β-Endorphin stimulates rat T lymphocyte proliferation

Journal of Neuroimmunology, 29 (1990) 239-248

239

Elsevier JNI 00996

fl-Endorphin stimulates rat T lymphocyte proliferation Lucinda M. H e m m i c k * and Jean M. Bidlack Department of Pharmacology, University of Rochester, School of Medicineand Dentistry, Rochester, NY 14642, U.S.A. (Received 17 January 1990) (Revised, received 3 May 1990) (Accepted 4 May 1990)

Key words: fl-Endorphin; T cell proliferation; Prostaglandin, fl-Endorphin receptor; Neuroimmunomodulation

Summary fl-Endorphin 1-31 and several structurally related peptides were tested for their ability to alter mitogen-induced T cell proliferation. Rat fl-endorphin 1-31 and human fl-endorphin 1-27 increased phytohemagglutinin (PHA)-induced [3H]thymidine incorporation into rat lymph node cells. However, when PHA-induced proliferation was suppressed by the inclusion of prostaglandin E1 (PGE1), human fl-endorphin 1-31 and a number of structurally similar peptides, including some peptides that did not alter mitogen-induced proliferation, significantly reduced the PGEx inhibition of PHA-stimulated T cell proliferation. Although the N-terminus of fl-endorphin was necessary for potency, inclusion of the opioid antagonist naloxone together with fl-endorphin 1-31 did not alter the blockage of PGE 1 inhibition of PHA-induced proliferation caused by fl-endorphin. The inhibition of mitogen-stimulated proliferation by either cholera toxin or forskolin, two additional compounds that like PGE 1 also elevate cyclic AMP levels, was not blocked by fl-endorphin. Verapamil suppression of proliferation was not modified by fl-endorphin, indicating that the fl-endorphin stimulatory effect was probably not due to Ca 2+ influx through verapamil-sensitive Ca 2÷ channels. These data suggest that fl-endorphin, acting through a nonopioid fl-endorphin receptor, may modulate immunocompetence by stimulating T cell proliferation and by counteracting the inhibitory effects of PGE~.

Introduction

fl-Endorphin (fl-END) has been reported to both enhance and suppress mitogen-induced

Address for correspondence: Dr. Jean M. Bidlack, Department of Pharmacology, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642, U.S.A. * Present address: Department of Pharmacology, Yale University, School of Medicine, New Haven, CT 06510, U.S.A.

lymphocyte proliferation (Gilman et al., 1982; McCain et al., 1982; Puppo et al., 1985; Fontana et al., 1987; Kusnecov et al., 1987; Gilmore and Weiner, 1989) and natural killer cell activity (Mathews et al., 1983; Kay et al., 1984; Shavit et al., 1984; Mandler et al., 1986; Prete et al., 1986). The nature of the fl-END receptor involved in this effects has not been resolved. While some investigators have reported that fl-END and other opioids act through opioid receptors, which can be blocked by antagonists such as naloxone and naltrexone (Mathews et al., 1983; Kay et al., 1984;

0165-5728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

240 Shavit et al., 1984; Mandler et al., 1986; Kusnecov et al., 1987), others have noted that B-END modulates immune functions by a naloxone-irreversible mechanism (Gilman et al., 1982; McCain et al., 1982; Puppo et al., 1985; Prete et al., 1986; Fontana et al., 1987). Binding studies have demonstrated both opioid (Lopker et al., 1980; Mehrisi and Mills, 1983; Madden et al., 1987; Fiorica and Spector, 1988; Ovadia et al., 1989) and nonopioid, naloxone-insensitive binding sites (Hazum et al., 1979; Ausiello and Roda, 1984; Schweigerer et al., 1985; Falke and Fischer, 1986) on lymphocytes. One report demonstrated that B-END bound to an opioid binding site on human polymorphonuclear leukocytes because the antagonist diprenorphine blocked the binding and internalization of human B-END (Bh-END) (Falke and Fischer, 1986). However, other studies have suggested that B-END binds to a nonopioid B-END binding site on cultured murine EL4 thymoma ceils (Schweigerer et al., 1985), murine splenocytes (Shahabi et al., 1990), cultured human lymphocytes, RPMI 6237 (Hazum et al., 1979) and U937 ceils (Shahabi and Sharp, 1989), and on the terminal complex of human complement (Schweigerer et al., 1982). Although the mechanism is currently unknown, studies have revealed that B-END can modify lymphocyte production of various lymphokines. B-END enhanced interleukin-2 (IL-2) production in murine lymphocytes (Gilmore and Weiner, 1988) and increased the surface expression of the T cell IL-2 receptor on human lymphocytes (Kleinhenz and Kilroy, 1986). B-END also stimulated production of leukocyte migration inhibitory factor by cells from cancer patients (Wolf and Peterson, 1986), but suppressed the production of interferon-~, in cultured human peripheral blood mononuclear cells (Peterson et al., 1987). B-END and other similar peptides may act as local accessory signals for activation (or inhibition) of lymphocyte functions. A subset of mouse spleen macrophages has been shown to contain immunoreactive B-END and other products of the pro-opiomelanocortin (POMC) parent hormone (Lolait et al., 1984). Subsequently, mRNA for POMC has been detected in mouse and rat spleen (Lolait et al., 1986; Westly et al., 1986) and in human lymphocytes (Oates et al., 1988). Prostaglandins of the E series (PGE) inhibit

both B and T lymphocyte functions (Pelus and Strausser, 1977; Goodwin et al., 1978; Simkin et al., 1987). PGE inhibition of T cell IL-2 production has been well established as one of its mechanisms of suppressing mitogen-stimulated T cell proliferation (Rappaport and Dodge, 1982; Tilden and Balch, 1982; Chouaib et al., 1987). PGE, by raising cyclic AMP levels in T cells, may also cause suppression of mitogen-induced proliferation by interacting in a complex way with intracellular Ca 2+ levels or phosphatidylinositol turnover (Lerner et al., 1988). In contrast to PGE, B-END increased lymphocyte IL-2 production (Gilmore and Weiner, 1988) and IL-2 receptor expression (Kleinhenz and Kilroy, 1986), and decreased cyclic AMP levels in brain (Law et al., 1981) and a neuroblastoma × glioma hybrid cell line (Law et al., 1983). B-END also stimulated concanavalin A (ConA)-induced Ca z + uptake into thymocytes (Hemmick and Bidlack, 1987). This current study was undertaken to determine whether B-END could alter mitogen-induced T cell proliferation and/or block PGE inhibition of proliferation.

Materials and methods

Cell preparation Cervical and mesenteric lymph nodes from a 150-175 g male Sprague-Dawley rat were pooled. Lymph node cells were released into 15 mM Hepes-balanced salt solution (BSS), pH 7.2, by pressing the nodes between the ends of frosted glass slides. Cells were washed 3 times in 15 mM Hepes-BSS, pH 7.2, by centrifugation at 200 × g for 10 rain at 5 ° C, and the suspension was passed over a glass wool column. Trypan blue dye exclusion was used as a measure of viability. Cells were counted and resuspended in Ex-Cell 300 medium, supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and 100/~g/ml streptomycin, to yield a final concentration of 2 × 105 cells/well.

Proliferation assay Cells were added in triplicate sets to flat-bottomed wells of microtiter plates. Cells were incubated with 50 # g / m l phytohemagglutinin (PHA) in the presence or absence of peptides in a

241 final volume of 200 #1. Except where indicated, all peptides were added to cultures at time 0 and were present for the entire 72 h time course of culturing at 3 7 ° C in a 5% CO2 incubator. Cultures were pulsed with 0.5/~Ci/well [3H]thymidine for 12 h before filtering the cells onto G F / C filters and washing with distilled, deionized water. Filters were counted in 5 ml Liquiscint scintillation fluid. Mitogen-induced proliferation was determined by subtracting the [3H]thymidine cpm of cells without mitogen from cells with mitogen. Statistical significance was determined by comparing treated samples to control samples using a two-tailed Student's t-test. Experimental values differing from control at p < 0.05 were considered significant. Trypan blue dye exclusion was used to assure that the highest concentrations of P G E 1, cholera toxin, forskolin, verapamil, peptides, and alkaloids used, were not toxic to the lymph node cells after the 72 h incubation. No compound used caused artifactual changes in [3H]thymidine adsorption to the filters during harvesting.

Materials Male Sprague-Dawley rats were obtained from Charles River Breeding Laboratories. Ex-Cell 300 medium was obtained from J.R. Scientific Co., Woodland, CA, U.S.A. PHA was purchased from ICN Immunobiologicals. [3H]Thymidine (185 G B q / m m o l ) was obtained from Amersham. Liquiscint scintillation fluid was purchased from National Diagnostics. Peptides were purchased from Peninsula laboratories and Bachem. Naloxone-HC1 was supplied by Endo Laboratories. Cholera toxin, forskolin, and verapamil were purchased from Sigma Chemical Co.

Results

Determining if fl-END could alter PHA-stimulated proliferation and block PGE 1 inhibition of proliferation Stimulating rat lymph node cells with 50 # g / m l PHA for 72 h resulted in approximately 70,000 clam of [3H]thymidine being incorporated into 2 × 105 cells, as is shown in Fig. 1. Basal [3H]thymidine uptake was always less than 500 cpm and was not altered by any peptide or alkaloid tested.

84" A m 0 x

70 ~

E 56Z 0 Fn~ 0 n {E 0

Ill

4228-

CONTROL

I-END 1-31

PGE I

PGEI+ B - E N D 1-31

Fig, 1. Effect of fib-END 1-31, PGE], and PGE] with fib-END

1-31 on the incorporation of [3H]thymidineinto lymph node cells in the presence of PHA. In a final volume of 200 /~l of Ex-Cell 300 medium containing 5% fetal bovine serum, 2 × 105 lymphocytes were incubated with 50 #g/ml PHA, and either 10/~M fib-END 1-31, 6/~M PGE1, or both, for 72 h at 37°C in a 5% CO2 incubator. Cells were pulsed with 0.5 #Ci/well of [3H]thymidine 12 h before the cells were collectedby filtration on GF/C glass fiber filters. Bars represent the mean percent of control [3Hlthymidineincorporation+ SEM from three or more separate experimentsperformed in triplicate. Tdr, thymidine.

When the lymphocytes were incubated with 10 /~M flh-END 1-31 in addition to PHA, no significant change in the incorporation of [3H]thymidine was observed. P G E 1 inhibition of PHA-stimulated [ 3H]thymidine incorporation into lymphocytes was titrated from 1 nM to 10 #M. Half-maximal inhibition was noted at 4.5 /~M PGE1, and 6 /LM P G E 1 caused a 75% suppression of PHA-induced [3H]thymidine incorporation into lymph node cells, as is shown in Fig. 1. PGE1 at 6 # M was not toxic to the cells, as determined by trypan blue dye exclusion. When 10/~M flh-END 1-31 was added to the cultures at the same time as PGE1 and PHA, an enhancement in [3H]thymidine incorporation was observed relative to the uptake obtained with PHA and P G E 1. By including 10 # M fib-END 1-31 together with PGE], [3H]thymidine uptake was increased from 25% of the PHA-alone control value to 65%. While flh-END 1-31 had no effect on T cell proliferation by itself or in the presence of the lectin mitogen PHA, /3h-END 1-31 significantly suppressed the ability of PGEx to inhibit proliferation ( p < 0.01).

242 TABLE I Z

THE ABILITY OF RAT r-END 1-31 TO STIMULATE PHA-INDUCED [3H]THYMIDINE INCORPORATION INTO RAT LYMPHOCYTES IN THE ABSENCE AND PRESENCE OF PGE 1

BO

0 ~60 ocuJ

o ~

z_ ~ 4 o

~

zo

i

3

K~

30

I00

5O0

000

3OOO ,o.ooo

~J-END 1-31 ( n M )

Fig. 2. Titration of fin-END 1-31 in blocking 6 gM PGE 1 inhibition of PHA-stimulated proliferation. Varying concentrations of fib-END 1-31 were added to the lymph node cells 72 h before harvesting the cells and measuring [3H]thymidine incorporation as described in Fig. 1. Points represent mean percent increase in [3H]thymidine incorporation relative to PHA+ PGE 15: SEM from three or more separate experiments performed in triplicate. Significant (p < 0.01) enhancement was noted at concentrations of 100 nM and above. Tdr, thymidine.

f l h - E N D 1 - 3 1 e n h a n c e m e n t of [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n i n t o l y m p h n o d e cells i n the presence of P H A a n d 6 g M P G E 1 was titrated from 1 n M to 10 g M of the peptide, a n d the results are shown in Fig. 2. Significant e n h a n c e m e n t ( p < 0.01) occurred with f i E N D 1 - 3 1 c o n c e n t r a t i o n s of 100 n M a n d greater. T h e P G E x c o n c e n t r a t i o n used i n the experim e n t s was n o t critical i n that rat r - E N D 1 - 3 1 a n d other E N D peptides suppressed P G E 1 i n h i b i t i o n of [3H]thymidine u p t a k e over a wide range of P G E 1 c o n c e n t r a t i o n s , as is s h o w n i n T a b l e 1. U n l i k e 1 0 / t M or lower c o n c e n t r a t i o n s of f i b - E N D 1-31, 1 #M rat r - E N D 1-31 enhanced [3H]thymidine i n c o r p o r a t i o n i n t o P H A - s t i m u l a t e d lymphocytes. At equirnolar c o n c e n t r a t i o n s , rat fiE N D 1 - 3 1 was more p o t e n t t h a n f i b - E N D 1 - 3 1 i n blocking the i n h i b i t i o n of [3H]thyrnidine u p t a k e caused b y P G E v

Determining if opioid antagonists couM block fiEND suppression of PGE1 inhibition of T cell proliferation As shown in Fig. 3, the opioid a n t a g o n i s t n a l o x o n e at a final c o n c e n t r a t i o n of 10 g M , a 10-fold greater c o n c e n t r a t i o n t h a n fin-END 1 - 3 1 a n d a c o n c e n t r a t i o n that would occupy all opioid receptors, did n o t alter the ability of f i b - E N D 1 - 3 1 to reduce the i n h i b i t i o n of [ 3 H ] t h y m i d i n e

Rat lymph node cells, 2×105 cells in 200 /tl Ex-Cell 300 medium, were cultured for 72 h at 37 o C in a 5% Co2 incubator in the presence of 50 gg/ml PHA, varying concentrations of PGE 1, and 1 gM rat B-END 1-31. [3H]Thymidine, 0.5 /~Ci/well, was added to the cultures 12 h before filtering the cells onto GF/C glass fiber filters. Data are the mean cpm 5: SD from a representative experiment, performed in triplicate. Condition

cpm

+ 1 gM rat r-END 1-31 cpm

PHA alone PHA+0.06 gM PGE] PHA +0.6/zM PGE 1 PHA+ 6/tM PGE]

643005:5800 58100±9400 50500±3700 21100±1800

78600+2800 86600±8900 72500± 600 34300+_5000

i n c o r p o r a t i o n caused b y P G E ] . N a l o x o n e alone h a d n o effect o n either P H A - i n d u c e d [3H]thymid i n e u p t a k e or the suppression of p r o h f e r a t i o n caused b y P G E v T h e p u t a t i v e e n d o g e n o u s b r a i n opioid a n t a g o n i s t f l h - E N D 1 - 2 7 (Nicolas a n d Li, 1985) was also tested for its ability to block the f i b - E N D 1 - 3 1 e n h a n c e m e n t . W h e n lymphocytes were c u l t u r e d with 10 g M f i b - E N D 1 - 2 7 together with 1 g M f i b - E N D 1 - 3 1 , only a 570 i n h i b i t i o n of

~00

Z

ga

CL ~ 6 0

~ 4 0

+E3-ENOPSi

+~-END I-St +NALOXONE

+{3-ENDI-51 +{]-END l-2T

Fig. 3. Effect of 10 laM naloxone or 10/tM flu-END 1-27 on the ability of 1 gM fin-END 1-31 to suppress 6 gM PGE 1 inhibition of 50 gg/ml PHA-stimulated [3H]thymidine incorporation into lymph node cells. [3H]Thymidine incorporation into 2 × 105 lymphocytes was measured as described in Fig. 1. Bars represent mean percent enhancement relative to PHA + PGE 15: SEM from three or more separate experiments performed in triplicate. Tdr, thymidine.

243 TABLE 2 EFFECT OF B-END AND STRUCTURALLY SIMILAR PEPTIDES ON PHA-STIMULATEDT CELL PROLIFERATION AND ON PGEl INHIBITION OF PHA-STIMULATED PROLIFERATION [3H]Thymidine incorporation into 2 x 10s lymph node cells in response to 50/~g/ml PHA was measured as described in Fig. 1. END peptides were tested at a final concentration of 1 laM. DAME, DADLE, DAGO, and DSLET were tested at a final concentration of 10 #M. The amount of [3H]thymidineincorporated into 50 lag/ml PHA-treated cells (PHA control) or the amount of [3H]thymidine incorporated into 50 #g/ml PHA and 6 #M PGEl-treated cells (PHA+ PGE1control) was set at 100%. Data are expressed as mean percent of control+ SEM from three or more separate experiments performed in triplicate. Peptide

%PHA control

%PHA + PGE1 control

Rat B-END 1-31 fib-END 1-31 ]3h-END 1-27 N-acetyl fib-END 1 - 3 1 yh-END flh-END 6-31 DAME DADLE DAGO DSLET

122±4 * 99+5 113+3" 101+3 103 ± 3 100±4 110±7 101 ± 2 101±1 101±3

164±18 * 134± 8 * 127± 1" 98± 1 95 ± 3 65+ 5 * 129± 6 * 105 ± 3 94± 6 90± 3

Asterisks denote significant (p < 0.05) enhancement or inhibition relative to the PHA control, or PHA+PGE1 control.

[3H]thymidine uptake was observed with 6 # M P G E 1, as is shown in Fig. 3. This almost complete blockage of PGE 1 inhibition of proliferation with 10 # M fib-END 1-27 together with 1 # M Bh-END 1-31 was similar to the value obtained with 10 # M Bh-END 1-31 alone. In the suppression of P G E 1 inhibition of PHA-induced proliferation of lymph node cells, flh-END 1-27 acted as an agonist with a potency similar to Bh-END 1-31.

Determining the receptor selectivity A number of peptides structurally related to Bh-END 1-31 were tested for their ability to affect either PHA-stimulated [3H]thymidine incorporation itself, or PGE 1 inhibition of PHA-stimulated proliferation. Table 2 shows that rat B-END 1-31 was the most potent peptide in blocking PGE~ inhibition of PHA-induced proliferation. Of the peptides which significantly suppressed the

PGE1 inhibition of PHA-induced proliferation, only rat B-END 1-31 and Bh-END 1-27 also stimulated proliferation in the presence of P H A alone. [D-AlaE]met-enkephalin (DAME), which contains the first five amino acids of ]3-END 1-31, with the exception of an Ala at the second position, to render the peptide resistant to proteases, slightly blocked the P G E 1 inhibition. However, D A M E was less potent than flh-END 1-31. A final concentration of 10/~M D A M E was necessary to achieve a significant suppression of the PGE~ effect, fib-END 6-31 exacerbated the PGE1 suppression, but did not affect PHA-stimulated proliferation in the absence of P G E 1. Bh-END 6-31 did not block the enhancement observed with Bh-END 1-31, suggesting that Bh-END 1-31 may be acting at more than one site. N-acetylation of Bh-END 1-31 completely blocked the activity of the peptide, indicating that the Tyr in the first position was critical for Bh-END 1-31 to suppress the PGE~ inhibition. The protease-resistant peptides, [D-Ala2,(Me)Phe4,Gly(OH)5]en kephalin (DAGO), specific for #-opioid binding sites, [D-Ser2,Leu~,Thr6]enkephalin (DSLET) and [o-Ala2,D-LeuS]enkephalin (DADLE), specific for 8-opioid binding sites, were not able to mimic B-END's stimulatory effect on T cell proliferation. N o peptide listed in Table 2 significantly altered basal rat lymph node cell [3H]thymidine incorporation.

Determining the time course for B-END 1-27 to suppress PGE 1 inhibition of proliferation The time course for fib-END 1-27 to block PGE 1 suppression of proliferation is shown in Fig. 4. Bh-END 1-27 was added at various times from 0 to 48 h during the 72 h incubation. If flh-END 1-27 was added up to 12 h after the addition of PHA and PGE1, the P G E 1 inhibition was partially blocked. However, when fin-END 1-27 was added at 24 or 28 h, the partial rescue of the proliferative response was progressively diminished.

Ability of B-END 1-31 to block the suppression of T cell proliferation caused by other compounds Experiments were conducted to determine whether fin-END 1-31 could reverse the inhibition of lymph node cell proliferation caused by other agents, besides PGE1, which are also known

244 ~oo

~

z-4O

,

[]

+ B-ENDF31 (I~M)

I 4

[]

NO I~-ENDI-31

i

Z

o_

80

<

_J_~ J-

~30 0

~20

\\\\\1

i

~,o I 6

z_o g 40

, ~2~

_

~2

18 TrME

24 (hr)

~

Fig. 4. Time course of the ability of fib-END 1-27 to reverse P G E 1 inhibition of PHA-stimulated [3H]thymidine incorporation into lymphocytes, fib-END 1-27, 10 ttM, was added to the cultures at the time points indicated, with time 0 representing the initiation of the cultures. [3H]Thymidine incorporation into lymphocytes was measured in the presence of 50 # g / m l PHA, as described in Fig. 1. Points represent mean percent increase of [3H]thymidine incorporation relative to P H A + P G E ~ from a representative experiment performed in triplicate. The average error of the individual points was 7%. Tdr, thymidine.

to raise lymphocyte cyclic AMP levels. Cholera toxin, which ADP-ribosylates the GTP-binding protein that regulates adenylate cyclase (Gill and Meren, 1978), and forskolin, which directly activates the catalytic subunit of adenylate cyclase (Seamon et al., 1981), inhibited PHA-stimulated [3H]thymidine incorporation in lymph node ceils as is shown in Fig. 5. In contrast to PGE 1 inhibition of proliferation, 1/~M fib-END 1-31 did not

[~]÷~ END I-3t(I,uM) ~]No ~-ENDI 51

C•

20

0

,

6.25

12.5

25

VERAPAMIL (pM) Fig. 6. Effect of 1 #M fib-END ]-31 on verapami] inhibition of PHA-stimulated proliferation. [3H]Thymidine incorporation into lymphocytes was measured as described in Fig. 1. All c o m p o u n d s were present for the entire 72 h culture. Bars represent m e a n percent of control PHA-stimulated [3H]thymidine incorporation into lymph node cells + SEM from three or more separate experiments performed in triplicate. Tdr, thymidine.

suppress cholera toxin or forskolin inhibition of PHA-stimulated lymph node cell proliferation. Verapamil, an agent which blocks Ca 2+ entry into certain voltage-dependent Ca z+ channels (Wagner et al., 1988), was used to inhibit PHAstimulated proliferation. As shown in Fig. 6, #M fib-END 1-31 did not alter verapamil suppression of PHA-stimulated [3H]thymidine incorporation into lymphocytes.

Discussion

8C

cb~

~_ ~ 6 6

g~ z -

ff

~

~4c 20 00l 0 iO I0 CHOLERA TOXIN (~Jglrnl)

010

I0 I00 FORSKOLIN (,uM)

Fig. 5. Effect of 1 /~M Bh-END 1-31 on cholera toxin or forskolin inhibition of PHA-stimulated [3H]thymidine incorporation into lymph node cells at varying concentrations of the inhibitors. [3H]Thymidine incorporation was measured as described in Fig. 1. All c o m p o u n d s were present for the entire 72 h culture. Bars represent mean percent of control PHA-stimulated proliferation _+SEM from three or more separate experiments performed in triplicate. Tdr, thymidine.

With the exception of morphine, opioid alkaloids did not stimulate proliferation of rat lymphocytes in the absence or presence of the mitogen PHA. Only morphine at a concentration of 10 /~M or greater enhanced PHA-induced [3H]thymidine uptake (Bidlack and Hemmick, 1990). None of the alkaloids, including morphine, blocked the PGEa-induced suppression of PHAstimulated proliferation (Hemmick and Bidlack, unpublished results). This current study has demonstrated that fiEND and several structurally related peptides enhanced PHA-induced proliferation and blocked PGE t inhibition of PHA-stimulated lymphocyte

245

proliferation. Table 2 illustrates that fib-END 131, rat B-END 1-31, flh-END 1-27, and DAME suppressed PGE 1 inhibition of PHA-induced [3H]thymidine incorporation into lymphocytes. Of these peptides, only rat B-END 1-31 and Bh-END 1-27, the two most potent peptides in inhibiting PGE~ suppression of proliferation, were also able to stimulate PHA-induced proliferation. The fact that rat B-END 1-31 was more effective than flh-END 1-31 is probably related to their differing amino acid residues at positions 26, 27, and 31 of the carboxyl-terminus. Whether the same mechanism is responsible for the stimulation of PHAinduced proliferation and the suppression of PGE 1 inhibition of proliferation by rat B-END 1-31 and flh-END 1-27 is unclear. Suppression of mitogen-induced proliferation by PGE~ may enhance the signal that can be obtained with the END peptides. A similar situation has been observed in brain and the neuroblastoma × glioma hybrid cell line NG108-15, where B-END and opioids have been shown to inhibit the production of cyclic AMP (Law et al., 1981, 1983). However, this inhibition can only be measured after the production of cyclic AMP had been stimulated by compounds such as PGE or forskolin. In addition to suppressing PGE-stimulated adenylate cyclase and blocking PGE inhibition of T cell proliferation, B-END has also been shown to block PGE~ inhibition of rat mast cell [14C]serotonin release (Yamasaki et al., 1982), to inhibit PGE 2 suppression of the autologous mixed lymphocyte reaction (Froelich, 1987), and to reduce PGE 2 inhibition of both IL-2-stimulated CT6 cell proliferation and human PHA-induced proliferation of lymphocytes (Farrar, 1984). These reports suggest that B-END antagonism of PGE-induced processes may be a phenomenon which is general to several systems. The result that might argue for different mechanisms being responsible for B-END stimulation of PHA-induced proliferation, and suppression of PGE~ inhibition of proliferation was that B-END peptides could block the inhibition of proliferation caused by PGE~ but not by forskolin or cholera toxin, two compounds that also elevate cyclic AMP levels. Bh-END 6-31 significantly exacerbated PGE 1 suppression of PHA-stimulated proliferation. This effect may be related to the carboxyl-terminal

character of Bh-END 6-31. McCain et al. (1987) noted that glycyl-L-glutamine, the carboxyl-terminal dipeptide of Bh-END, suppressed PHA-induced proliferation of human lymphocytes. This finding also suggests that B-END 1-31 and peptide fragments of B-END 1-31 may be acting at more than one site with opposite actions. If B-END is acting at multiple sites with opposing effects, this finding may explain why the actions of B-END 1-31 are not very prominent when the peptide is given alone. Multiple B-END binding sites may also account for the discrepancy seen between binding studies and physiological studies. For example, Bh-END 6-31 inhibited [1251]B hEND 1-31 binding to cultured murine lymphocytes, while Bh-END 1-27 was ineffective in blocking binding (Shahabi et al., 1990). However, in this study, Bh-END 6-31 and Bh-END 1-27 had opposite effects on proliferation. The potency of Bh-END 1-27 in inhibiting PGE~ suppression contrasts with reports that this peptide acted as an opioid antagonist in brain (Nicolas and Li, 1985). However, its mimicry of the fib-END 1-31 effect in this study supports the suggestion that the END peptides were acting through a nonopioid B-END receptor, similar to the ~-receptor described in rat vas deferens (Schulz et al., 1981) and brain (Goodman et al., 1983). Using murine splenocytes, Gilmore and Weiner (1989) reported that Bh-END 1-27 was ineffective at enhancing ConA-induced proliferation. This difference in the effect of Bh-END 1-27 upon these two systems is not clear, but could reflect differences in cell populations. The activity of DAME implies that the presence of the amino terminus of B-END is important for potency. However, DAME was an order of magnitude less potent than Bh-END 1-31, and 10 /tM DAME was necessary to obtain an effect. N-acetylation of Bh-END 1-31 abolished the ability of the peptide to block PGE 1 suppression. These data demonstrate the importance of the amino terminus, although it seems equally likely that the remainder of the peptide is somehow involved, since 1 #M ~,-END (Bh-END 1-17) did not alter the PGE 1 inhibition. B-END amino acid residues 1-21 were necessary to achieve a physiological response in the rat vas deferens (Schulz et al., 1981). The pentapetide DAGO, specific for #-opioid recep-

246 tors (Handa et al., 1981), and DSLET and DADLE, with the highest affinity for 3-opioid receptors (David et al., 1982), had no effect on T cell proliferation. This finding and the fact that naloxone did not reverse r-END's suppression of PGEa inhibition of T cell proliferation support the hypothesis that B-END and related peptides were not acting through an opioid receptor on lymphocytes, but instead were acting through a specific B-END receptor. Possible mechanisms by which B-END blocked PGE 1 inhibition of PHA-induced proliferation were examined to determine if B-END was acting by altering the effect of compounds that specifically raised cyclic AMP levels or if B-END was acting by stimulating Ca 2+ transport through verapamil-sensitive Ca 2+ channels. While no information is available concerning the effects of B-END on lymphocyte cyclic nucleotide levels, B-END is known to inhibit adenylate cyclase in brain (Law et al., 1981). The concentration of cyclic AMP is a major modulator of lymphocyte proliferation (Wang et al., 1978). Since PGE increases cyclic AMP levels in lymphocytes, B-END may have suppressed the PGE 1 inhibition of proliferation by inhibiting lymphocyte adenylate cyclase. This possibility was tested by examining the ability of/3-END to block either cholera toxin or forskolin inhibition of PHA-stimulated proliferation. B-END did not block the suppression caused by either compound across a range of concentrations. These data suggest that direct inhibition of lymphocyte adenylate cyclase is probably not the mechanism underlying /3-END inhibition of PGE~ suppression of proliferation. Another possible mechanism for/3-END inhibition of PGE~ suppression of proliferation is induction of Ca 2+ influx. Increases in intracellular Ca 2+ are known to stimulate lymphocyte proliferation (Lichtman et al., 1983), and proliferation can be blocked by calcium channel blockers (Birx et al., 1984). B-END has been shown to increase 45Ca2+ uptake into ConA-stimulated rat thymocytes (Hemmick and Bidlack, 1987). However, B-END did not affect the verapamil inhibition of mitogen-induced proliferation. It should be noted, however, that verapamil, although a classical blocker of voltage-dependent Ca 2+ channels, does not appear to block all forms of voltage-depen-

dent Ca 2+ channels (Wagner et al., 1988). There is the additional possibility that B-END may promote Ca 2+ entry through voltage-independent lymphocyte Ca a+ channels, as have been described in cloned T-helper cells (Kuno et al., 1986). B-END has been reported to enhance lymphokine production (Wolf and Peterson, 1986; Gilmore and Weiner, 1988) and to stimulate IL-2 receptor expression (Kleinhenz and Kilroy, 1986). The mechanism(s) by which B-END stimulates lymphokine and IL-2 receptor levels is currently unknown. In this study, the time course for /3hEND 1-27 to inhibit PGE 1 suppression of proliferation revealed that if the addition of Bh-END 1-27 to cultures treated with PGE x at time 0 was delayed 24 h or more, the suppression was markedly diminished. These results indicate that B-END may act by preventing PGEx inhibition of a relatively early event in the proliferative response. One possibility concerns activation of protein kinase C. PGE~ suppression of T cell activation has recently been linked to inhibition of protein kinase C activation (Chouaib et al., 1987). Since early protein kinase C activation has been found vital to the proliferative response (Friedrich and Gullberg, 1988), it is possible that B-END acts by blocking PGE 1 suppression of protein kinase C activation. While the mechanism remains unknown, this study has demonstrated that in rat lymph node cells, B-END increased mitogen-induced proliferation and diminished PGE~ inhibition of proliferation. B-END stimulated proliferation by acting through a nonopioid B-END receptor. Binding and functional studies using purified T cell subsets will allow the further characterization of this ]3END receptor and its role in modulating immunocompetence.

Acknowledgements This work was supported by grants DA03742, DA04355, and DA07232 from the National Institute on Drug Abuse.

247

References Ausiello, C.M. and Roda, L.G. (1984) Leu-enkephalin binding to cultured human T lymphocytes. Cell Biol. Int. Rep. 8, 97-106. Bidlack, J.M. and Hemmick, L.M. (1990) Morphine enhancement of mitogen-induced T-cell proliferation. In: F. Quirion, K. Jhamandas and C. Gianoulakis (Eds.), The International Narcotics Research Conference (INRC) 1989, Alan R. Liss, New York, pp. 405-408. Birx, D.L., Berger, M. and Fleischer, T.A. (1984) The interference of T cell activation by calcium channel blocking agents. J. Immunol. 133, 2904-2909. Chouaib, S., Robb, R.J., Welte, K. and Dupont, B. (1987) Analysis of prostaglandin E 2 effect on T lymphocyte activation. Abrogation of prostaglandin E 2 inhibitory effect by the tumor promoter 12-O-tetradecanoylphorbol 13acetate. J. Clin. Invest. 80, 333-340. David, M., Moisand, C., Meunier, J.-C., Morgat, J.-L., Gacel, G. and Roques, B.P. (1982) [3H]Tyr-D-Ser-Gly-Phe-LeuThr: a specific probe for the 8-opiate receptor subtype in brain membranes. Eur. J. Pharmacol. 78, 385-387. Falke, N.E. and Fischer, E.G. (1986) Opiate receptor mediated internalization of 12sI-fl-endorphin in human polymorphonuclear leucocytes. Cell Biol. Int. Rep. 10, 429-435. Farrar, W.L. (1984) Endorphin modulation of lymphokine activity. In: F. Fraioli, A. Isidori and M. Mazzetti (Eds.), Opioid Peptides in the Periphery, Elsevier, Amsterdam, pp. 159-165. Fiorica, E. and Spector, S. (1988) Opioid binding site in EL-4 thymoma cell line. Life Sci. 42, 199-106. Fontana, L., Fattorossi, A., D'Amelio, R., Migliorati, A. and Perricone, R. (1987) Modulation of human concanavalin A-induced lymphocyte proliferative response by physiological concentrations of /~-endorphin. Immunopharmacology 13, 111-115. Friedrich, B. and Gullberg, M. (1988) The role of protein kinase C in early activation vs. growth of T lymphocytes. Eur. J. Immunol. 18, 489-492. Froelich, C.J. (1987) Modulation of the autologous mixed lymphocyte reaction by ~8-endorphin. J. Neuroimmunol. 17, 1-10. Gill, D.M. and Meren, R. (1978) ADP-ribosylation of membrane proteins catalyzed by cholera toxin: basis of the activation of adenylate cyclase. Proc. Natl. Acad. Sci. U.S.A. 75, 3050-3054. Gilman, S.C., Schwartz, J.M., Milner, R.J., Bloom, F.E. and Feldman, J.D. (1982) /~-Endorphin enhances lymphocyte proliferative responses. Proc. Natl. Acad. So. U.S.A. 79, 4226-4230. Gilmore, W. and Weiner, L.P. (1988) /3-Endorphin enhances intefleukin-2 (IL-2) production in murine lymphocytes. J. Neuroimmunol. 18, 125-138. Gilmore, W. and Weiner, L.P. (1989) The opioid specificity of beta-endorphin enhancement of routine lymphocyte prohferation. Immunopharmacology 17, 19-30. Goodman, R.R., Houghten, R.A. and Pasternak, G.W. (1983) Autoradiography of [3H]~8-endorphin binding in brain. Brain Res. 288, 334-337.

Goodwin, J.S., Messner, R,P. and Peake, G.T. (1978) Prostaglandin suppression of mitogen-stimulated lymphocytes in vitro. Changes with mitogen dose and preincubation. J. Clin. Invest. 62, 753-760. Handa, B.K., Lane, A.C., Lord, J.A.H., Morgan, B.A., Rance, M,J. and Smith, C.F.C. (1981) Analogues of fl-LPH61_64 possessing selective agonist activity at /z-opiate receptors. Eur. J. Pharmacol. 70, 531-540. Hazum, E., Chang, K.-J. and Cuatrecasas, P. (1979) Specific nonopiate receptors for B-endorphin. Science 205, 10331035. Hemmick, L.M. and Bidlack, J.M. (1987) ~8-Endorphin modulation of mitogen-stimulated calcium uptake by rat thymocytes. Life Sci. 41, 1971-1978. Kay, N., Allen, J. and Morley, J.E. (1984) Endorphins stimulate normal human peripheral blood lymphocyte natural killer activity. Life So. 35, 53-59. Kleinhenz, M.E. and Kilroy, T.E. (1986) Modulation of the T cell IL-2 receptor by ~-endorphin. J. Leukocyte Biol. 38, 129A. Kuno, M., Goronzy, J., Weyand, C.M. and Gardner, P. (1986) Single-channel and whole-cell recordings of mitogen-reguiated inward currents in human cloned helper T lymphocytes. Nature 323, 269-273. Kusnecov, A.W., Husband, A.J., King, M.G., Pang, G. and Smith, R. (1987) In vivo effects of fl-endorphin of lymphocyte proliferation and intedeukin 2 production. Brain Behav. Immun. 1, 88-97. Law, P.Y., Wu, J., Koeltler, J.E. and Loh, H.H. (1981) Demonstration and characterization of opiate inhibition of the striatal adenylate cyclase. J. Neurocbem. 36, 1834--1846. Law, P.Y.,~Hom, D.S. and Lob, H.H. (1983) Opiate regulation of adenosine 3:5-cyclic monophosphate level in neuroblastoma ×glioma NG108-15 hybrid cells. Relationship between receptor occupancy and effect. Mol. Pharmacol. 23, 26-35. Lichtman, A.H., Segel, G.B. and Lichtman, M.A, (1983) The role of calcium in lymphocyte proliferation (an interpretive review). Blood 61, 413-422. Lolait, S.J., Lira, A.T.W., Toh, B.H. and Funder, J.W. (1984) Immunoreactive fl-endorphin in a subpopulation of mouse spleen macrophages. J. Clin. Invest. 73, 277-280. Lolait, S.J., Clements, J.A., Markwick, A.J., Cheng, C., McNally, M., Smith, A.I. and Funder, J.W. (1986) Proopiomelanocortin messenger ribonucleic acid and posttranslational processing of beta endorphin in spleen macrophages. J. Clin. Invest. 77, 1776-1779. Lopker, A., Abood, L.G., Hoss, W. and Lionetti, F.J. (1980) Stereoselective muscarinic acetylcholine and opiate receptors in human phagocytic leukocytes. Biochem. Pharmacol. 29, 1361-1365. Madden, J.J., Donahoe, R.M., Zwemer-Collins, J., Shafer, D.A. and Falek, A. (1987) Binding of naloxone to human T lymphocytes. Biochem. Pharmacol. 36, 4103-4109. Mandler, R.N., Biddison, W.E., Mandler, R. and Serrate, S.A. (1986) fl-Endorphin augments the cytolytic activity and interferon production of natural killer cells, J. Immunol. 136, 934-939. Mathews, P.M., Froelich, C.J., Sibbitt, Jr., W.L. and Bank-

248 burst, A.D. (1983) Enhancement of natural cytotoxicity by /3-endorphin. J. Imrnunol. 130, 1658-1662. McCain, H.W., Lamster, I.B., Bozzone, J.M. and Grbic, J.T. (1982) /3-Endorphin modulates human immune activity via non-opiate receptor mechanisms. Life Sci. 31, 1619-1624. McCain, H.W., Bilotta, J. and Lamster, I.B. (1987) Endorphinergic modulation of immune function: potent action of the dipeptide glyeyl-l-glutamine. Life Sci. 41, 169176. Mehrisi, J.N. and Mills, I.H. (1983) Opiate receptors on lymphocytes and platelets in man. Clin. Immunol. Immunopathol. 27, 240-249. Nicolas, P. and Li, C.H. (1985) fl-Endorphin-(1-27) is a naturally occurring antagonist to etorphine-induced analgesia. Proc. Natl. Acad. Sci. U.S.A. 82, 3178-3181. Oates, E.L., Allaway, G.P., Armstrong, G.R., Boyajian, R.A., Kehrl, J.H. and Prabhaker, B.S. (1988) Human lymphocytes produce pro-opiomelanocortin gene-related transcripts. Effects of lymphotropic viruses. J. Biol. Chem. 263, 10041-10044. Ovadia, H., Nitsan, P. and Abramsky, O. (1989) Characterization of opiate binding sites on membranes of rat lymphocytes. J. Neuroimmunol. 21, 93-102. Pelus, L.M. and Strausser, H.R. (1977) Prostaglandins and the immune response. Life Sci. 20, 903-914. Peterson, P.K., Sharp, B., Gekker, G., Brummitt, C. and Keane, W.F. (1987) Opioid-mediated suppression of interferon--t production by cultured peripheral blood mononuclear cells. J. Clin. Invest. 80, 824-831. Prete, P., Levin, E.R. and Pedram, A. (1966) The in vitro effects of endogenous opiates on natural killer cells, antigen-specific cytolytic T cells, and T-cell subsets. Exp. Neurol. 92, 349-359. Puppo, F., Corsini, G., Mangini, P., Bottaro, L. and Barreca, T. (1985) Influence of/3-endorphin on phytohemagglutinin-induced lymphocyte proliferation and on the expression of mononuclear cell surface antigens in vitro. Imrnunopharmacology 10, 119-125. Rappaport, R.S. and Dodge, G.R. (1982) Prostaglandin E inhibits the production of human interleukin 2. J. Exp. Med. 155, 943-948. Schulz, R., Wiister, M. and Herz, A. (1981) pharmacological characterization of the c-opioid receptor. J. Pharmacol. Exp. Ther. 216, 604-606. Schweigerer, L., Bhakdi, S. and Teschemacher, H. (1982) Specific non-opiate binding sites for human /3-endorphin

on the terminal complex of human complement. Nature 296, 572-574. Schweigerer, L., Schmidt, W., Teschemacher, H. and Gramsch, C. (1985)/3-Endorphin: surface binding and internalization in thymoma cells. Proc. Natl. Acad. Sci. U.S.A. 82, 57515755. Seamon, K.B., Padgett, W. and Daly, J.W. (1981) Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc. Natl. Acad. Sci. U.S.A. 75, 3050-3054. Shahabi, N.A., Peterson, P.K. and Sharp, B. (1990) /3-Endorphin binding to naloxone-insensitive sites on a human mononuclear cell line (U937): effects of cations and guanosine triphosphate. Endocrinology 126, 3006-3015. Shahabi, N.A., Linner, K.M. and Sharp, B.M. (1990) Murine splenocytes express a naloxone-insensitive binding site for fl-endorphin. Endocrinology 126, 1442-1448. Shavit, Y., Lewis, J.W., Terman, G.W., Gale, R.P. and Liesbeskind, J.C. (1984) Opioid peptides mediate the suppressive effect of stress on natural killer cell cytotoxicity. Science 223, 188-190. Simkin, N.J., Jelinek, D.F. and Lipsky, P.E. (1987) Inhibition of human B cell responsiveness by prostaglandin E 2. J. Immunol. 138, 1074-1081. Tilden, A.B. and Balch, C.M. (1982) A comparison of PGE 2 effects on human suppressor cell function and on interleukin 2 function. J. Immunol. 129, 2469-2473. Wagner, J.A., Guggino, S.E., Reynolds, I.J., Snowman, A.M., Biswas, A., Olivera, B.M. and Snyder, S.H. (1988) Calcium antagonist receptors. Clinical and physiological relevance. Ann. N.Y. Acad. Sci. 522, 116-133. Wang, T., Sheppard, J.R. and Foker, J.E. (1978) Rise and fall of cyclic AMP required for onset of lymphocyte DNA synthesis. Science 201, 155-157. Westly, H.J., Kleiss, A.J., Kelley, K.W., Wong, P.K.Y. and Yuen, P.-H. (1986) Newcastle disease virus-infected splenocytes express the proopiomelanocortin gene. J. Exp. Med. 163, 1589-1594. Wolf, G.T. and Peterson, K.A. (1986) Beta endorphin enhances in vitro lymphokine production in patients with squamous carcinoma of the head and neck. Otolaryngol. Head Neck Surg. 94, 224-229. Yamasaki, Y., Shimamura, O., Kizu, A., Nakagawa, M. and Ijichi, H. (1982) IgE-mediated 14C-serotonin release from rat mast cells modulated by morphine and endorphins. Life Sci. 31,471-478.