Prostaglandin-mediated regulation of the mixed lymphocyte culture and generation of cytotoxic cells

CELLULAR

IMMUNOLOGY

183 (1980)

56,172-

Prostaglandin-Mediated Regulation of the Mixed Lymphocyte Culture and Generation of Cytotoxic Cells1 L. DARROW

TIMOTHY Departments

of Clinical

Pathology

Received

and Microbiology, Center, Syracuse,

January

AND RUSSELL State University New York 13210

18, 1980; Accepted

February

H. TOMAR of New

26,

York

Upstate

Medical

I980

We examined the role of prostaglandins or prostaglandin-producing cells in the regulation of proliferation and generation of specific cytotoxicity in one-way mixed lymphocyte cultures of mouse spleen cells. Cultures treated with indomethacin or other prostaglandin synthesis inhibitors resulted in enhanced proliferation and cytotoxicity. The level of prostaglandins produced in vitro, as measured by RIA, was 10e8 M and was found to be completely blocked by indomethacin. Adding back low8 M PG restored baseline (control) proliferative responses. Kinetics of the enhanced MLC response were unchanged from controls as were the specificities of the cytotoxic cells. Cells from indomethacin-treated cultures were more efficient at killing targets than those from control cultures. Prostaglandins appear to have a preferential effect on the induction of cytotoxic cells.

INTRODUCTION Prostaglandins (PG)2 play a role in regulation of the immune response. Human and murine mononuclear cells produce and release PG in response to in vitro mitogen and antigen stimulation (l-3). Furthermore, prostaglandins of the E series have been shown to inhibit lymphokine production (4), antibody production (5), mitogen-induced proliferation (6, 7), and direct cytolysis by mouse spleen cells in vitro (8). In vivo, PG produced by tumors can inhibit antibody production (9), generation of a tumor-specific cytotoxic response (lo), or even the rejection of a tumor allograft (11). Webb and Nowowiejski in mice (2) and Goodwin et al. in man (7) have shown that blocking the production of prostaglandins in cultures of mitogen-stimulated lymphocytes results in marked increases in the proliferative response. Webb has demonstrated that blocking PG production in viva or in vitro can result in an increased production of antibody (3). He has also reported * This work has been supported in part by New York State Health Resources Grants 355 and 1159 and the Immunology Education, Research, and Development Fund. 2 Abbreviations used: MLC, mixed lymphocyte culture; AA,,,, MLC using stimulating cells syngeneic to responding cells: AB,, MLC using stimulating cells allogeneic to responding cells; PBS, phosphate-buffered saline; FCS, fetal calf serum; Con A, concanavalin A; PG, prostaglandin; PGE,, prostaglandin El; Indo., indomethacin; [3H]TdR, tritiated thymidine; cpm + SE, counts per minute 2 standard error; T-X- 100, Triton X-100 in culture medium (10% v/v); SI, stimulation index; CI, cytotoxic index. 172 0008-8749/80/150172-12$02.00/O Copyright 0 1980 by Academic press, Inc. AI1 rights of reproduction in any form reserved.

PROSTAGLANDIN

REGULATION

OF CM1

173

enhancement of the proliferative response in two-way mixed lymphocyte cultures when PG synthesis is blocked (3). While prostaglandins appear to modulate in vitro lymphocyte responses (2,3,7), there have been no investigations which determine the antigen specificity, effects on kinetics, and cellular preferences of this regulatory mechanism. Therefore, we report here our findings on the role of endogenous prostaglandins in autoregulation of in vitro cell-mediated immune responses. MATERIALS

AND METHODS

Animals. C57BL/6 (H-2b) or DBA/2 (H-2d) male mice were purchased from Microbiological Associates (Walkersville, Md.) at 6 weeks of age. C,H (H-2k) male animals were raised in our own colony. All animals were from 8 to 12 weeks of age when used. The P-815 (H-2d) mastocytoma was maintained as an ascites tumor by weekly intraperitoneal passage in CDF, (DBA/2 x BALB/c)F, mice. Drugs. Indomethacin (Merck Sharpe & Dohme), an irreversible inhibitor of prostaglandin synthesis (12) was dissolved in 95% ethanol at 4 mg/ml and diluted to 20 &ml in culture medium just prior to use. PGE, (Sigma and a gift from Dr. J. Pike, Upjohn) was dissolved in 95% ethanol at 4 x 10e3 M and diluted in culture medium just before use. Tolmetin (Tolectin, McNeil) and ibuprofen (Motrin, Upjohn), both inhibitors of PG synthesis (13), were also dissolved in ethanol and diluted just before use. It was determined that addition of ethanol at concentrations equal to those present in the dissolved drugs had no detectable effect on our cultures. Cell suspensions. Single-cell suspensions were prepared by removing spleens aseptically and pressing them through fine stainless-steel mesh into ice-cold phosphate-buffered saline (PBS). Any remaining clumps were broken up by repeated pipetting with a small bore Pasteur pipet and the suspensions transferred to glass culture tubes. The cells were pelleted by centrifugation and suspended in Tris-buffered NH&l to lyse contaminating red cells (14). After incubating for 2 min at 37”C, 10 ml of ice-cold PBS was added and the ceils were washed three times. After the third wash, the cells were suspended in culture medium which consisted of RPM1 1640 with penicillin and streptomycin (Associated Biomedic Systems) supplemented with 5 x lo-” M 2-mercaptoethanol (Calbiochem), 50 pgiml gentamicin (Schering), and 20 mM Hepes buffer (Gibco). The cell suspensions were counted in a hemocytometer and diluted in culture medium at a final concentration lo6 cells/ml. Responsing cells were suspended in medium with 40% fetal calf serum (FCS) (Gibco). Stimulating cells were incubated after the first wash for 20 min at 37°C with mitomycin C (Sigma) at 50 ~g/ml/SO x lo6 cells in PBS. Following three washes, they were suspended in medium, counted, and diluted to 8 x lo6 cells/ml. Mixed lymphocyte cultures (Micro). Mixed lymphocyte cultures were carried out in triplicate wells of flat-bottom microculture plates (Costar No. 3596) by combining 0.05 ml (4 x loj) responding cells with 0.05 ml (4 x l(Y) mitomycin-treated syngeneic (AAm) or allogeneic (ABm) stimulating spleen cells. The final volume of 0.2 ml was made up with added medium or medium containing drugs. The cultures were incubated at 37°C in 5% COz/air. The proliferative response was determined at 120 hr by measuring uptake of

174

DARROW

AND

TOMAR

[3H]thymidine ([3H]TdR) (specific activity, 5.0 Ci/mmol Amersham) following an 18-hr pulse with 1 &i/well. Cultures were harvested onto glass fiber filters using an Otto Hiller automatic cell harvester. The dried filters were added to scintillation vials containing 6 ml of Betafluor (National Diagnostics) and counted in a Beckman LS-355 counter. Thymidine uptake is expressed as mean counts per minute (cpm) +/- standard error (SE) from triplicate walls. Responses are expressed as net cpm (mean cpm ABm cultures-mean cpm AAm cultures). Stimulation index (SI) is determined as follows: SI =

mean cpm ABm mean cpm AAm *

Mixed lymphocyte cultures (macro). MLCs were also prepared in flat-bottom 15 x 30-mm glass vials containing 4 x lo6 responding C57BL/6 spleen cells and 4 x IO6 mitomycin C-treated syngeneic (AAm) or DBA/2 allogeneic (ABm) spleen cells in a final volume of 2 ml culture medium with 10% FCS. Indomethacin or control medium was added at initiation, Assay for cytotoxicity. Cytotoxic activity present in cultures on Day 5 was measured using a 51Cr release assay (15). Target cells were either P-815 (H-2d) mastocytoma cells or concanavalin A (Con A)-induced blasts from C3H (H-2k) spleen cells. The C3H blasts were prepared by incubating 4 x lo6 cells in 2 ml culture medium containing 10% FCS and 5 CLg/ml Con A for 72 hr. All target cells were labeled by incubating 20 x lo6 cells with 200 &i 51Cr (as Sodium 51Chromate [Yr], Amersham) in a final volume of 0.4 ml containing 10% FCS. The cells were incubated at 37°C in a shaking water bath for 40 min. Labeled target cells were washed three times in PBS with 10% FCS, suspended in RPM1 with FCS, counted, and diluted to 2 x lo5 cells/ml. To each well of the MLC-microculture plates was added 0.05 ml (1 x lo4 cells). Three wells were included which contained only 0.2 ml medium and 0.05 ml target cells for determination of spontaneous 51Cr release. Three wells were also included which contained 0.1 ml media, 0.1 ml 10% Triton X-100 (Packard) in RPM1 and 0.05 ml target cells for determination of total experimental release (T-X-100). The plates were spun at 1OOg for 5 min and incubated for 4 hr in 5% CO,/air at 37°C. After incubation the plates were recentrifuged and 0.125-m] aliquots of the supernatant fluid removed from each well and placed in tubes for counting on a Searle model 1185 gamma counter. Cytotoxic index (CI) was calculated as follows:

cI =

(cpm ABm - cpm AAm) (cpm T-X-100 - cpm AAm)

*

When cytotoxic cells were prepared in macrocultures, they were assayed on day 5. Responding cells were recovered from the vials, washed three times in fresh medium and resuspended in culture medium containing 10% FCS. Viability was determined and graded numbers of viable cells were combined with lo4 radiolabeled targets in a 4-hr assay. Determination of viability. Viability of culture cells was determined by trypan blue dye exclusion according to the method of Boyse et al. (16). Total immunoreactive prostaglandin Radioimmunoassay of prostaglandins. (IPG) was extracted from 1 ml of the MLC supernate by adding 3 ml 3:3:1

PROSTAGLANDIN

REGULATION TABLE

1

Effect of Adding Indomethacin Experiment

Indo. addedb (/4

175

OF CM1

to MLC”

[3H]TdR uptake (cpm + SE) SI’

Responsed (%)

1,549 1,288

5.7 12.3

100 265

57,540 t 8,283 216,041 + 17,666

6.4 20.5

100 424

40,846 t 6,285 70,792 2 5,587

4.7 8.5

100 195

AAm

ABm

1

1.0

7,139 * 1,175 7,852 k 3,926

2

1.0

9,023 t 1,498 10,521 + 2,246

3

1.0

8,757 i8,287 2

40,626 k 96,497 f

175 758

a MLC consisted of 4 x 1tP C57BL/6 spleen cells responding to 4 x lo5 mitomycin C-treated DBA/2 stimulating spleen cells. b Indomethacin or control medium was added at initiation of cultures. c SI = stimulation index = cpm ABmicpm AAm. d Percentage response = net cpm + drug/net cpm - drug x 100%.

ethy1acetate:isopropanol:O.l N HCl. Two phases were generated by adding 3 ml saline and 2 ml ethyl acetate. Duplicate l-ml aliquots of the upper, organic, phase were dried under nitrogen in 12 x 75mm glass tubes. IPG levels were determined using the RIA method of Zusman er al. (17). [3H]PGE2 (specific activity 160 CVmmol, Amersham) and rabbit antibody to PGE, PGA, and PGB (Calbiochem) were used in the assay. For calculation of IPG molarity, we assumed a molecular weight of 348 daltons. Preparation of data. All experiments were done in triplicate and the data expressed as the mean cpm +/- SE. Each experiment was repeated at least three times. RESULTS Effects of indomethacin on proliferation and induction of cytotoxicity in the MLC. Indomethacin (1 pg) or control medium was added to the one-way MLC at initiation. Indomethacin had no significant effect on [3H]TdR uptake by AAm syngeneic control cultures but significantly increased that response by stimulated (ABm) cultures (Table 1). These results indicated that indomethacin at the concentration used is not itself mitogenic for mouse spleen cells but enhances the response of stimulated cells and are in agreement with the work of Webb and Osheroff (3). We next examined the effects of indomethacin on the development of cytotoxicity in the MLC. Blocking PG synthesis enhanced the development of cytotoxic cells (Table 2). The effect was on the induction of cytotoxicity and not on the cytotoxic effector cells themselves since there was no enhancing effect by the drug when added at the time of the present during the chromium release assay. Effect of varying doses of indomethacin. Concentrations of indomethacin from 0.01 to 5.0 pg enhanced the [3H]TdR uptake in ABm cultures and had no effect on AAm controls (Fig. 1). At the highest concentration tested, 10 pg per culture, the

176

DARROW AND TOMAR TABLE Indomethacin

2

also Increases Generation of Cytotoxic Cells in MLC”

Indomethacin addedb (/a)

Experiment 1

Cytotoxic indexC (%) 2 SE

Response* (%I

1.0 1.0’

44 ” 3 62 2 5 43 k 3

100 141 98

0 1.0

23 2 4 50 c 5

100 217

0

2

,JCytotoxic cells were generated in MLC using 4 x lo5 C57BL/6 spleen cells responding to 4 x lo5 mitomycin C-treated DBA/2 stimulating spleen cells. 5LCr-labeled P815 target cells (1 x 10’) were added to the cultures on Day 5 and 51Cr released after 5 hr was measured. b Drug was added at initiation of cultures except where indicated. c Cytotoxic index (CI) = cpm AB, - cpm AA&pm TX 100 - cpm AA,,, x 100%. d Percentage response = CI (+ drug)/CI (- drug) x 100%. p Indomethacin was added just prior to the Vr release assay and was present during the procedure.

drug was inhibitory for both stimulated and control responses. At 0.001 pg or below, there was no significant effect on any of the cultures. Therefore, we used 0.1 to 1.0 pg (1.4 x 10e5 to 1.4 x lop6 M) indomethacin in all subsequent studies. Effects of other PG synthetase inhibitors. If inhibition of PG production were responsible for MLC enhancement, other drugs with the same activity should also enhance MLCs. Tolmetin, ibuprofen, and phenylbutazone, all inhibitors of PG synthesis (13), were tested and all enhanced MLCs (Table 3). Levels of immunoreactive prostaglandin (IPG) in the MLC supernates. PGE and

40 ", +I ? i-

,t=

50 -

f;~

f'III I I

\

&++

I 0

+-

. 0.0001

0.001

0.01

0.1

1.0

5.0

10

INDONETHAClN ADDED (JIG)

FIG. 1. Effect of varied amounts of added indomethacin on proliferation in MLC. 4 x lo5 C57BIJ6 spleen cells were cultured with 4 x 105 mitomycin C-treated syngeneic (AA,) or allogeneic (AB,) spleen cells. Indomethacin or control medium was added at initiation. [3H]TdR uptake at 96 hr. (0 0) AA,,, cultures. (0 - - - 0) AB, cultures.

PROSTAGLANDIN

REGULATION TABLE

Effect of Adding Four Nonsteroidal

177

OF CM1

3 Antiinflammatory

Drugs to MLC”

[3H]TdR uptake (cpm k SE) Experiment

SI

Responsec (%I

216

3.9

100

10,983 ?z 1,283

103,003 + 10,663

9.4

210

11,532 r 1,992

94,059 + 3,043

8.2

242

12,420 +- 1,346

98,493 k

9,651

7.9

253

11,224 ?z 1,131

64,817 2 2,535

5.8

100

Drug added*

AAm

None Indomethacin (1 x 10-S M) Tolmetin (1 x 10-S M) Ibuprofen (1 x lo-5M)

10,865 2

None Indomethacin (1 x lo-5M) Phenylbutazone (5 x 10-S M)

Ah 129

42,095 k

17,364 k 2,078

194,079 k

5,082

11.2

331

16,516 k 2,439

229,247 k

3,690

13.9

397

a MLC prepared as described under Materials and Methods. * Added at initiation. c Percentage response = net cpm (with drug)/net cpm (without drug)

x

100%.

PGA plasma levels in man are in the range of 5 x lo+’ to 1 x lo-* M (18). We compared levels of PG produced in the MLC to those present in vivo to help establish a role in immune regulation by PG. We therefore measured immunoreactive prostaglandin (IPG) in the MLC supernates and found that as early as 24 hr following culture initiation, IPG in the MLC supernates were already at the reported in vivo levels (Table 4). At 48 and 72 hr, supernate IPG remained at or above this level. The primary action of indomethacin is inhibition of PG production (12). In Table 5 we demonstrate that addition of the drug to the MLC inhibited the production of IPG and maintained supemate IPG at the background level. Reestablishment of “normal” response by adding PGE,. We reasoned that if PG produced in the culture play a role in regulating the response, then adding back PG to enhanced cultures to reestablish PG levels should “normalize” the response. We therefore added graded concentrations of PGE, to cultures in which PG synthesis TABLE Immunoreactive

4

Prostaglandins (IPG) in MLC Supernates

IPG (nglml) (mean -+ SE) Time” (W

AAm

A&n

24 48 72

1.83 -c 0.36 1.56 % 0.34 1.23 k 0.20

2.57 ? 1.05 3.91 c 0.93 3.21 2 0.56

Net (rig/ml) (AB,-AA& 0.74 2.35 2.04

NS” * **

n IPG was determined at the indicated times in five experiments using a radioimmunoassay. b Significance: P value represents comparison of AA,,, and AB, supemate IPG levels. *P < 0.025. **P < 0.01. NS, not significant.

178

DARROW AND TOMAR TABLE Effect of Added Indomethacin

5

on IPG Levels in Supernates of MLC

IPG (rig/ml) (mean ? SE) Time” (hr)

Net (rig/ml) (AB,-AA,,J

Indo.

AAm

Ah,

24

+

1.13 k 0.47 0.53 + 0.33

1.57 + 0.40 0.39 k 0.21

0.44 -

NS’ NS

48

+

1.46 * 0.22 0.44 t 0.30

3.48 k 1.07 0.15 t 0.47

2.02 -

* NS

72

+

1.17 k 0.25 0.18 k 0.53

2.74 2 0.25 0.11 f 0.32

1.57 ** NS

n IPG was determined in four experiments at times indicated using a radioimmunoassay. b Significance: P value represents comparison of AA,,, and AB, supernate IPG levels. *P < 0.05. **P < 0.01. NS, not significant.

had been blocked and found that 1 x IO-+ M added PGE, indeed restored the response to control levels (Fig. 2). This same concentration had only a slight inhibitory effect on the control cultures. Increased concentrations of added PGEz caused correspondingly greater inhibition of both control and enhanced cultures. Effects of indomethacin on the kinetics of proliferative and cytotoxic cell responses in the MLC. We explored the nature of the enhanced MLC response by comparing its kinetics with that of controls. Indomethacin or control medium was 150

25

FIG. 2. Effect of adding PGE, to control or indomethacin treated MLC. Control MLC with added medium (0); enhanced MLC with added indomethacin (0); PGE, was added to control or enhanced cultures at initiation. r3H]TdR uptake at 96 hr.

PROSTAGLANDIN

MLC

KINETICS

ARE

REGULATION

UNCHANGED

BY

179

OF CM1

INDOMETHACIN

100. /

+I

/

m

/

96 hours

FIG. 3. Kinetics of [3H]TdR uptake in MLC in the presence and absence of added indomethacin. 0) AB, without indomethacin; (0 - - - 0) AA,,, with 1 (0 0) AA,,, without indomethacin; (0 pg indomethacin; (0 - - - 0) AB, with 1 pg indomethacin; cpm (X 103) [3H]TdR 2 SE.

added at initiation of multiple cultures and [3H]TdR uptake was determined at 48, 72,96, 120, and 144 hr. Both proliferative responses peaked at 120 hr, although as early as 72 hr of incubation the enhanced culture showed significantly more

10

3

4 DAYS

5

6

FIG. 4. Kinetics of development of cytotoxicity in the MLC in the presence or absence of indomethacin. (0 0) cytotoxicity in control cultures; (0 0) cytotoxicity in indomethacin enhanced cultures. (All differences significant at P < 0.025 or better.)

180

DARROW AND TOMAR TABLE

6

Specificity of Enhanced Cytotoxicity in MLC” When Indomethacin

Is Added at Initiation Cytotoxic index targetb

Responding spleen cells C57BLl6 (H-2b)

Stimulating spleen cells (mit. C)

Indomethacin added b.4

DBA/2 (H-2d)

1.0 1.0

CIH (H-23

P815 (H-2d)

GH (H-2k)

23 t 4 48 + 2 3k1 11 +6

7*2 11 k 7 36 2 1 57 k 2

n MLC consisted of 4 x 105 C57BL/6 (H-2!‘) normal spleen cells responding to 4 x lo5 mitomycin C-treated DBA/2 (H-2d) or C,H (H-23 spleen cells. b Targets were YR-labeled P815 (H-2”) tumor cells or CIH (H-23 con A-induced blasts (5 hr 51Cr release from 1 x lo4 labeled targets added to MLC on Day 5).

[3H]TdR uptake (Fig. 3). The kinetics of cytotoxic cell development were also unchanged by added indomethacin (Fig. 4). Specijcity of enhanced cytotoxicity. We questioned if the increased cytotoxic response remained specific for the stimulating alloantigens. To determine this, cytotoxic cells were generated in MLC in which C57BW6 (H-2b) spleen cells were stimulated by DBA/2 (H-2d) or C,H (H-2k) spleen cells. Control medium or indomethacin was added to parallel cultures. Cytotoxic cells arising in each of these cultures were tested for their ability to kill labeled targets bearing H-2 antigens identical to those present on the stimulating (specific cytotoxicity) or different (nonspecific cytotoxicity) alloantigen. Table 6 demonstrates that killing is specific and remains so when enhanced by adding indomethacin at initiation. TABLE

7

Viable Cell Recovery, [3H]TdR Uptake, and Cytotoxicity Present in MLCs with and without Added Indomethacin”

Indomethacin (1 x IO-5 M)

No. viable cellsb (Xlfw

3

+

19.3 20.7

4

+ + +

Day

5 6

Control (%I

Net cpm [3H]TdR k SE

Control (%I

100 107

22,890 + 2126 36,349 k 1097

100 159

1 5

100 500

35.8 58.0

100 162

40,489 t 3542 110,400 + 1183

100 273

15 27

100 180

36.7 54.0

100 147

50,654 ” 6572 175,447 k 9207

100 346

23 65

100 283

28.3 43.0

100 152

30,170 k 4669 36,735 k 7011

100 122

14 53

100 379

a MLC prepared as described under Materials and Methods. b Viability by trypan blue dye exclusion. c Four-hour assay.

CIc (%I

Control (%I

PROSTAGLANDIN

REGULATION

181

OF CM1

60

50

40 s

Y :: z E 20

10

g 30 ELeo...

.

50

I

.

30

20

I..

.

10

EFFECTOR CELLWARGET

.I.

\

1

5

2

CELL

FIG. 5. Cytotoxicity by viable spleen cells recovered from 5-day normal or indomethacin enhanced MLC. Four-hour Wr release assay. (0 0) cytotoxicity by cells from normal MLC; (0 0) cytotoxicity by cells from enhanced MLC. An effectoritarget ratio of 5.3: 1 in the enhanced culture gave a cytotoxicity of 30% compared to 14: 1 in control cultures.

Correlation of viable cell recoveries with proliferative responses and development ofcytotoxic cells in MLC. It was reasonable to assume that the increased

responses seen in drug-treated MLCs reflected an increase in the number of viable cells. We therefore attempted to correlate the number of viable cells per culture with net cpm [3H]TdR uptake and cytotoxic activity present on different days. We found (Table 7) that by Day 3 there was no significant difference in the number of viable cells present in normal or treated cultures, although a significant difference in net [3H]TdR uptake existed. Cytotoxicity at this time was low but still showed enhancement with indomethacin. By Day 4 there were significant increases in all three measurements: viable cell number was 162%; thymidine uptake was 273%; cytotoxicity was 180% of control. These relationships were maintained throughout the remainder of the experiment. Selective suppression of cytotoxic responses by endogenous PG. The data in Table 7 indicated that significant increases in the number of viable cells per culture ranged from 147 to 162% of the controls, whereas increases in [3H]TdR uptake and cytotoxicity ran as high as 346 and 379%, respectively. This suggested that a relatively small population of cytotoxic precursor cells was selectively suppressed by endogenous PG. To test this, we prepared cytotoxic cells in macro MLCs with or without added indomethacin and recovered effector cells on Day 5. We then compared the ability of equal numbers of viable cells from indomethacin-treated and control cultures to kill labeled targets. Cells from cultures containing indomethacin demonstrated more killing per cell than those recovered from control cultures (Fig. 5). An effector-to-target ratio of 14: 1 was required to cause lysis of

182

DARROW

AND

TOMAR

30% of the target cells if the effecters were derived from normal cultures, whereas a ratio of only 5.3: 1 was required if the effecters were derived from indomethacinenhanced cultures. Expressing these results in lytic units per lo6 cells (19) and using a CI of 30% as representing one lytic unit, we see that there are 7.2 Lu/106 cells in control cultures and 18.9 Lu/106 cells in drug-treated cultures. DISCUSSION We have investigated the one-way MLC and generation of cytotoxic cells in the presence of PG and PG synthesis inhibitors. Inhibition of PG production by indomethacin was usually accompanied by highly significant (two- to threefold) increases in [3H]TdR uptake. By adding back 1O-8 M PG to indomethacin-treated cultures, we were able to restore the control level of proliferation. Endogenous PG primarily affects the magnitude of the response. The kinetics of [3H]TdR uptake and development of cytotoxic cells were the same for control and indomethacin-treated cultures. Similarly, indomethacin did not alter the specificity of the cytotoxic activity. Thus, endogenous, autoregulatory PG controls the magnitude of an immune response but not antigen specificity or kinetics of induction. Cytotoxic populations generated in the absence of endogenous PG production are two to three times more effective than cells from controls. This suggests that PG preferentially inhibit the induction of cytotoxic cells. In support of this, increases in [3H]TdR uptake in the presence of indomethacin correlated very well with increases in cytotoxicity. For example, Day 4 cytotoxic activity in indomethacin-enhanced cultures was 180% of controls, correlating very well with the Day 3 proliferative increase of 159%. This same correlation was closely followed throughout, i.e., Day 4 rH]TdR increased to 275% and Day 5 cytotoxicity to 283% (Table 7). This suggests that cells in the preproliferating stage during one 24-hr period gave rise to cytotoxic effector cells assayable during the next 24-hr period. The increased numbers of proliferating cells are probably the cytotoxic precursors themselves. However, we cannot rule out the possibility that T cells helping to differentiate cytotoxic precursors also are dividing. Endogenous PG may inhibit one or both populations. Previous investigations into the role of PG in control of cytotoxic responses were limited to evaluation of the effector phase of the response (8, 10). The concentrations of PG required to significantly inhibit the in vitro effector phase of in viva generated cytotoxic cells has been reported to be in excess of 1 x lop7 M. Furthermore, Plaut (20) demonstrated that even such high concentrations of PG or other CAMP elevating agents fail to inhibit in vitro generated cells. Shearer et al. (21) have shown that passing normal spleen cells ever insolubilized histamine columns removes cells required for generation of in vitro cytotoxic responses. Previously, Weinstein and Melmon (22) found that cells which bear receptors for histamine also bear receptors for epinephrine and prostaglandins, suggesting that cytotoxic precursors have such receptors. We have demonstrated significant enhancement of the induction of a cytotoxic response when endogenous PG synthesis was blocked. Levels as low as 10es M endogenous PG appeared to modulate this response. Thus, whereas the effector phase of cytotoxic cells is sensitive to inhibition by high concentrations of PGEz (in vivo generated cells), or

PROSTAGLANDIN

REGULATION

OF CM1

183

not at all sensitive (in vitro generated cells), the inductive phase of the in vitro response is highly sensitive to PG modulation. PG may act to directly inhibit responding cells, as suggested by Goodwin et al. (7) in man or they may act indirectly to induce a suppressor cell as suggested by Webb and Nowowiejski (2). Alternatively, PGs may be acting in concert with another factor(s) and play a modulator role in fine control of the immune response much as PGs appear to act in regulating epinephrine-induced lipolysis (23). While we do not know the source of in vivo serum levels of PG and its physiological role in regulation of immune responses, it is clear that 1 x lop8 M endogenous PG or 1 x lop8 M added PGE, exert profound effects on proliferation and generation of cytotoxic cells in vitro. The cell source and mechanism(s) of action of endogenous PGs in regulating this immune response await further clarification. ACKNOWLEDGMENT We wish to acknowledge Dr. Bertie F. Argyris for her help in the early stages of this work and for her critical review of the manuscript.

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