Mitogen-induced changes in lymphocyte prostaglandin levels: A signal for the induction of suppressor cell activity

CELLULARIMMUNOLOGY41, 72-85 (1978)

Mitogen-lnduced Changes in Lymphocyte Prostaglandin Levels: A Signal for the Induction of Suppressor Cell Activity* DAVD R. WEBB z AND IRENE NOWOWIEySKI

Roche Institute of Molecular Biology, Nutley, New Jersey 07110 Received March 30, 1978 Glass-wool adherent T lymphocytes suppress P H A stimulated DNA synthesis in non-adherent lymphocytes. This suppression is blocked by adding inhibitors of prostaglandin synthetase to the cultures. P H A stimulates the appearance of prostaglandin in cultures of both non-adherent and glass-wool-adherent lymphocytes between 24 to 48 hr after stimulation. Since the non-adherent lymphocytes are sensitive to prostaglandin induced suppression of DNA synthesis only when the prostaglandin is added between 0 to 24 hr after P H A stimulation, it is unlikely that prostaglandin directly blocks DNA synthesis in the non-adherent lymphocytes. The glass-wool-adherent lymphocytes can be stimulated directly by the addition of PGE2 to become suppressor cells and to release a soluble suppressor product. P H A added in the presence or absence of prostaglandin synthetase inhibitors does not directly activate the glass-wooladherent suppressor cell. These data have led us to suggest a model for the interaction of the nonadherent, PHA-sensitive lymphocyte with the glass-wooI-adherent suppressor ceil in which the non-adherent lymphocyte is stimulated by P H A to make prostaglandin which in turn stimulates the glass-adherent suppressor cell to produce its soluble suppressor product which then blocks P H A induced DNA synthesis in the non-adherent lymphocytes. Thus, these data suggest that some lymphocyte subpopulations may be stimulated directly by products of other lymphocyte populations in the absence of any mitogenic signal.

INTRODUCTION T h e r e g u l a t i o n of i m m u n o c o l n p e t e n t cell function by soluble m e d i a t o r s has come u n d e r intense investigation in recent y e a r s (for review see Ref. 1). T h e studies have c o n c e n t r a t e d on identification of m e d i a t o r s in t e r m s of their effects in v a r i o u s bioassays. R e l a t i v e l y little is k n o w n about their chemical nature, m o d e of synthesis or the m e c h a n i s m by which they e x e r t their effects. R e p o r t s f r o m this l a b o r a t o r y a n d others ( 2 - 9 ) have implicated the p r o s t a g l a n d i n s as i m p o r t a n t m e d i a t o r s of i m m u n o c o m p e t e n t cell function. A l t h o u g h it is k n o w n that e x o g e n o u s l y a d d e d p r o s t a glandins can s u p p r e s s v a r i o u s l y m p h o c y t e a n d m a c r o p h a g e functions, much less is k n o w n about the role of e n d o g e n o u s l y synthesized p r o s t a g l a n d i n in the control of l y m p h o c y t e or m a c r o p h a g e activity. I t has been shown p r e v i o u s l y that antigens and mitogens can induce increases in p r o s t a g l a n d i n in whole spleens or spleen cell cultures a n d t h a t these increases, in general, p l a y a negative role in r e g u l a t i n g antigen or m i t o g e n stimulated cell functions (2~4, 8 ) . I t has not been directly shown * In memory of our colleague Andrew Jameison. 1 To whom any correspondence should be addressed. 72

0008-8749/78/0411-0072502.00/0 Copyright © 1978 by AcademicPress, Inc. All rights of reproductionin any form reserved.

PROSTAGLANDIN-INDUCED

SUPPRESSOR CELL

73

whether lymphocytes are capable of synthesizing prostaglandins in response to a stimulus although indirect evidence has been presented previously from this laboratory (4). In experiments already reported (3) from this laboratory, it was established that a glass-adherent splenic T cell could inhibit DNA synthesis in non-adherent spleen cells stimulated with P H A or concanavalin A. It was shown that this suppression was not the result of cytotoxicity or due to cold thymidine competition. The inhibition was blocked if inhibitors of prostaglandin synthesis were added to mixtures of the two populations stimulated with PHA. In this report, we present evidence that lymphocyte populations make prostaglandin in response to PHA. The function of this endogenously synthesized prostaglandin appears in part to regulate the activity of a suppressor cell population which responds to this prostaglandin stimulus by making a soluble suppressor substance° The resulting interaction is a "flywheel" type regulatory mechanism which has been hypothesized to operate in the control of some aspects of immune responses (10). MATERIALS AND METHODS

Animals. C57B1/6J males 6 to 10 weeks of age were obtained from Jackson Laboratories, Bar Harbor, Maine. Mitogens. Phytohemagglutinin (PHA) was purchased from Burroughs-Wellcome (Research Triangle Park, N. C.) in lyophilized form. It was suspended in RPMI1640 and kept frozen at - 20°C until use. Prostaglandins ( PG ) and prostaglandin synthetase inhibitors ( PG SI) . Prostaglandins E2 and F2~, a gift of Hoffmann-LaRoche (Nutley, N. J.), were stored either in lyophilized form at 5°C or were dissolved in 95% ethanol and stored at --20°C. The prostaglandin synthetase inhibitors, indomethacin (Sigma, St. Louis, Mo.) a non-reversible inhibitor, D,L-6-chloro-a-methylcarbazole-2-acetic acid (Ro20-5720) a reversible inhibitor, and octadeca-9,12-diynoic acid (Ro-3-1314) a nonreversible inhibitor (both gifts of Hoffmann-LaRoche), were stored in dry form at either room temperature or at - 2 0 ° C and dissolved in a bicarbonate solution immediately before use. Preparation and culture of spleen cells. The separation of spleen cells into adherent (GAL) or non-adherent (NAL) lymphocyte fractions on glass-wool columns has been described previously (3). Analysis of the cell populations using cytotoxie antisera shows that NAL are composed of 75 to 85% T cells, 15 to 25% B cells with less than 5 % macrophage contamination. GAL populations are made up of 50 to 70% B cells and 30 to 50% T cells with less than 5% contaminating macrophages. GAT lymphocytes were rountinely prepared by incubating GAL with the appropriate dilution of a rabbit anti-mouse ~ chain specific antisera (Microbiological Associates, Bethesda, Md.) in the presence of guinea pig complement for 1 hr at 37°C on a rotating device. The remaining cells were washed extensively in HBSS and counted using trypan blue staining as a measure of viability. These cells are > 95 % T cells as measured using specific B cell mitogens and anti-thymocyte antisera. Splenic macrophages were prepared by incubating whole spleen cell suspensions in MEM + 10% heat inactivated fetal calf serum for 30 rain on plastic petri plates at 37°C. The plates were then rinsed three times with several-fold volumes of warm HBSS. The adherent cells, composed of > 95% macrophages, were removed

74

WEBB AND NOWOWIEJSKI

using a rubber policeman and counted using a hemocytolneter and a viable stain (trypan blue). The subfractionation of NAL on G-10 Sephadex was carried out using the procedure of Ly and Mishell (11). The presence of macrophages was assayed on the basis of carbon ingestion and adherence to glass. Splenic lymphocytes were cultured in microtiter trays in RPMI-1640 + penicillin-streptomycin, glutamine. In some experiments, 0.1 M Hepes was also added. The concentrations of lymphocytes varied between 5 × 10~ or 1 × 106 per well. For measurement of P H A stimulated PG levels the cell concentration used was 1 × 10Z/mI which previously had been found to be within optimal range for mitogen stinmlation of PG and DNA synthesis (data not shown). The dose range for optimal stimulation of DNA synthesis was found to be between 0.62 t~g to 2.5/~g/ml. The lowest dose giving optimal (maximum) [3H]dT incorporation varied somewhat frona experiment to experiment; therefore, the data presented here were selected on the basis of that dose of P H A which gave optimum stimulation in a given experiment. This procedure helps to insure that effects being observed are not due to fluctuations in mitogen responsiveness. Measurement of DNA synthesis. [SH]thymidine ([aH]dT) incorporation was used as a measure of DNA synthesis (12). The microtiter cultures were harvested using a sample harvester (Mash II, Microbiological Assoc., Bethesda, Md.) as previously described (3). Measurement of prostaglandin. Immunoreaetive PG (iPG) was measured using an antibody to PGE1 prepared by Miles Laboratories using methods previously described (2, 13, 14). The antibody used for the radioimmunoassay of PGFz~ was a gift of Dr. J. Paulsrud (Hoffmann-LaRoehe). Its specificity and activity have been determined to be limited strictly to prostaglandins F ~ and F ~ and structurally similar analogues exclusive of the A, B, and E series (J. Paulsrud, personal communication). The assay of PGF2~ has also been previously described (2, 13, 14). The range of sensitivity of these assays is 10 to 1000 picograms PG. RESULTS

Stimzdatlon of prostaylandin synthesis by PHA. In a previous report (3), experiments were presented using PGSI which suggested that PG played a role in the GAL mediated suppression of P H A stimulated DNA synthesis in NAL cultures. This made it important to determine the amount of PG generated in culture by mitogen stimulated NAL, GAL, and N A L / G A L mixtures. In the experiments presented in Table 1, the separated subpopulations of non-adherent lymphocytes (NAL) and glass-adherent T cells (GAT) and the recombined N A L / G A T were cultured in the presence or absence of PHA, and the amount of PG made by the different populations was assessed at different times. The results show that iPG appears in the cultures between 24 to 72 hr in NAL, GAT, and N A L / G A T (1 : 1 mixture). Also, most of the PG made does not seem to be PGF2~. Although > 90% of the macrophages are removed from NAL during glass wool fractionation, it was still conceivable that macrophages could be involved in mitogen stimulated PG synthesis. To assess this possibility, NAL were additionally fractionated over Sephadex G-10. NAL were chosen because they are the principal mitogen responsive population in terms of stimulation of DNA synthesis and the removal of macrophages on G-10 Sephadex did not result in significant loss of lymphocytes

PROSTAGLANDIN-INDUCED

SUPPRESSOR CELL

75

(as was observed with GAT). NAL (5 × 106 cellsflnl) were cultured with PHA for 24 or 48 hr. No macrophages could be detected in these cultures as measured by adherence to glass and uptake of colloidal carbon. As may be seen by the data in Table 2, the amount of PG appearing in the cultures following G-10 purification is comparable to the same preparation of NAL not passed over G-10. Since we had previously reported that PGSI could reverse GAL suppression of NAL DNA synthesis stimulated by PHA, it was important to determine the degree of inhibition of PG synthesis obtained. Although any of the cell populations may be used, we chose NAL which were cultured in the presence of PHA--PGSI. The iPG was assayed 24, 48, or 72 hr after culture initiation (Table 3). Additionally, cultures were exposed first to P H A and then PGSI 24 hr after PHA TABLE 1 Stimulation of Prostaglandin Levels by PHA in Spleen Cell Subpopulations Cell population

NAL

GAT

NAL/GAT (l:l)

Culture time

Average net iPG (ng/culture)~

24 48 72

(5)

2.74 4- 0.9 P < 0.005 6.82 4- 2.0 -P < 0.005 5.0 4- 3.1 P < 0.005

24 48 72

(3)

1.27 4- 0.1 P < 0.05 8.17 4- 2.1 P < 0.005 5.4 =k 2.1 P < 0.005

24 48 72

(2)

1.4 4- 0 . 0 1 P < 0.05 10.0 4- 4.2 P < 0.005 11.1 4- 0.1 P < 0.005 Average net P G F ~ (ng/culture) ~

NAL

GAT

N A L / G A T (1:1)

24 48 72

(4)

24 48 72

(2)

24 48 72

(2)

0.45 4- 0.1 N.S. 0.55 4- 0.1 N.S. 0.15 4- 0.01 N.S. No net change No net change No net change 3.9 4- 0.2 _P < 0.05 2.6 4- 0.I P < 0.05 4.9 4- 0.5 P < 0 . 0 5

These values represent the mean PG levels from several experiments. In each experiment, cultures were prepared in duplicate or triplicate and incubated with or without mitogen for varying times. At the end of the incubation period, the cultures were assayed for PG as described in Materials and Methods. The net PG value was obtained by subtracting the mean PG level in control (nonmitogen-treated) cultures from the mean PG level in mitogen-treated cultures for each individual experiment. The PG levels depicted here represent the average net PG levels of the several experiments 4. standard error. The number in parenthesis indicates the number of separate experiments performed from which the average values were obtained. The average basal levels of iPG in NAL (24-72 hr) was 0.62 4- 0.18 ng; GAT basal levels were 5.56 4. 1.07 ng; and N A L / G A T basal level averaged at 4.23 4- 1.61 ng; basal level of N / G A T PGF=~ was 1.06 4- 0.07 ng/culture. Calculation of levels of significance was performed using analysis of variance (ANOVA) (28). N.S. = not significant.

76

WEBB AND NOWOWIEJ-SKI TABLE 2 Effect of G-10 Sephadex Filtration on Glass Wool Purified, Mitogen Responsive Splenic L y m p h o c y t e s - - M e a s u r e m e n t of Prostaglandin Levels

Time

24 48

NAL

NAL-G-10

iPG ~

PGF2~ ~

iPG •

PGF2~ ~

1.4 4- 0.1 P < 0.05 2.2 4- 0.6 P < 0.05

0.4 4- 0.02 0.0 --

0.9 4- 0.5 N.S. 2.0 4- 0.6 _P < 0.05

0.4 4- 0.02 0.5 4- 0.05

These values represent the net values of P G in ng/culture 4- standard error, the control levels of the relevant P G having been subtracted. The basal level of iPG (24-48 hr) was 0.37 4- 0.07 in NAL, and 0.39 4- 0.09 in NAL-G-10. The basal level of PGF2~ was 0.27 4- 0.10 in NAL and 0.18 4- 0.03 in NAL-G-10. The level of significance was determined as in Table 1. The number of NAL cells/culture was 5 X 106. exposure and iPG measured

48 hr after culture initiation. The data are reported

as t h e i n c r e m e n t of s u p p r e s s i o n of i P G i n c u l u r e s r e c e i v i n g P H A cultures receiving PHA

+ PGSI

versus

alone. As can be seen when the inhibitors are added at the

t i m e of c u l t u r e i n i t i a t i o n , i P G levels a r e e f f e c t i v e l y r e d u c e d b y 4 8 h r . H o w e v e r , if t h e P G S I a r e a d d e d 2 4 h r a f t e r c u l t u r e i n i t i a t i o n , t h e d e g r e e of i n h i b i t i o n is e i t h e r absent or greatly diminished.

Effects of exogenous addition of PG on P H A stimulated D N A synthesis in N A L . S e v e r a l i n v e s t i g a t o r s h a v e p r e v i o u s l y s h o w n t h a t w h e n P G E 1 o r E2 is a d d e d a t t h e t i m e of c u l t u r e i n i t i a t i o n m i t o g e n i n d u c e d t r a n s f o r m a t i o n c o u l d b e i n h i b i t e d ( 1 5 - 1 7 ) . I n T a b l e 4 is s h o w n t h e r e s u l t s of e x p e r i m e n t s i n w h i c h P G E 2 w a s TABLE 3 Prostaglandln Levels in NAL Cells After Exposure to Prostaglandin Synthetase Inhibitors and P H A Culture time (hr.)

Drug

E / C (iPG) ~

24

5720 1428 Indo

10-6 Nr 10 -7 M 10 -7 M

0 addition 0 addition 0 addition

0.70 N.S. 0.40 P < 0.05 0.30 P < 0.005

48

5720 1428 Indo

10-8 M 10-7 M 10-7 M

0 addition 0 addition 0 addition

0.20 P < 0.005 0.33 P < 0.005 0.25 P < 0.005

72

5720 1428 Indo

10 -6 M 10-~ M 10-7 M

0 addition 0 addition 0 addition

0.19 P < 0.005 0.50 P < 0.005 0 . 1 9 P < 0.005

48

5720 1428 Indo 1314

10 -6 10 -7 10 -7 10-v

M 24 addition 3// 24 addition M 24 addition M 24 addition

1.28 3.75 1.0 4.8

N.S. P < 0.05 N.S. P < 0.05

The E / C values were calculated by dividing the average amount of iPG measured in triplicate cultures in groups receiving PGSI + P H A (0.5 vg/ml) by the average amount of iPG in control cultures receiving P H A only. The n u m b e r of cells/culture was 1 X 107. Average base levels of iPG (24-72 hr) were 0.38 4- 0.09. Statistical analysis was carried out using analysis of variance (28). N.S. = not significant.

PROSTAGLANDIN-INDUCED

SUPPRESSOR CELL

77

a d d e d at the time of culture initiation or at various times thereafter. T h e d a t a shows that P G E 2 m a y inhibit D N A synthesis ( m e a s u r e d after 72 h r culture) w h e n it is a d d e d up to 48 h r after culture initiation. T h e degree of inhibition, however, diminishes w h e n P G E 2 is a d d e d after 24 hr. T h i s is m o r e clearly shown w h e n a P G S I is p r e s e n t in the cultures to p r e v e n t e n d o g e n o u s synthesis of P G . I n this instance, P G E ~ i n d u c e d inhibition of D N A synthesis d i s a p p e a r s w h e n it is a d d e d after 24 hr. Delayed addition of PGSI to PHA stimulated cultures of N A L / G A L . I n conj u n c t i o n with e x p e r i m e n t s showing t h a t P G S I cannot block P G synthesis w h e n a d d e d 24 h r after culture initiation, e x p e r i m e n t s were p e r f o r m e d to m e a s u r e the effects of P G S I on D N A synthesis in N A L / G A L cultures. I n T a b l e 5 a r e shown the results of a n e x p e r i m e n t in which indomethacin was a d d e d to cultures of N A L / G A T in which the relative concentration of G A T to N A L was varied. T h e data show t h a t the ability of i n d o m e t h a c i n to block G A T suppression is related to the concentration of G A T cells present. A t lower concentrations of G A T which still give 30 to 50% suppression, an almost complete reversal of s u p p r e s s i o n is achieved w h e n indomethacin is a d d e d to the cultures. T h e d a t a in T a b l e 6 in which a high ratio of G A L / N A L was used show that w h e n Ro-20-5720 or i n d o m e t h a c i n is a d d e d 24 or 48 h r after culture initiation, no reversal of G A L suppression is observed. P G S I a d d e d at 0 time p a r t i a l l y reverses s u p p r e s s o r effects. T h i s e x p e r i m e n t also suggests that the P G S I are not non-specifically enhancing [SH] d T inc o r p o r a t i o n since such e n h a n c e m e n t w o u l d be likely to occur w h e n e v e r the P G S I TABLE 4 Effects of the Exogenous Addition of Prostaglandin on DNA Synthesis in NAL Stimulated by PHA Culture conditions

PHA PHA PHA PHA PHA PHA PHA PHA PHA

(0.5 ~g/ml) -[- PGE 10-6 M + PGE 10-7 M + PGE 10-6 M + PGE 10-7 M + PGE 10-6 M + PGE 10-7 M + PGE 10-~ M + PGE 10-7 M

PHA PHA PHA PHA PHA

(0.25 ~g/ml) (0.125 ~g/ml) qq- (0.125 gg/ml) + (0.125 ~g/ml) q- (0.125 gg/ml)

Time of addition

0 0 24 24 48 48 70 70

Ro-20-5720 (10 6 M) + PGE2 10-6 M + PGE2 10-6 M + PGE2 10-6 M

0 24 48

CPM 4- SE a

35,293 6,664 8,458 4,651 9,475 15,849 26,849 33,239 32,657

Expt. 1 ± 2,878 -4- 1,015 (0.18) -4- 88 (0.23) -4- 743 (0.13) 4- 1,721 (0.27) -4- 3,084 (0.45) ± 1,727 (0.76) -4- 1,136 (0.94) 5:3,769 (0.93)

1o < _,o < .P < 2" < _P < P < N.S. N.S.

0.001 0.001 0.001 0.001 0.02 0.1

Expt. 2 138,948 4- 4,611 189,640 5:4,736 103,5435:1,839 (0.55) P < 0.05 114,139 4- 6,102 (0.60) P < 0.05 180,5754- 3,437 (0.95) N.S.

These values represent the mean CPM of triplicate cultures exposed to [*H]dT for 4 hr (Expt. 1) or 24 hr (Expt. 2) =1=standard error. The values in parenthesis represent the E/C value obtained by comparing the control group (PHA + Ro-20-5720) with groups receiving PHA + PGSI + PGEe. Levels of significance were determined by using Students' t test (28). N.S. = not significant.

78

WEBB AND N O W O W I E J S K I

TABLE 5 Effects of Indomethacin on GAT Cell Suppression NAL DNA Synthesis Stimulated by PHA Cultures

[aH]dt incorporation Without indomethacin

NAL NAL/GAT 4/1 8/1 16/1 32/1 64/1

41,134 ± 4,528 5,650 4- 194 12,349 4- 4,077 19,847 ± 4,422 24,342 ± 1,437 26,804 ± 3,706

With indomethacin 54,336 4- 5,870 8,889 4- 1,335 P < 17,673 4- 2,942 N.S. 32,752 4- 5,909 P < 38,931 ± 3,195 P < 44,725 -4- 4,510 P <

0.1 0.05 0.05 0.05

Cultures received indomethacin (10.7 3A')at the beginning of the incubation period. The PHA concentration used was 0.125 ~g/mI. The values represent the mean total DH]dT incorporation after 72 hr in culture (24 br labeling period) ± standard error. Levels of significance were determined using analysis of variance (28) comparing groups which received indomethacin to groups which did not receive indomethacin. The number of NAL/culture was kept constant (1 X 10~ cells/well) and GAT were added in varying dilutions as indicated. N.S. = not significant. are added. The P G S I have little or no effect on D N A synthesis in isolated N A L or GAL. The effect of macrophages and PGSI on D N A synthesis in N A L . Macrophages are known to contribute to the regulation of lymphocyte function and they produce prostaglandin (1, 7). Therefore, experiments were performed to check the possibility that in spite of the low level of macrophages present in our cultures they might still be influencing the results. Accordingly, N A L were prepared and cultured with varying concentrations of splenic macrophages in the presence or absence of Ro-20-5720. The results of a typical experiment are shown in Table 7. It is clear that macrophages have no effect on P H A stimulated D N A synthesis in N A L cultures in the presence or absence of Ro-20-5720. Also, the ratio of N A L / m a c r o phages ( 1 0 0 / 1 - 5 / 1 ) is comparable to ratios of N A L / G A T where very good suppression is observed (see Table 5). This suggests that lnacrophages are not a significant component of this system. The role of PC in the control of mitogen activated suppressor function. The experiments presented in the preceding sections as well as data published previously (3) suggests that P G plays a role in modulating suppressor function. One possible explanation of how this occurs is that both N A L and G A T cells are producing P G which, in turn, could be regulating the functioning of the suppressor cell. To test this hypothesis, an experiment was designed in which an isolated population containing suppressor T cells ( G A T ) were exposed to P H A , PGE2, or P H A and PGE2 for 48 hr in the presence or absence of P G S I . Then, either the supernatants or the cells were tested for their ability to suppress N A L D N A synthesis. The purpose of these experiments was to test whether or not P G added exogenously would have any effect on suppressor function. The results of two such experiments are shown in Fig. la, b. In the experiment depicted in Fig. la, G A T cells were exposed to P H A , PGE2 (10 -5 M ) or both for 48 hr; the cells were then centrifuged, resuspended in fresh medium, counted using a viable stain (trypan blue), and m i x e d in various ratios with fresh N A L and exposed to P H A for 72 hr, then assayed for [ ~ H ] d T incorporation. The exposure

PROSTAGLANDIN-INDUCED

SUPPRESSOR

CELL

79

of G A T to either PGE2 or P H A + PGE2 markedly improves the suppressive capacity of '&e G A T cells. In Fig. lb, the data are derived from an experiment in which G A T cells were exposed to P H A , PGE=, or both in the presence of Ro-20-5720. A between-groups analysis of variance for both experiments showed that G A T exposed to P G E or P H A + P G E were significantly more suppressive at 1/40-1/80 ratio ( P < 0.001) than G A T controls or G A T receiving P H A alone. Use of a P G S I was done to eliminate any contribution of the P G synthetic pathway to the suppressor cell activity being measured. The results are essentially identical to that obtained using G A T cells which have not been treated with P G S I . In addition to using suppressor cells, supernatants were collected from G A T cells exposed to P H A , PGEz or both in the presence or absence of P G S I after 48 hr in culture (2a, b). These supernatants were extensively dialyzed against phosphatebuffered saline ( p H 7.2) and mixed with N A L cells stinmlated with P H A . Here, too, supernatants obtained from G A T ceils exposed to PGE2 or PGE2 + P H A show the most suppressor activity. A between-groups analysis of variance indicates that supernatants from G A T exposed to P G E or P H A + P G E are significantly more suppressive ( P < 0.001) at a dilution of 1/8 than supernatants from G A T exposed to saline or PI-IA alone. It should be noted that PGE2 may be added to G A T and the supernatants collected after 24 hr in culture; such G A T supernatants are also highly suppressive (data not shown). Recent experiments have shown that the suppressor product is sensitive to proteolysis and highly reTABLE 6 Effects of the Delayed Addition of Prostaglandin Synthetase Inhibitors on GAL Suppression of NAL DNA Synthesis Cultures

Time of addition

Expt. 1

Expt. 2 I-3H]dT Incorporation

Ro-20-5720 (10 6 M) NAL

GAL

-0 24 48 -

-

0 24 48 NAL/GAL (2 : 1)

-

-

0 24 48

Indomethacin (10-7 M)

94,976 4- 7,897 92,283 4- 7,523 79,398 4- 5,885 73,689 4- 7,921

71,574 4- 2,334 82,806 4- 5,534 84,057 -4- 5,769 89,153 4- 3,566

8,460 :t: 843 13,944 4- 3,127 11,086 4- 1,634 8,950 4- 37

27,115 4- 2,319 36,877 4- 2,177 32,788 4- 1,324 33,291 4- 1,220

25,225 4- 4,039 (74.0) 32,809 4- 1,535 (55.0) 46,793 4- 9,679 (49.2) P < 0.05 44,970 4- 4,040 (38.0) P < 0.05 28,699 4- 506 (72.0) N.S. 31,178 4- 2,469 (57.0) N.S. 34,723 4- 2,567 (62.9) N.S. 33,285 =t: 2,838 (54.0) N.S.

PHA dose used was 1.25 #g/ml for Expt. 1 and 0.625/zg/ml for Expt. 2.24 hr pulse with [-~H]dT from 48 to 72 hr. The numbers in parentheses represent the % suppression. Each value depicts the mean CPM of triplicate cultures 4- standard error. The level of significance was determined using analysis of variance (28) comparing NAL/GAL control (no additions) to NAL/GAL receiving Ro-20-5720. The number of NAL/culture was 1 X 108;the number of GAL/culture was 0.5 X 106. N.S. = not significant.

80

WEBB AND NOWOWIEJSKI

TABI,E 7 The Effect of Macrophagcs and l'rostaglandin Synthctasc Inhibitor on NAI, DNA Syul.hcsis Cultures containing

Ro-20-5720

Average CPM Triplicate culture

NAL NAL q- 1% Macrophages NAL + 10% Macrophages NAL + 20% Macrophages

q-+ --t--}-

46,403 ± 610 58,266 ± 1,431 48,365 ± 2,815 63,846 -4- 2,218 45,740 ± 7,256 56,557 -4- 907 38,989 ± 1,687 58,866 ± 952

The concentration of N A L / c u l t u r e was 1 X 10 ". The concentration of macrophages varied from 1 X 104 to 2 X 105/culture. The concentration of PHA used was 2.5 #g/ml. Incorporation of [3H]Tdr into macrophage cultures was negligible ( < 100 cpm).

sistant to heating suggesting that it is a heat-stable protein (T. Rogers, I. Nowowiejski, and D. R. Webb, in press). These experiments demonstrated that G A T could be activated by PGE2 in the presence of PGSI. That being the case, an experiment was designed to assess the capacity of N A L to stimulate G A T suppressor cell function under circumstances in which only the N A L were exposed to P I I A ~ PGSI. In this experiment N A L were placed in the inner chamber of a double chamber culture vessel (Marbrook type) and mixed with a high dose of Pt-IA (5 ~g/ml) ---Ro-20-5720 (10 -5 M ) . This dose of P H A was chosen because experiments showed that increasing the dose of P H A increased the amount of iPG which accumulates in the culture (data not shown). A dialysis membrane separated the N A L from G A T cells placed in the outer chamber. 1°HA could not pass through the dialysis membrane to affect the G A T ceils and as shown earlier, Ro-20-5720 should have no effect on G A T activation. Thus, this experiment could assess in a direct way the ability of P G synthesized by N A L cells to activate G A T suppressor ceils. The G A T cells were exposed to N A L for 48 hr, then collected, washed and incubated with fresh N A L stimulated with a dose of P H A (0.625 ~g/ml) which results in minimal iPG accumulation. The cultures were pulsed with [ 3 H ] T d r {roln 48 to 72 hr to assess D N A synthesis. The results are shown in Table 8. Using the dilution analysis technique, it is clear that G A T exposed to N A L + P I t A are far more active suppressors than G A T exposed to N A L + Pt-IA + Ro-20-5720. This strongly implies that P t I A stimulated N A L release a product of the P G pathway which can activate G A T suppressor cells. G A T exposed to N A L treated with saline had no suppressive capacity (data not shown). DISCUSSION Several investigators have presented evidence that antigens or mitogens may induce the synthesis of P G in the spleen (2, 8), or in spleen cell cultures (18) or in macrophage-rieh cultures (6, 7). Direct evidence as to the capacity of lymphoeytes to make P G has not appeared although indirect evidence has been presented (4). The data shown here using highly purified mouse splenic lymphocytes

PROSTAGLANDIN-INDUCED SUPPRESSOR CELL

81

as well as a recent report by Rapoport et al. (using human peripheral blood lymphocytes) (19) strongly suggest that lymphocytes can synthesize P G in response to mitogenic or antigenic stimuli. Tile increases in P G synthesis reported here using purified lymphocytes also correlate very precisely with mitogen-induced increases in P G in whole spleen cells reported by Ferraris and DeRubertis (18). That macrophages are not an active participant in this particular system is suggested by the data in Table 7. In our experiments, the addition of macrophages has a minimal effect on P H A stimulated D N A synthesis in N A L cultures in the presence or absence of a prostaglandin synthetase inhibitor. Having established that activated lymphocytes could apparently make PG, it was of interest to know what role P G synthesis played in regulating lymphocyte activation a n d / o r function. W e had previously established that the addition of P G S I could reverse to a significant degree the suppression of D N A synthesis observed when P H A stimulated N A L were mixed with G A T ceils (3). The data presented here show that significant amounts of iPG accmnulate after 24 hr in cultures of both NAL, GAT, and N A L / G A T . Thus, it is possible that P G could be suppressing D N A synthesis directly. Recent experiments of Goodwin et al. (20), as well as data from this laboratory (Rogers and Webb, unpublished observations) have shown that P G E concentrations as low as 10-8 M may give some suppression ( 1 0 - 3 0 % ) of P H A induced D N A synthesis. Thus, the nanogram quantities of P G could account for a portion of the suppression observed here. However, the amounts of endogenous P G present in the isolated populations of NAL, for example, seem to have little effect on D N A synthesis. If P G synthesis in N A L is blocked by PGSI, very little enhancement of the P H A induced D N A synthesis is observed (Ref. 3 and Tables 4-7) suggesting a limited role for endogenous PG in the direct suppression of N A L D N A synthesis. Another important point is the time element of suppression. P H A stimulated D N A synthesis begins 36 hr after culture initiation in this system (A. T. Jamieson and D. R. Webb, unpublished observations). Previous studies (3) had shown TABLE 8 Effect of NAL on GAT Suppressor Cell Activation Ratio of NAL/GAL 5/1

GAT exposed to NAL -k PHA

10/1

20/1 40/1 5/1 10/1 20/1 40/1 NAL control

NAL q- PHA q- Ro-20-5720

[-aH]Tdr incorporation 29,661 4- 2,516 (66%) P < 0.0005 52,737 4- 2,234 (40%)/' < 0.001 60,923 4- 4,3O5 (3O%) P < 0.01 64,437 -4- 4,485 (26%) P < 0.01 25,095 4- 4,567 (72%) P < 0.0005 70,787 4- 8,273 (19%) N.S. 76,216 4- 5,389 (13%) N.S. 99,083 -4- 1,161 (0%) 86,751 4- 4,429

These values represent the mean CPM of triplicate cultures 4- the standard error. The numbers in parentheses depict the percent suppression. Levels of significancewere established using Students t test (28). The concentration of NAL was held constant at 1 X 106 cells/well and the GAT concentration varied from 2 X 105 to 2.5 X 104 cells/well. GAT cells alone incorporated <200 CPM. N.S. = not significant.

82

WEBB A N D

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FIG. 1. The effect of PGE on suppressor activity in GAT cells. GAT cells were incubated with PHA, PGE or both for 48 hr. The GAT cells were then centrifuged, resuspended in fresh medium and mixed with fresh N A L cells in lhe presence of P H A and cultured for 72 hr. [~H]dT was added 24 hr prior to cell harvesting. (A) The values represent the mean CPM of triplicate cultures. The mean CPM -+ standard error in NAL cultures treated only with P I I A is depicted at the left hand side of each figure. GAT treated with: saline - - O ; PHA (0.125 ~g/ml) • ; POE2 (10 -~ M) ~ ; or P H A and PGE - - - A (B) GAT treated with Ro-20-5720 and exposed to: saline O ; P t I A (0.125 ~g/ml) 0--; PGE2 (10 -~ M) /k ; or P H A and PGE - - - A - - - . GAT ceil DNA synthesis at the time of assay in all groups was negligible ( < 200 cpm/culture). PGE and PGE + P H A treated GAT showed significant (P < 0.001) suppression at all doses; control and PI-IA treated GAT were suppressive only at 1/10 as measured by Student's t test (28). GAT cell D NA synthesis was minimal (<200 cpm). The number of NAL/culture was 1 × 100 and the number of GAT varied as indicated. that G A L h a d no effect on N A L until D N A synthesis h a d been initiated. I n fact, m e a s u r e m e n t s of R N A m e t a b o l i s m a n d p r o t e i n synthesis in N A L s t i m u l a t e d with P H A as opposed to N A L / G A L revealed little s u p p r e s s i o n until D N A synthesis h a d begun. T h e 36 h r time point also coincides to the p e r i o d w h e n i P G accumulation is rising. T h u s , the delay in s u p p r e s s o r effectiveness could reflect a t h r e s h o l d r e q u i r e m e n t for P G or a cell cycle event r e q u i r e d by N A L or G A T or both before s u p p r e s s i o n could be detected. I n this r e g a r d , it is i m p o r t a n t to p o i n t out that, in e x p e r i m e n t s to be p r e s e n t e d elsewhere ( T . I . Rogers, I. N o w o w i e j s k i , and D. R. W e b b , s u b m i t t e d for p u b l i c a t i o n ) , P G E can stimulate the a p p e a r a n c e of s u p p r e s s o r w i t h i n 2 h r of addition. T h u s , as soon as the p r o p e r level of P G is reached, supp r e s s o r cells can be induced. T h i s is consistent with d a t a p r e v i o u s l y r e p o r t e d ( 3 ) which showed that s u p p r e s s o r cells could be a d d e d 24 or 48 h r after culture initiation a n d still suppress D N A synthesis. Since we k n o w n that P G E 2 can stimulate r a p i d

PROSTAGLANDN IN I-DUCED SUPPRESSOR

83

CELL

release of suppressor, this suggests that P G levels at 24 or 48 hr may be high enough to activate the suppressor cells. Based on these considerations, experiments were begun to examine the effects of P t l A and PGE2 on G A T suppressor cells. The data presented in Figs. 1 and 2 show that PGE2 alone can stimulate an increase in the activity of the suppressor population. The fact that P H A - t r e a t e d G A T cells are no more active than untreated control cells suggests that these suppressor cells may be indifferent to the presence of the mitogen in so far as suppressor function is concerned. The lack of activation by P H A of G A T suppressors is important. The data in Table 1 shows that P H A stimulates an increase in iPG in G A T yet the amount of i P G which accumulates is not sufficient to activate the suppressor cells. Either it is below the threshold required for activation (e.g., perhaps a contribution from N A L is important, Table 8) or G A T do not produce the proper product of the P G pathway required for activation (see below). The dose of PGE2 added to these cultures (10 -~ M ) was selected because it was the amount of P G E which gives optimal stimulation of cyclic A M P levels in G A T (D. Webb, submitted for publication). The question may be raised as to whether this is a realistic physiological dose with respect to what is observed in culture. T w o points should be made in this regard. First, the experiment presented in Table 8 suggests that the amount of P G or products of the P G pathway generated by N A L between 0 to 48 hr is sufficient to activate G A T in a manner similar to that observed using exogenously added PGE2. Second, there is no reason to assume that P G E is necessarily the substance produced by N A L which activates G A T . There are, for example, other products of the P G synthetase pathway (e.g., endoperoxides and prostacyclin) which are many times more effective at stimulating cyclic A M P levels than P G E (21). The data presented here simply show that one product of the P G synthetase pathway, 20 __I

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FIG. 2. Effect of PGE on the production of suppressor "factor" by GAT cells. GAT ceils were prepared as in Fig. 1 and cultm-e supernatants collected at 48 hr. The supernatants were dialyzed against phosphate buffer saline (pH 7.2) overnight and added to fresh cultures of NAL stimulated with PHA for 72 hr. The values represent the mean CPM of triplicate cultures labeled from 48 to 72 hr with [SH]dT. The --II represents NAL exposed to PHA only.

84

WEBB AND N O W O W I E J S I ( I

PGE2, is effective in activating GAT. It does not prove that PGE2 is, in fact, responsible for the activation of G A T in the N A L / G A T mixture. It is important to note that at high cell concentrations all G A T cells are equally suppressive. This is consistent with the observation (Table 1) that unstinmlated G A T may accumulate a high level of iPG ( ~ 5 rig) and thus in high concentrations may be suppressive. Alternatively, it may be argued that since these G A T do not come from immunologically naive mice, the G A T suppressor population has already achieved a certain minimal activation which becomes detectable at high concentrations or ratios of G A T / N A L . This is made more plausible by estimating that in the normal mouse spleen the proportion of N A L is probably 50% of the total lymphocyte population whereas the total for G A T may be estimated to be approximately 5% of the total lymphocyte population (these estimates being based on the relative proportion of N A L and G A T recovered from the isolation procedures). Thus, very high ratios of N A L / G A T (e.g., 5/1 to 10/1) may not reflect a realistic physiological state. The possibility that lymphocytic or monocytic suppressor cells act either via direct release of P G or via P G mediated mechanism has received support from the work of Mertin et al. (22) and particularly from data presented by Goodwin et al. (20, 23). In the latter case, human peripheral blood lymphocytes were obtained and fractionated over glass wool columns. A weakly glass adherent suppressor cell was detected whose effects on responder D N A synthesis was blocked in the presence of indomethacin. This confirms our earlier observations made in the mouse system (3) and extends them to a human model. A number of laboratories have reported mitogen activation of non-specific suppressor cells (24-27). Such suppressor cells have been identified in both lymphocyte and macrophage enriched cell populations. Very little data has been developed concerning the activation of these suppressor cells although in most cases it appears the activation was assumed to occur via direct contact with the mitogen. The data presented in this report shows that some suppressor cells may be induced by direct exposure to PG. This, apparently, represents activation by a nonantigenic, non-mitogenic signal. The data also suggest that under certain circumstances the target of the suppressor cell can contribute to suppressor cell activation and this may involve P G or a product of the P G pathway. REFERENCES 1. 2. 3. 4. 5. 6. 7.

Waksman, }3. t:I., and Namba, T., Cell Immu~zoL 21, 161, 1976. Webb, D. R., and Osheroff, P. L., P~'oc. Nat. dcad. Sci. U S A 73, 1300, 1976. Webb, D. R., and Jamieson, A. T., Cell Immit1~ol. 24, 45, 1976. Zimeeki, M., and Webb, D. R., ]. Immm~ol. 117, 2158, 1976. Webb, D. R., and Nowowiejski, I., Cell Immu~ol. 33, 1, 1977. Gordon, D., Bray, ~. A., and Morley, J., Nature 262, 401, 1976. t~Iumes, J. L., 13onney, R. J., Pelus, L., Dahlgreen, M. E., Sadowski, S. J., Kuehl, Jr., F. A., and Davies, P., IVature 269, 149 1977. 8. Plescia O. J., Smith, A., and Greenwhich, K., P~'oe. Nat. Acad. Sci. USA 72, 1848, 1975. 9. Zurier, R. B., and Sayadoff, D. M., Inflammatio~ 1, 93, 1975. 10. Eardley, D. D., al~d Gershon, R. K., Y. Exp. Med. 142, 524, 1975. 11. l~y, I., and Mishell, R. I., ]. I~zmuJ~oI. Methods 5, 239, 1974. 12. Cave, M. D., J. Cell Biol. 29, 209, 1966. 13. Caldwell, ]3. U., Burnstein, S., Brock, W. A., and Sperott, J., J. Clilz. E~docri~tol. 33, 71, 1971.

PROSTAGLANDIN-INDUCED SUPPRESSOR CELL

g5

14. Hansen, J. H., Jacob, E., and Frenz, G. D., I~, "Methods in Enzymology" (D. B. McCormack and L. D. Wright, Eds.), Vol. 18, p. 130. Academic Press, New York, 1971. 15. Pelus, L. M., and Strausser, H. R., Li/e Sciences 20, 903, 1977. 16. Parker, C. W., Bauman, M. L., and Huber, M. G., J. Cliu. I~vest. 52, 1,336, 197,3. 17. Toth, M., Zakar, T., and Antoni, F., Actc~ Biochim. Biophys. Acad. Sci. H,mg. 10, 161, 1973.

18. Ferraris, V. A., and DeRubertis, F. R., :. Cli~. Invest. 54, 378, 1974. 19. Rapoport, B., Pellarisetty, R. J., Herman, E. A., and Congco, E. G., Biochem. t~iophys. Res. Co~.nm. 77, 1245, 1977. 20. Goodwin, J. S., Bankhurst, A. D., and Messner, R. P., Y. Exp. Med. 146, 1719, 1977. 21. Gorman, R. R., Bunting, S., and Miller, O. V., Prostaala~ldi~*s 13, 377, 1977. 22. Mertin, J., Meade, C. J., Hunt, R., and Sheena, J., I~.~t. Arch. o: Allergy and Applied Imm~*~¢ol. 53, 469, 1977. 23. Goodwin, J. S., Messner, R. P., Bankhurst, A. D., Peake, G. T., Saiki, J. I-t., and Williams, R. C., New E~.gla~d J. Med. 297, 963, 1977. 24. Folch, I-I., and Waksman, B. H., :. I~.~.~nu~ol. 113, 127, 1974. 25. Folch, H., and Waksman, B. I-I., Cell. I~mm~ol. 9, 12, 1973. 26. Rich, R. R., and Pierce, C. W., Y. Immu,zol. 112, 1360, 1974. 27. Dutton, R. W., J. E:cp. Med. 136, 1445, 1972. 28. Steel, R. G., and Torrie, J. H., I~z, "Principles and Procedures of Statistics." McGraw-Hill Book Co. Inc., New York 1960.