million responder cells (usually C3H spleen cells) were incubated in a total volume of 4.5 ml culture medium (RPM1 1640, GIBCO medium supplemented with 10 mM L-glutamine (GIBCO), streptomycin/penicillin (GIBCO, 100 units/ml), 0.5% Hepes (GIBCO), 10% fetal calf serum (GIBCO), and 3 X low5 M 2-mercaptoethanol together with 0.4 ml EL-4 supematant and the indicated numbers of irradiated (1500 rad) stimulator cells for 5 days at 37°C in 5% CO2 if not indicated otherwise. Responder and stimulator cells were unfractionated spleen cells if not indicated otherwise. The cultures were tested after 5 days for cytotoxic activity as previously described (16, 17). Target cells. The target cells were prepared by culturing spleen cells with 2.2 &ml concanavalin A (Pharmacia, Uppsala, Sweden) for l-3 days. Irradiation of stimulator cells. The stimulator cells were irradiated with 1500 rad from a Gammacell 1000 D (Atomic Energy of Canada Limited, Commercial Products) equipped with a 2300 Ci 13’Cssource (1700 rad/min). The preparation of IL-2-containing supernatants from EL-4 thymoma cells. Supematants were obtained from an IL-Zproducing EL4 subline (kindly provided by Dr. J. Farrar) after induction with PMA as previously described (18). Briefly, lo6 EL-4 cells/ml were incubated together with 10 rig/ml of phorbol myristic acetate (PMA, Sigma) for 48 hr. The supematants were collected and stored frozen at -20°C. They usually contained 100-300 units/ml of IL-2. Preparation of T-cell-enriched nylon-wool-nonadherent spleen cells. Lymphoid cells were incubated in nylon-wool columns and eluted as described (19). The preparation of “plastic adherent cells” (AC). The term “plastic adherent spleen cells” is used in this paper for a fraction of spleen cells which was prepared by a procedure that has been reported to enrich especially for dendritic cells (20, 21). Spleen cells from three mice (approx. 3 X lo8 cells) were incubated in 10 ml culture medium in plastic petri dishes of 10 cm diameter (Greiner, Niirtingen, West Germany, Type TC94/ 16) at 37°C. After 2 hr, the supematant was discarded, the nonadherent cells were washed off carefully by extensive rinsing with warm culture medium or BSS, and the adherent cells were again incubated with 10 ml of fresh culture medium for another 18 hr. The cells which detached spontaneously during this time period (usually l-2 X lo6 cells) were then collected in the supematant and used as the “plastic adherent spleen cell population” in these
PGE2 AUGMENTS CYTOTOXIC
RESPONSES
45
studies. According to reports from another laboratory (20, 21), this population is a mixture of dendritic cells @O-95%) and macrophages. Preincubation of stimulator cells. In some experiments the stimulator cells were cultured at a density of 1.5-2 X 10’ cells/45 ml culture medium for 24 hr with 0.02 mg of concanavalin A (Con A, Pharmacia) per culture. The application of indomethacin and prostaglandins. Indomethacin is a nonsteroidal anti-inflammatory drug and a potent irreversible inhibitor of prostaglandin synthesis (22-24). A stock solution of indomethacin (Sigma) was prepared by dissolving 10 mg/ml in ethanol. The cell cultures usually contained 0.5 pg indomethacin/ml, i.e., 1:20,000 dilution of the stock solution in culture medium. The various types of prostaglandins were obtained from Sigma. Stock solutions ( 10e2M in ethanol) were stored at -20°C. The prostaglandins were applied to cultures at concentrations of 2 X lo-’ M if not indicated otherwise. Control cultures with corresponding concentrations of ethanol were also routinely tested. RESULTS Augmentation of cytotoxic responsesin vivo. The cytotoxic activity in the spleen after in vivo immunization of C3H mice with allogeneic spleen cells ip was usually 30
20 10
----Q@dWddElYO q aededday-l
-----
o-L-4 2
irL I
20
p 0 E 0 $ .s
10 0
40-
I
I
PGIp
3020lo-
o-ty
,
1x10-6 mm.
I
3x10-6 1x10-5 3x10-5 of prostaglandins (Molll)
ethanol
FIG. 1. Augmentation of cytotoxic responsesagainst allogeneic cells by graded dosesof prostaglandins. C3H mice were immunized by injection of 5 X IOr C57BL/6 cells in 0.2 ml BSS ip. The mice also received ip injections of 0.5 ml BSS containing the indicated concentrations of prostaglandins. Control groups received 0.5 ml of a 0.3% solution of ethanol in BSS, because the prosmglandin solutions were routinely prepared from lo-* M stock solutions in ethanol. The cytotoxic activity of the spleen cells was tested 5 days after immunization at attacketltarget cell ratios 100~1and 20: 1. The figure shows the data at the ratio 200: 1. The SE values for the individual points (51Cr-releasedata) were in all our experiments less than 3%, usually even less than 2%.
46
HACKER-SHAHIN
AND DRijGE
very weak (see Fig. 1). The cytotoxic responseswere markedly augmented, however, by the simultaneous injection of prostaglandins (PGEz, PGE, , or PGIz). The doseresponse was similar for all three types of prostaglandins (Fig. 1). There was, however, a difference with respect to the optimal time of administration. The extremely labile PG12had to be administered at the time of immunization (Day 0) and was ineffective if given the day before. The more stable compound PGE, produced the most effective augmentation on Day -1 and slightly weaker effects on Day 0; and PGEz expressed an intermediate behavior (Fig. 1). A possible explanation for these differences is that the relatively more stable compound PGE, might have persisted in sufficient concentration into the period after immunization and might have inhibited the ongoing activation of the CTL. Repeated injections of PGEz or PGE, on the day of immunization and on several subsequent days were indeed found to suppress the cytotoxic response (data not shown). The time course of the cytotoxic response after immunization in combination with PGE2 or PGEl was similar to primary cytotoxic responses in vitro: The peak activity was found on Day 5 (Fig. 2). The experiment in Table 1 illustrates two points: First, PGDz, PGF,, and PGF2, did not augment the cytotoxic response in vivo. (Similar results were also obtained
B) PGE1 day 0 P I e
lo-
b 6 0 so
o
8 3
0
3
5
7
10
day after immunization
FIG. 2. Time course of the cytotoxic response in vivo alter treatment with PGEz or PGE,. C3H mice were immunized with 5 X 10’ irradiated (1500 tad) C57BL/6 spleen cells ip and sacrificed 3, 5, 7, or 10 days later. The cytotoxic activity of the spleen cells against C57BL/6 target cells was then tested at the attacker:target cell ratios 200: 1 and 40: 1. The mice in panels A and B also received 0.5 ml of an 1 X IO-* M PGEr or PGE, solution, respectively, on the day of immunization (Day 0). The mice in panel C received the same amount of PGE, on the day before immunization.
PGEr AUGMENTS CYTOTOXIC
47
RESPONSES
TABLE 1 The Effect of Different Types of Prostaglandins on the Activation of Cytotoxic Activity in Viva Immunized with C57BL/6 cells
Without immunization
Prostaglandins injected
200: 1
40: 1
200: 1
40: 1
None (ethanol in BSS) PGEr PG12
0.8 29.0 14.3 0.9 2.8 0.5
-1.6 24.1 12.6 0.6 1.8 1.0
0.6 3.8 3.1 5.7 5.0 3.6
-1.5 0.1 0.3 -1.3 0.3 -3.0
=D2
PGFI, EF2.7
Note. BSS (0.5 ml) containing 1 X 10e5M prostaglandins of the indicated type and 0.1% ethanol was injected into C3H mice ip. Some of the mice were immunized 1 day later with 5 X 10’ irradiated (1500 tad) C57BL/6 spleencells in 0.2 ml BSSip. The mice were sacrificed6 days after the injection of prostaglandins (i.e., 5 days after immunization). The data show the cytotoxic activity (% specific “Cr release)of the spleen cells against C57BL/6 target cells at the attacker:tatget cell ratios 200: 1 and 40: 1. The cytotoxic activity against DBA/Z and A.TH target cells was also tested but was always less than 2%.
in vitro as discussed later.) Second, PGE;, and PGI2 did not induce a polyclonal response. The cytotoxic response required the combination of prostaglandins and immunizing antigen (C57BL/6) and was specific for that antigen. There was no cytotoxic activity against the unrelated target cells DBA/2 and A.TH. Augmentation of cytotoxic responses in vitro. In order to study the augmenting effect of PGE2 in vitro, we routinely provided the cultures with EL4 supernatant as a source of helper factor, since PGEz has been reported to inhibit the production of IL-2 (10, 13, 14). The EL-4 supernatant also contained at least one additional helper factor (25) and a small amount of PMA. The response of C3H spleen cells against irradiated BALB/c stimulator cells in such cultures was found to be strongly augmented by the addition of 10e6A4 PGE2 and reduced to almost background levels by the addition of 0.5 &ml of indomethacin (Fig. 3, left panel). The response was still specific for BALB/c, indicating that PGE2 did not trigger a polyclonal activation of CTL precursor cells. Since we have recently observed that different types of stimulator cells differ strongly in their ability to stimulate cytotoxic responses (26), we studied the hypothesis that the exceptionally immunogenic stimulator cells, i.e., an adherent cell preparation enriched for dendritic cells (AC) and concanavalin A-activated spleen cells (Con A-spl) (26), stimulate most effectively because they produce prostaglandins. The hypothesis predicted that responses against these types of stimulator cells would show a less stringent requirement for externally added PGE2. Our experiments (Fig. 3, middle and right panels) did not support this hypothesis: the cytotoxic responsesagainst AC or Con A-spl stimulator cells were also strongly augmented by PGE2. Similar effects were observed with nylon-wool nonadherent splenic T cells as stimulator cells (data not shown), which were previously found to be exceptionally weak stimulator cells (26). The augmenting effect of PGE2 was observed if PGE2 was added at the initiation
48
HACKER-SHAHIN
AND DRiiGE stim:
A) Target: c57 BL16
40
stim: 2 x 105 BALBlc
3
4
5
stim: spl 5
6
7
3
2 x to5 BALBlc + 2 x lO-‘BL/G-AC
4
5
2 x 105 BALE/c spl f + 2 x I+ BL/6-Con A st9 j
spl ‘r f
6
7 day of culture
FIG. 3. The effect of PGE2 and indomethacin on the development of the cytotoxic response against different types of stimulator cells in vitro. C3H spleen cells (2 X 10’) were incubated in 4.5ml cultures with 2 X 10’ irradiated BALB/c splenic stimulators and with or without 2 X 10’ irradiated C57BL/6 AC or 2 X IO4irradiated C57BL/6 Con A-spl. All cultures also received 0.4 ml EL-4 supematant (containing 80 units of IL-2), and some of the cultures also received F’GE2at a final concentration of low6M or 2 pg indomethacin per culture. The cytotoxic activity against C57BL/6 and BALB/c target cells was determined at the indicated time intervals after the initiation of the culture. Cytotoxic activity was determined at attacker:target cell ratios 25: I, 5:1, and I:l. The figure shows the data at the ratio of 25:l.
of the cultures but not if added 1 day later (Table 2). The addition of PGE2 on Day 1 produced a slight suppression. The comparison of different types of prostaglandins revealed that the in vitro responseswere augmented by PGEz and PGE, , but not by several other prostaglanTABLE 2 PC& Augments Cytotoxic Responsesonly if Added to the Culture on Day 0. No PC&
F’GEz Day 0
PGEt Day I
Stimulator cells
Target cells
25:l
5:1
25:l
5:l
25:1
5:1
C57BL/6
C57BL/6 BALB/c BALB/c C57BL/6
12.0 -1.3 12.4 -4.5
0.9 -1.3 3.2 -3.5
33.0 2.5 40.1 -0.1
11.8 -1.1 16.2 -2.1
5.9 -2.3 7.6 -1.8
-1.9 -1.5 4.4 -4.1
DBA/2
Note. C3H responder cells (2 X IO’) were incubated with 2 X lo6 irradiated C57BL/6 or DBA/Z stimulator cells and 0.6 ml EL-4 supematant for 5 days in 4.5-ml cultures. Some of the cultures received additionally 0.5 ml of a 3 X low6 M PGEz solution either at the start of the culture (Day 0) or 1 day later (Day 1). The data show the % specific 5’Cr release of C57BL/6 and BALB/c target cells at the attackerztarget cell ratios 25: 1 and 5: I.
PGEz AUGMENTS CYTOTOXIC
49
RESPONSES
dins, including prostacyclin (PG12, Fig. 4). The discrepancy between this and our in vivo results (Fig. 1) reflects, possibly, the extreme instability of PG12. DISCUSSION The major result from these experiments was that some prostaglandins including PGE2 augment cytotoxic responsesagainst allogeneic cells in vivo and, under certain conditions, also in vitro. Whereas PGEz has previously been reported to inhibit cytotoxic responses (4, 5), this is the first report that we are aware of about a potentiating effect on cytotoxic responses. The expression of the augmenting effect of PGE2 in vitro was only observed when the cultures were provided with an IL-Zcontaining helper factor. Control cultures without the helper factor were usually suppressedby PGE2 (data not shown). This is compatible with previous observations that PGE2 inhibits the production of IL-2 (10, 13, 14). IL-2-containing EL-4 supernatants have been added to our in vitro experiments primarily in order to avoid an indirect effect of PGE2 via its effect on the IL-2-producing helper cells. The EL-4 supematant contained also at least one additional helper factor (25) and a small amount of PMA, which might also serve as a cofactor in the stimulation of the CTL precursor cells. However, the supematant did not stimulate a substantial cytotoxic response in the absence of the corresponding antigen (see Fig. 3, BL/6-Target). Moreover, our observation that the in vivo activation of CTL is augmented by PGE2 indicates that our culture system with the IL-Zcontaining EL4 supematant represents a physiologically relevant situation. Positive regulatory effects of PGE have already been observed with other immune responses (1-3, 15, 27, 28); most of these reports have been dealing with in viva observations. This fact may underscore the biological significance of these effects. The disadvantage of the in vivo systems is, however, that complex interpretations such as altered migration properties of lymphocytes in vivo cannot be excluded. Our demonstration of an immune potentiating effect of PGEp in vitro excludes such an interpretation and may be useful for the further analysis of this regulatory effect. 30
1
no PG
no PG. indomethacin
PGE,
d PGE,
PGD,
1
PGFp
FIG. 4. Effect of different types of prostaglandins and indomethacin on the cytotoxic response. C3H spleen cells (2 X 10’) were incubated with 2 X lo6 irradiated C57BL/6 spleen cells in 4.5-ml cultures containing 0.4 ml EL4 supematant with or without 2.0 pg indomethacin and with the indicated types of prostaglandin at a final concentration of 2 X lo-’ M. The data show the cytotoxic activity against C57BL/6 target cells at the attacker:target cell ratio 5: 1.
50
HACKER-SHAHIN
AND DROGE
There are several possible mechanisms that could explain why PGE2 augments T-cell functions in some experimental situations and inhibits them in others: (i) PGEZis known to increase the cellular concentrations of cyclic AMP in lymphocytes ( 11,29) and this may be important for the late cyclic events in activated lymphocytes but inhibitory in the early phase of the cell cycle (29). The in vivo situation may be characterized by suboptimal concentrations of cyclic AMP. (ii) Another interpretation is that PGEl inhibits not only the activation of lymphokine-producing cells and CTL precursor cells, but also the action of some types of suppressor T cells. It is quite conceivable that a slight inhibition of the effector precursor cell population together with an inhibition of a suppressor cell system can provide a positive net effect on the effector cell activation. (iii) It is also conceivable that PGEz is one of the activating signals which are produced by the stimulator cells and delivered to effector precursor cells during the stimulation process. It has been reported that stimulator cells induce proliferative responses at relatively low doses and suppress at higher cell numbers (30, 31). PGE2 may be responsible for both, the inducing and the inhibitory effect, in a dose dependant way. It should be noted, however, that PGE2 does not induce a polyclonal response (see Fig. 3 and Tables 1 and 2). This would have been expected if PGE2 would function as the only relevant signal of the stimulator cells. (iv) The possibility that PGE2 activates the immunogenic potential of the stimulator cells appearsunlikely, since several attempts to demonstrate an increased immunogenic potential after preincubation of the stimulator cells in PGEs-containing cultures have failed (W.D., unpublished). (v) The alternative possibility that PGE2 may inhibit previously activated cells in the responding lymphocyte preparation and thereby reduce the background of proliferating cells, which may compete for lymphokines or other positive signals with the antigenspecific response under test, seemsalso unlikely. Experiments on the effect of PGEz on the antigen-driven proliferation in relation to the background proliferation did not confirm this possibility (W.D., unpublished). (vi) At present there are no data that support or exclude the possibility that PGEz induces the maturation of CTL by stopping the proliferative phase of the response. The observation that the augmenting effect of PGE2 is only seen if PGE2 is present early in the course of the response (Table 2) could be explained by the assumption that it induces the production of a late maturation factor. ACKNOWLEDGMENTS The expert technical assistanceof Mrs. Heike Schmidt and Mrs. Sabine Panknin and the assistanceof 1. Schmoch-Fryson and Mrs. J. Rami in the preparation of this manuscript is gratetilly acknowledged.
REFERENCES 1. Mertin, J., and Stackpoole, A., Nature (London) 294, 456, 1981. 2. Favalli, C., Garaci, E., Etheredge, E., Santoro, M. G., and Jaffe, B. M., J. Immunol. 125, 897, 1980. 3. Garaci, C. R., Favalli, C., del Gobbo, V., Garaci, E., and Jtie, B. M., Science (Washington, D.C.) 220, 1163, 1983. 4. Leung, K. H., and Mihich, E. Nature (London) 288, 597, 1980. 5. Wolf, M., and Driige, W., Cell. Immunol. 72, 286, 1982. 6. Goodwin, J. S., Bankhurst, A. D., and Messner, R. P., J. Exp. Med. 146, 1719, 1977. 7. Webb, D. R., and Nowowiejski, M. D., Cell. Immunol. 41, 12, 1978. 8. Darrow, T. L., and Tomar, R. H., Cell Immunol. 56, 172, 1980. 9. Baker, P. E., Fahey, J. V., and Munck, A., Cell. Immunol. 61, 52, 1981.
PGE2 AUGMENTS CYTOTOXIC
RESPONSES
51
IO. Tilden, A. B., and Balch, Ch. M., J. Immunol. 129, 2469, 198 1. 11. Gemsa, D. In “Lymphokines: A Forum for Immunoregulatory Cell Products” (E. Pick, Ed., and M. Landy, Advisory Ed.), Vol. 4, p. 335. Academic Press,New York, 1981. 12. Bray, M. A., Immunol. Today 80,65, 1980. 13. Rappaport, R. S., and Dodge, G. R., Med. 155,943, 1982. 14. Walker, C., Kristensen, F., Bettens, F., and deWeck, A. L., J. Immunol. 130, 1770, 1983. 15. Weider, K. J., and Webb, D. R., Prostaglandins Med. 7, 79, 1981. 16. Reddehase,M., Suessmuth, W., Moyers, C., Falk, W., and Driige, W., J Immunol. 128, 61, 1982. 17. Galli, P., and Driige, W., Eur. J. Immunol. 10, 87, 1980. 18. Farrar, J. J., Fuller-Farrar, J., Simon, P. L., Hilfiker, M. L., Stadler, B. M., and Farrar, W. L., J. Immunol.
125, 2555, 1980.
19. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. 20. Nussenzweig M. C., and Steinman, R. M., J. Exp. Med. 151, 1196, 1980. 2 1. Steinman, R. M., Gutchinov, B., Witmer, M. D., and Nussenzweig, M. C., J. Exp. Med. 157, 613, 1983. 22. Vane, J. R., Nat. New Biol. 231, 232, 1971. 23. Thomas, D. R., Philpott, G. W., and Jaffe, B. M., Exp. Cell Res. 84, 40, 1974. 24. Gaut, 2. N., Baruth, H., Randall, L. O., Ashley, C., and Paulsrud, J. R., Prostaglandins 10, 59, 1975. 25. Mtinnel, D. N., Falk, W., and Droge, W., J. Immunol. 130, 2508, 1983. 26. Droge, W., Moyers, C., Wehrmaker, A., Schmidt, H., Panknin, S., Miinnel, D., and Falk, W., J. Immunol.
27. 28. 29. 30.
132, 2749, 1984.
Mertin, J., Stackpoole, A., and Shumway, S. J., Transplantation 37, 4, 1984. Stobo, J. D., Kennedy, M. S., and Goldyne, M. E. J. Clin. Invest. 64, 1188, 1979. Hadden, J. W., and Coffey, R. G., Immunol. Today It, 299, 1982. Lamb, J. R., Skidmore, B. J., Green, N., Chiller, J. M., and Feldmann, M., J. Exp. Med. 157, 1434, 1983. 31. Matis, L. A., Glimcher, L. H., Pam, W. E., and Schwartz, R. H., Proc. Natl. Acad. Sci. USA 80, 6019, 1983.
91, 43-5 1 (1985)
IMMUNOLOGY
Augmentation
of Cytotoxic
Responses
BARBEL HACKER-SHAHIN
by Prostaglandin
E2
AND WULF DROGE
Institut fir Immunologie und Genetik, DeutschesKrebsforschungszentrum, D-6900 Heidelberg, West Germany Received August 6, 1984; accepted October 4, I984 The cytotoxic-T-lymphocyte activity in the spleen cells after in vivo immunization of C3H mice with allogeneic spleen cells ip was consistently very weak. Substantial cytotoxic responses were obtained, however, when prostaglandins (PGE,, PGE, , or PG12)were injected ip together with or prior to the immunization. An augmentation of cytotoxic responsesagainst allogeneic stimulator cells was also observed in mixed lymphocyte cultures which were provided with an interleukin 2-containing helper factor. This augmentation was observed when PGEz was added at the start of the culture but not if added 1 day later. Indomethacin was found to be suppressive in these cultures. 0 1985 Academic PBS, Inc.
INTRODUCTION Prostaglandin E2 (PGE2)’ has been reported to mediate positive (l-3) and negative (4-14) regulatory effects on the immune system. In particular, PGE2 has been shown to inhibit the activation of cytotoxic T lymphocytes (CTL) (4, 5). Prostaglandins have been shown to inhibit the proliferative response of lymphocytes including IL-2-dependent cytolytic-T-cell lines [(6- 10); reviewed in ( 11, 12)]. Indomethacin and other inhibitors of prostaglandin synthesis have been shown to enhance the induction of cytotoxic responses against allogeneic cells (8); and finally, PGE2 has been reported to inhibit the production of IL-2 in human lymphocyte preparations (10, 13, 14). Immune-stimulator-y effects of PGEp have been mainly observed in vivo. AntiPGE antibodies have been reported to inhibit the in vivo development of several types of T-cell-mediated immune reactions (1). A PGE2 derivative (diM-PGE*) was found to increase T-cell-mediated immune responses in B16 melanoma-bearing C57BL/6 mice and delayed the development of the tumor, whereas indomethacin was found to enhance tumor development (2). PGE2 has also been implicated as a possible mediator of the biological activity of thymic factor (3). In vitro, PGEz was also shown to augment the immunoglobulin production by primed spleen cell populations (15). We now report an immune stimulatory effect of PGE2 on the cytotoxic response against allogeneic cells in vivo and in vitro. The augmenting ’ Abbreviations used: AC, adherent cells; Con A-spl, concanavalin A-activated spleen cells; CTL, cytotoxic T lymphocytes; IL-2, interleukin 2; PGE,, prostaglandin Es; PG12,prostacyclin; TNP, trinitrophenyl; Hepes, 4-(2-hydroxyethyl> 1-piperazineethanesulfonic acid; BSS, balanced saline solution; PMA, phorbol myristic acetate. 43 0008-8749185$3.00 Copyright 0 1985 by Academic Ras, Inc. All rights of reproduction in any form reserved.
44
HiiCKER-SHAHIN
AND
DRijGE
effect of PGE2 in vitro was observed in cultures with additional helper factor(s). The suppressive effect of PGE2 was usually observed in cultures without an external source of helper factor(s) (4). The demonstration of the augmenting effect of PGE2 in cultures excludes certain interpretations, such as altered migration pattern of lymphocytes in vivo, and it may be useful to analyze the mechanism of the immune stimulatory effects of PGEz in detail. MATERIAL AND METHODS Animals. The mice were obtained from Bomholtgard, Ry, Denmark. Most of the experiments were performed with 8- to lo-week-old male animals. Activation of cytotoxic T lymphocytes in vivo. C3H mice were immunized by injection of 5 X 10’ irradiated (1500 t-ad) allogeneic spleen cells ip. The mice were sacrificed 5 days later, and their spleen cells were tested for cytotoxic activity against “0-labeled target cells at the indicated attacker:target cell ratios. Activation of cytotoxic T lymphocytes in vitro. Twenty million responder cells (usually C3H spleen cells) were incubated in a total volume of 4.5 ml culture medium (RPM1 1640, GIBCO medium supplemented with 10 mM L-glutamine (GIBCO), streptomycin/penicillin (GIBCO, 100 units/ml), 0.5% Hepes (GIBCO), 10% fetal calf serum (GIBCO), and 3 X low5 M 2-mercaptoethanol together with 0.4 ml EL-4 supematant and the indicated numbers of irradiated (1500 rad) stimulator cells for 5 days at 37°C in 5% CO2 if not indicated otherwise. Responder and stimulator cells were unfractionated spleen cells if not indicated otherwise. The cultures were tested after 5 days for cytotoxic activity as previously described (16, 17). Target cells. The target cells were prepared by culturing spleen cells with 2.2 &ml concanavalin A (Pharmacia, Uppsala, Sweden) for l-3 days. Irradiation of stimulator cells. The stimulator cells were irradiated with 1500 rad from a Gammacell 1000 D (Atomic Energy of Canada Limited, Commercial Products) equipped with a 2300 Ci 13’Cssource (1700 rad/min). The preparation of IL-2-containing supernatants from EL-4 thymoma cells. Supematants were obtained from an IL-Zproducing EL4 subline (kindly provided by Dr. J. Farrar) after induction with PMA as previously described (18). Briefly, lo6 EL-4 cells/ml were incubated together with 10 rig/ml of phorbol myristic acetate (PMA, Sigma) for 48 hr. The supematants were collected and stored frozen at -20°C. They usually contained 100-300 units/ml of IL-2. Preparation of T-cell-enriched nylon-wool-nonadherent spleen cells. Lymphoid cells were incubated in nylon-wool columns and eluted as described (19). The preparation of “plastic adherent cells” (AC). The term “plastic adherent spleen cells” is used in this paper for a fraction of spleen cells which was prepared by a procedure that has been reported to enrich especially for dendritic cells (20, 21). Spleen cells from three mice (approx. 3 X lo8 cells) were incubated in 10 ml culture medium in plastic petri dishes of 10 cm diameter (Greiner, Niirtingen, West Germany, Type TC94/ 16) at 37°C. After 2 hr, the supematant was discarded, the nonadherent cells were washed off carefully by extensive rinsing with warm culture medium or BSS, and the adherent cells were again incubated with 10 ml of fresh culture medium for another 18 hr. The cells which detached spontaneously during this time period (usually l-2 X lo6 cells) were then collected in the supematant and used as the “plastic adherent spleen cell population” in these
PGE2 AUGMENTS CYTOTOXIC
RESPONSES
45
studies. According to reports from another laboratory (20, 21), this population is a mixture of dendritic cells @O-95%) and macrophages. Preincubation of stimulator cells. In some experiments the stimulator cells were cultured at a density of 1.5-2 X 10’ cells/45 ml culture medium for 24 hr with 0.02 mg of concanavalin A (Con A, Pharmacia) per culture. The application of indomethacin and prostaglandins. Indomethacin is a nonsteroidal anti-inflammatory drug and a potent irreversible inhibitor of prostaglandin synthesis (22-24). A stock solution of indomethacin (Sigma) was prepared by dissolving 10 mg/ml in ethanol. The cell cultures usually contained 0.5 pg indomethacin/ml, i.e., 1:20,000 dilution of the stock solution in culture medium. The various types of prostaglandins were obtained from Sigma. Stock solutions ( 10e2M in ethanol) were stored at -20°C. The prostaglandins were applied to cultures at concentrations of 2 X lo-’ M if not indicated otherwise. Control cultures with corresponding concentrations of ethanol were also routinely tested. RESULTS Augmentation of cytotoxic responsesin vivo. The cytotoxic activity in the spleen after in vivo immunization of C3H mice with allogeneic spleen cells ip was usually 30
20 10
----Q@dWddElYO q aededday-l
-----
o-L-4 2
irL I
20
p 0 E 0 $ .s
10 0
40-
I
I
PGIp
3020lo-
o-ty
,
1x10-6 mm.
I
3x10-6 1x10-5 3x10-5 of prostaglandins (Molll)
ethanol
FIG. 1. Augmentation of cytotoxic responsesagainst allogeneic cells by graded dosesof prostaglandins. C3H mice were immunized by injection of 5 X IOr C57BL/6 cells in 0.2 ml BSS ip. The mice also received ip injections of 0.5 ml BSS containing the indicated concentrations of prostaglandins. Control groups received 0.5 ml of a 0.3% solution of ethanol in BSS, because the prosmglandin solutions were routinely prepared from lo-* M stock solutions in ethanol. The cytotoxic activity of the spleen cells was tested 5 days after immunization at attacketltarget cell ratios 100~1and 20: 1. The figure shows the data at the ratio 200: 1. The SE values for the individual points (51Cr-releasedata) were in all our experiments less than 3%, usually even less than 2%.
46
HACKER-SHAHIN
AND DRijGE
very weak (see Fig. 1). The cytotoxic responseswere markedly augmented, however, by the simultaneous injection of prostaglandins (PGEz, PGE, , or PGIz). The doseresponse was similar for all three types of prostaglandins (Fig. 1). There was, however, a difference with respect to the optimal time of administration. The extremely labile PG12had to be administered at the time of immunization (Day 0) and was ineffective if given the day before. The more stable compound PGE, produced the most effective augmentation on Day -1 and slightly weaker effects on Day 0; and PGEz expressed an intermediate behavior (Fig. 1). A possible explanation for these differences is that the relatively more stable compound PGE, might have persisted in sufficient concentration into the period after immunization and might have inhibited the ongoing activation of the CTL. Repeated injections of PGEz or PGE, on the day of immunization and on several subsequent days were indeed found to suppress the cytotoxic response (data not shown). The time course of the cytotoxic response after immunization in combination with PGE2 or PGEl was similar to primary cytotoxic responses in vitro: The peak activity was found on Day 5 (Fig. 2). The experiment in Table 1 illustrates two points: First, PGDz, PGF,, and PGF2, did not augment the cytotoxic response in vivo. (Similar results were also obtained
B) PGE1 day 0 P I e
lo-
b 6 0 so
o
8 3
0
3
5
7
10
day after immunization
FIG. 2. Time course of the cytotoxic response in vivo alter treatment with PGEz or PGE,. C3H mice were immunized with 5 X 10’ irradiated (1500 tad) C57BL/6 spleen cells ip and sacrificed 3, 5, 7, or 10 days later. The cytotoxic activity of the spleen cells against C57BL/6 target cells was then tested at the attacker:target cell ratios 200: 1 and 40: 1. The mice in panels A and B also received 0.5 ml of an 1 X IO-* M PGEr or PGE, solution, respectively, on the day of immunization (Day 0). The mice in panel C received the same amount of PGE, on the day before immunization.
PGEr AUGMENTS CYTOTOXIC
47
RESPONSES
TABLE 1 The Effect of Different Types of Prostaglandins on the Activation of Cytotoxic Activity in Viva Immunized with C57BL/6 cells
Without immunization
Prostaglandins injected
200: 1
40: 1
200: 1
40: 1
None (ethanol in BSS) PGEr PG12
0.8 29.0 14.3 0.9 2.8 0.5
-1.6 24.1 12.6 0.6 1.8 1.0
0.6 3.8 3.1 5.7 5.0 3.6
-1.5 0.1 0.3 -1.3 0.3 -3.0
=D2
PGFI, EF2.7
Note. BSS (0.5 ml) containing 1 X 10e5M prostaglandins of the indicated type and 0.1% ethanol was injected into C3H mice ip. Some of the mice were immunized 1 day later with 5 X 10’ irradiated (1500 tad) C57BL/6 spleencells in 0.2 ml BSSip. The mice were sacrificed6 days after the injection of prostaglandins (i.e., 5 days after immunization). The data show the cytotoxic activity (% specific “Cr release)of the spleen cells against C57BL/6 target cells at the attacker:tatget cell ratios 200: 1 and 40: 1. The cytotoxic activity against DBA/Z and A.TH target cells was also tested but was always less than 2%.
in vitro as discussed later.) Second, PGE;, and PGI2 did not induce a polyclonal response. The cytotoxic response required the combination of prostaglandins and immunizing antigen (C57BL/6) and was specific for that antigen. There was no cytotoxic activity against the unrelated target cells DBA/2 and A.TH. Augmentation of cytotoxic responses in vitro. In order to study the augmenting effect of PGE2 in vitro, we routinely provided the cultures with EL4 supernatant as a source of helper factor, since PGEz has been reported to inhibit the production of IL-2 (10, 13, 14). The EL-4 supernatant also contained at least one additional helper factor (25) and a small amount of PMA. The response of C3H spleen cells against irradiated BALB/c stimulator cells in such cultures was found to be strongly augmented by the addition of 10e6A4 PGE2 and reduced to almost background levels by the addition of 0.5 &ml of indomethacin (Fig. 3, left panel). The response was still specific for BALB/c, indicating that PGE2 did not trigger a polyclonal activation of CTL precursor cells. Since we have recently observed that different types of stimulator cells differ strongly in their ability to stimulate cytotoxic responses (26), we studied the hypothesis that the exceptionally immunogenic stimulator cells, i.e., an adherent cell preparation enriched for dendritic cells (AC) and concanavalin A-activated spleen cells (Con A-spl) (26), stimulate most effectively because they produce prostaglandins. The hypothesis predicted that responses against these types of stimulator cells would show a less stringent requirement for externally added PGE2. Our experiments (Fig. 3, middle and right panels) did not support this hypothesis: the cytotoxic responsesagainst AC or Con A-spl stimulator cells were also strongly augmented by PGE2. Similar effects were observed with nylon-wool nonadherent splenic T cells as stimulator cells (data not shown), which were previously found to be exceptionally weak stimulator cells (26). The augmenting effect of PGE2 was observed if PGE2 was added at the initiation
48
HACKER-SHAHIN
AND DRiiGE stim:
A) Target: c57 BL16
40
stim: 2 x 105 BALBlc
3
4
5
stim: spl 5
6
7
3
2 x to5 BALBlc + 2 x lO-‘BL/G-AC
4
5
2 x 105 BALE/c spl f + 2 x I+ BL/6-Con A st9 j
spl ‘r f
6
7 day of culture
FIG. 3. The effect of PGE2 and indomethacin on the development of the cytotoxic response against different types of stimulator cells in vitro. C3H spleen cells (2 X 10’) were incubated in 4.5ml cultures with 2 X 10’ irradiated BALB/c splenic stimulators and with or without 2 X 10’ irradiated C57BL/6 AC or 2 X IO4irradiated C57BL/6 Con A-spl. All cultures also received 0.4 ml EL-4 supematant (containing 80 units of IL-2), and some of the cultures also received F’GE2at a final concentration of low6M or 2 pg indomethacin per culture. The cytotoxic activity against C57BL/6 and BALB/c target cells was determined at the indicated time intervals after the initiation of the culture. Cytotoxic activity was determined at attacker:target cell ratios 25: I, 5:1, and I:l. The figure shows the data at the ratio of 25:l.
of the cultures but not if added 1 day later (Table 2). The addition of PGE2 on Day 1 produced a slight suppression. The comparison of different types of prostaglandins revealed that the in vitro responseswere augmented by PGEz and PGE, , but not by several other prostaglanTABLE 2 PC& Augments Cytotoxic Responsesonly if Added to the Culture on Day 0. No PC&
F’GEz Day 0
PGEt Day I
Stimulator cells
Target cells
25:l
5:1
25:l
5:l
25:1
5:1
C57BL/6
C57BL/6 BALB/c BALB/c C57BL/6
12.0 -1.3 12.4 -4.5
0.9 -1.3 3.2 -3.5
33.0 2.5 40.1 -0.1
11.8 -1.1 16.2 -2.1
5.9 -2.3 7.6 -1.8
-1.9 -1.5 4.4 -4.1
DBA/2
Note. C3H responder cells (2 X IO’) were incubated with 2 X lo6 irradiated C57BL/6 or DBA/Z stimulator cells and 0.6 ml EL-4 supematant for 5 days in 4.5-ml cultures. Some of the cultures received additionally 0.5 ml of a 3 X low6 M PGEz solution either at the start of the culture (Day 0) or 1 day later (Day 1). The data show the % specific 5’Cr release of C57BL/6 and BALB/c target cells at the attackerztarget cell ratios 25: 1 and 5: I.
PGEz AUGMENTS CYTOTOXIC
49
RESPONSES
dins, including prostacyclin (PG12, Fig. 4). The discrepancy between this and our in vivo results (Fig. 1) reflects, possibly, the extreme instability of PG12. DISCUSSION The major result from these experiments was that some prostaglandins including PGE2 augment cytotoxic responsesagainst allogeneic cells in vivo and, under certain conditions, also in vitro. Whereas PGEz has previously been reported to inhibit cytotoxic responses (4, 5), this is the first report that we are aware of about a potentiating effect on cytotoxic responses. The expression of the augmenting effect of PGE2 in vitro was only observed when the cultures were provided with an IL-Zcontaining helper factor. Control cultures without the helper factor were usually suppressedby PGE2 (data not shown). This is compatible with previous observations that PGE2 inhibits the production of IL-2 (10, 13, 14). IL-2-containing EL-4 supernatants have been added to our in vitro experiments primarily in order to avoid an indirect effect of PGE2 via its effect on the IL-2-producing helper cells. The EL-4 supematant contained also at least one additional helper factor (25) and a small amount of PMA, which might also serve as a cofactor in the stimulation of the CTL precursor cells. However, the supematant did not stimulate a substantial cytotoxic response in the absence of the corresponding antigen (see Fig. 3, BL/6-Target). Moreover, our observation that the in vivo activation of CTL is augmented by PGE2 indicates that our culture system with the IL-Zcontaining EL4 supematant represents a physiologically relevant situation. Positive regulatory effects of PGE have already been observed with other immune responses (1-3, 15, 27, 28); most of these reports have been dealing with in viva observations. This fact may underscore the biological significance of these effects. The disadvantage of the in vivo systems is, however, that complex interpretations such as altered migration properties of lymphocytes in vivo cannot be excluded. Our demonstration of an immune potentiating effect of PGEp in vitro excludes such an interpretation and may be useful for the further analysis of this regulatory effect. 30
1
no PG
no PG. indomethacin
PGE,
d PGE,
PGD,
1
PGFp
FIG. 4. Effect of different types of prostaglandins and indomethacin on the cytotoxic response. C3H spleen cells (2 X 10’) were incubated with 2 X lo6 irradiated C57BL/6 spleen cells in 4.5-ml cultures containing 0.4 ml EL4 supematant with or without 2.0 pg indomethacin and with the indicated types of prostaglandin at a final concentration of 2 X lo-’ M. The data show the cytotoxic activity against C57BL/6 target cells at the attacker:target cell ratio 5: 1.
50
HACKER-SHAHIN
AND DROGE
There are several possible mechanisms that could explain why PGE2 augments T-cell functions in some experimental situations and inhibits them in others: (i) PGEZis known to increase the cellular concentrations of cyclic AMP in lymphocytes ( 11,29) and this may be important for the late cyclic events in activated lymphocytes but inhibitory in the early phase of the cell cycle (29). The in vivo situation may be characterized by suboptimal concentrations of cyclic AMP. (ii) Another interpretation is that PGEl inhibits not only the activation of lymphokine-producing cells and CTL precursor cells, but also the action of some types of suppressor T cells. It is quite conceivable that a slight inhibition of the effector precursor cell population together with an inhibition of a suppressor cell system can provide a positive net effect on the effector cell activation. (iii) It is also conceivable that PGEz is one of the activating signals which are produced by the stimulator cells and delivered to effector precursor cells during the stimulation process. It has been reported that stimulator cells induce proliferative responses at relatively low doses and suppress at higher cell numbers (30, 31). PGE2 may be responsible for both, the inducing and the inhibitory effect, in a dose dependant way. It should be noted, however, that PGE2 does not induce a polyclonal response (see Fig. 3 and Tables 1 and 2). This would have been expected if PGE2 would function as the only relevant signal of the stimulator cells. (iv) The possibility that PGE2 activates the immunogenic potential of the stimulator cells appearsunlikely, since several attempts to demonstrate an increased immunogenic potential after preincubation of the stimulator cells in PGEs-containing cultures have failed (W.D., unpublished). (v) The alternative possibility that PGE2 may inhibit previously activated cells in the responding lymphocyte preparation and thereby reduce the background of proliferating cells, which may compete for lymphokines or other positive signals with the antigenspecific response under test, seemsalso unlikely. Experiments on the effect of PGEz on the antigen-driven proliferation in relation to the background proliferation did not confirm this possibility (W.D., unpublished). (vi) At present there are no data that support or exclude the possibility that PGEz induces the maturation of CTL by stopping the proliferative phase of the response. The observation that the augmenting effect of PGE2 is only seen if PGE2 is present early in the course of the response (Table 2) could be explained by the assumption that it induces the production of a late maturation factor. ACKNOWLEDGMENTS The expert technical assistanceof Mrs. Heike Schmidt and Mrs. Sabine Panknin and the assistanceof 1. Schmoch-Fryson and Mrs. J. Rami in the preparation of this manuscript is gratetilly acknowledged.
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RESPONSES
51
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Mertin, J., Stackpoole, A., and Shumway, S. J., Transplantation 37, 4, 1984. Stobo, J. D., Kennedy, M. S., and Goldyne, M. E. J. Clin. Invest. 64, 1188, 1979. Hadden, J. W., and Coffey, R. G., Immunol. Today It, 299, 1982. Lamb, J. R., Skidmore, B. J., Green, N., Chiller, J. M., and Feldmann, M., J. Exp. Med. 157, 1434, 1983. 31. Matis, L. A., Glimcher, L. H., Pam, W. E., and Schwartz, R. H., Proc. Natl. Acad. Sci. USA 80, 6019, 1983.