Modulation of the autologous mixed lymphocyte reaction by β-endorphin

Journal of Neuroimmunology, 17 (1987) 1-10

Elsevier JNI 00532

Modulation of the autologous mixed lymphocyte reaction by fl-endorphin Christopher J. Froelich Section of Rheumatology, Department of Medicine, University of Illinois at Chicago, Chicago, IL, U.S.A.

(Received 29 January 1987) (Revised, received29 May 1987) (Accepted 28 May 1987)

Key words: Autologousmixed lymphocytereaction; fl-Endorphin; a-Endorphin; Neuroimmunomodula-

tion Summary The effect of the opiate peptide, fl-endorphin ( r - E N D ) , on the autologous and allogeneic mixed lymphocyte reaction was examined. Physiologic concentrations of r - E N D augmented proliferation of the autologous mixed lymphocyte reaction (AMLR) but the allogeneic M L R was not altered. Alpha-endorphin ( a - E N D ) was ineffective. Pre-incubation of the stimulator subset (i.e., B cells and macrophages) with 10 -8 M r - E N D followed by addition to A M L R culture without additional opiate peptide did not produce augmentation. The r - E N D - i n d u c e d augmentation of the A M L R was partially inhibited by the opiate antagonist naloxone, r - E N D augmentation was not due to increased secretion of interleukin-2. When pros° taglandin E 2 (PGE2) was added to A M L R cultures wherein the stimulator cell fraction was vigorously depleted of adherent cells, suppression was observed which could be reversed by the addition of r - E N D (10 -8 M). The potential mechanisms producing the increased proliferative response during the A M L R are discussed.

Introduction

The immune system can no longer be considered an autonomously functioning 'organ'. Rather it is intimately connected and therefore modulated by the central Address for correspondence:ChristopherJ. Froelich, M.D., Department of Medicine/Rheumatology, University of Illinois at Chicago, 840 South Wood Street, Rm 1006 CSB, Chicago, IL 60612, U.S.A. 0165-5728/87/$03.50 © 1987 ElsevierScience Publishers B.V. (Biomedical Division)

nervous system (CNS) via a number of neurohormones and by innervation of lymphoid structures (Ader 1981). Moreover it appears that lymphoid cells elaborate a number of peptides which act on the CNS (Blalock 1985). Thus a complete lymphoid/CNS nexus exists. One neuropeptide which influences a number of human immune functions is fl-END. Previous investigations show that fl-END depresses some T cell functions, such as active T cell rosette formation (Wybran et al. 1979) and lymphocyte proliferation (McCain et al. 1982). However, fl-END may augment other lymphoid functions. For example, the opiate peptide enhances natural killer cell activity (Matthews et al. 1983). Currently, very little is known about the possible effects of opiate peptides on the AMLR. The AMLR has been postulated to represent a group of cellular interactions critical for the maintenance of self-tolerance and thus important in the prevention of autoimmunity and also may play a pivotal role in tumor immunosurveillance (Talal and Smith 1982). In this report, fl-END has been shown to enhance the AMLR. The augmentation may be partially attributable to the ability of fl-END to counteract the inhibitory effect of PGE 2 release by adherent cells present in the stimulator cell fraction.

Materials and methods

Isolation of mononuclear cell subsets Peripheral blood was obtained from fasting donors who were not taking medication (8:00-9 00 a.m.). Mononuclear cells were prepared by density gradient centrifugation on Ficoll-Hypaque, washed three times in phosphate-buffered saline (PBS, pH 7.2) and resuspended in RPMI-1640 plus 10% heat-inactivated pooled AB sera (NHS, 5 x 106/ml) and antibiotics. This is designated as complete medium. For certain experiments macrophage depletion was performed by plastic Petri dish adherence. Plastic culture dishes (100 mm, Falcon No. 3003, Becton Dickinson, Oxnard, CA) were coated with 10% heat-inactivated AB serum for 30 rain at 37 ° C. The mononuclear cells (5 ml at 5 × 106/1111) were dispensed onto the plates and incubated at 37 ° C for 1 h. Nonadherent cells were removed and the adherent cells were discarded. The mononuclear cells were isolated into a stimulator cell fraction (B cell and macrophage) and responder cell fraction (i.e., T cells) by sheep red blood cell (SRBC) rosetting technique. SRBC were washed three times with PBS and resuspended as a 5% suspension of PBS/5% NHS containing neuraminidase (50 U / m l , Calbiochem, LaJolla, CA). The SRBC were incubated at 37 °C for 1 h and were washed thrice with complete medium. Ceils were mixed with SRBC at a 1 : 100 ratio for 15 rain at 37 ° C in 15 ml conical polystyrene tubes (Coming No. 25311-15). The tubes were centrifuged at 200 X g for 5 rain and these tubes were incubated on ice for 1 h. Following incubation the cells were gently resuspended by rotation, layered on FicoU-Hypaque and centrifuged at 400 × g for 20 rain. The B cell-enriched subset was retrieved from the interface and washed incomplete medium. The T cells were freed from the SRBC by lysis with 0.85% Tris-NH4C1 buffer. T cells were washed three times in complete medium. The B cell-enriched population was

70-85% surface immunoglobulin positive, 15-20% esterase positive (i.e., no macrophage depletion step) and < 5% SRBC positive cells whereas the T cell subset was 95-97% SRBC positive. For certain experiments, adherent cells were vigorously depleted by exposure to the plastic Petri dishes (e.g., 1% esterase-positive cells were recovered, see above). For the performance of the allogeneic M L R the nonadherent cell fraction was utilized.

Autologous and allogeneic mixed lymphocyte reaction A M L R and M L R were performed in parallel using two normal donors. To 98-well round-bottom microtiters plates, 1 x 105 T cells were added. Either autologous stimulator cells ( m a c r o p h a g e / B cell fraction) or allogeneic stimulator cells (nonadherent mononuclear cells) were added to the wells at various densities (see Results). The stimulator cells were pretreated with mitomycin C (50/~g/ml) for 30 min at 37 ° C and washed three times with complete medium before use. The A M L R and M L R combinations were incubated for 168 and 120 h respectively at 37 ° C in humidified a i r / 5 % CO 2. 18 h prior to termination the cultures were pulsed with [3H]thymidine (1 #Ci/well, in 20 #1, spec. act. 5 C i / m o l , Amersham, Arlington Heights, IL). Thereafter cells were harvested with a Mash apparatus and processed in a scintillation counter. All cultures were performed in triplicate and the data expressed as mean cpm. For certain experiments, the stimulator cell fraction underwent pre-incubation with fl-END (10 -8 M) or a - E N D (10 -8 M) for 24 h before set-up of A M L R cultures.

Opiate peptides and other reagents Immediately before each experiment, the protease-resistant opiates fl-[D-Ala2] endorphin and a-endorphin (Peninsula Labs., Belmont, CA) were added to RPMI (1 X 10 -6 M) and were diluted to the appropriate concentration when dispensed into the microwells. The D-Ala2-substituted form of the peptide was used because it is less susceptible to enzymatic degradation but maintains potent biologic activity (Walker et al. 1977). Naloxone (NAL) was purchased from Endolab (San Juan, PR), processed as described above and used at a final concentration of 10 -6 M. P G E 2 was purchased from Sigma (St. Louis, MO).

Mononuclear cell surface antigen expression Following the removal of adherent cells, mononuclear cells from five donors in the study were incubated in complete medium (1 x 106/ml) containing 10 -8 M fl-END for 24 h. Following incubation, the cells were washed twice with PBS in 16 x 100 mm polypropylene tubes and resuspended at 107 cells/ml. Aliquots of 100 /~1 were dispensed into 12 x 75 mm polypropylene tubes to which the appropriate monoclonal antibody was added. The cells were incubated on ice for 30 min, washed twice with PBS/0.1% Na azide and stained with FITC-conjugated goat anti-mouse Ig (1 : 10; Tago, Burlingame, CA). Following a 30 min incubation on ice the cells underwent two additional washes. Monoclonal lymphoreactivity was assessed either by flow cytometry or by direct visualization. For flow cytometric analysis, the cell pellet was resuspended in 1 ml PBS/0.01% Na azide and filtered

t h r o u g h n y l o n mesh. A n a l y s i s was p e r f o r m e d o n the F a c s II ( B e c t o n - D i c k i n s o n , M o u n t a i n View, C A ) a n d c o n s i s t e d o f 20000 cells using the following p a r a m e t e r s : scatter 4 x 5; fluorescence 16 x 1; t h r e s h o l d 10; a n d 900 V. U n d e r these c o n d i t i o n s the m a j o r i t y of a u t o f l u o r e s c e n t cells r e s i d e d in c h a n n e l s 1 - 2 0 . A s a control, a m o u s e I g G m y e l o m a was used. F o r direct visualization, cells were p e l l e t e d as d e s c r i b e d above, r e s u s p e n d e d in 100 ~tl P B S / 0 . 0 1 % N a azide, a n d 200 cells were c o u n t e d for fluorescence using a Zeiss fluorescence microscope.

Monoclonal antibodies and antisera T h e p a n e l of m o n o c l o n a l a n t i b o d i e s used for this s t u d y were O K T 3 ( p a n T cells), O K T 4 ( h e l p e r / i n d u c e r T cell subset), O K T 8 ( s u p p r e s s o r , c y t o t o x i c T cell subset), O K M I ( m o n o c y t e s , n a t u r a l killer cells). T h e s e reagents were o b t a i n e d f r o m O r t h o P h a r m a c e u t i c a l s ( R a r i t a n , N J). B cells were i d e n t i f i e d with F I T C - c o n j u g a t e d p e p s i n - d i g e s t e d g o a t a n t i - h u m a n p o l y c l o n a l I g ( C a p p e l , Cochranville, PA).

Interleukin-2 (IL-2) bioassay T h e m e t h o d of Gillis et al. was used, e m p l o y i n g the i n d i c a t o r cell line C T L L - 2 0 . T h e ' s t a n d a r d ' I L - 2 was p u r c h a s e d f r o m E l e c t r o n u c l e o n i c s (Silver Springs, M D ) . This p a r t i a l l y p u r i f i e d p r e p a r a t i o n possessed a n activity of 64 U / m l .

Results

A s shown in T a b l e 1, when the s t i m u l a t o r - r e s p o n d e r ratio was 1 : 1, / 3 - E N D in the range of 10 - 8 to 10 -1° a u g m e n t e d the A M L R w h e r e a s the M L R was u n a f fected. However, it should b e n o t e d t h a t this clear i n c r e a s e was o b s e r v e d a m o n g six of the eight volunteers e x a m i n e d ( d a t a n o t shown). T h e o p i a t e a n t a g o n i s t N A L p a r t i a l l y reversed the e n h a n c i n g effect of f l - E N D , f l - E N D a n d N A L , w h e n a d d e d to

TABLE 1 E F F E C T OF fl-END A N D N A L ON A M L R A N D M L R

Treatment

Concentration

AMLR a

MLR

NAL fl-END

10 -6 0 10- 8 10-10 10-12 10 -6 + 10 -8 10-6+10 -1° 10 6-t-10-12

12.8 + 9.4 13.4 + 6.8 36.4 +__10.4 * * 31.2 + 9.4 * * 28.4 + 10.3 * 24.4_+ 7.8 19.9+ 7.8 17.9+ 3.4

89.1 + 10.3 92.8 5:16.0 86.1 5:17.4

NAL+ fl-END

87.1 5 : 9 . 3

89.5 ± 16.8 ND h

a Values are expressed as mean cpm + SD × 10- 3 (n = 6). Statistical analysis was performed by using the Dunnet's t-test comparing sham-treated AMLR cultures versus fl-END-treated cells. * P < 0.05; • * P < 0.01. b Not done.

TABLE 2 EFFECT OF PULSING STIMULATORCELL FRACTION WITH OPIATE PEPTIDES ON THE AMLR Treatment

Concentration

AMLR a

B-END B-END a-END a-END

0 10-8 0 10-8

14.6±4.1 17.2+ 3.4 16.3 ± 5.2 15.8 ± 4.9

Values are expressed as mean cpm+SD (n = 2). Statistical analysis was performed by the Student t-test for paired samples comparing cpm in cultures with or without the described opiate peptide (P = 0.08). the responding T cell fraction did not augment proliferation; maximal cpm for B-END-supplemented cultures was 419 _+ 128 cpm and 391 _+ 156 cpm for NAL. a - E N D did not modify the A M L R (data not shown). It is quite plausible that B-END was not mediating a modulatory augmentation of the A M L R but rather the stimulation was due to antigen presentation of the opiate to reactive T cells. In this regard, experiments were performed to address this issue. In a series of five experiments the stimulator cell fraction was pulsed with B-END or a - E N D (10 -s M) for 4 h. Both B-END- and a-END-pulsed stimulator cells were cultured at stimulator/responder cell ratios of 1 : 1. As shown in Table 2, virtually no increased proliferation was observed, thus the role of antigen-induced augmentation of the A M L R by B-END is minimal. Moreover, as noted above, when a - E N D was added to A M L R cultures an augmented A M L R was not observed. In addition, when responder or stimulator cells were pulsed for 24 h, no increase in proliferation was noted (data not shown).

Effects of B-END on different stimulator / responder ratios in the A M L R and on the kinetics of proliferation The previous experiments were performed on a stimulator/responder ratio of 1 : 1. In order to determine if this was the ratio which optimally responded to the enhancing effects of B-END, the proliferative response of the A M L R was performed using different densities of stimulator cells. One of these experiments is shown in Table 3. Optimal proliferation was apparent at a ratio of 1 : 1 whereas it was less at 2 : 1 and the enhancing effect of B-END was nonexistent at a ratio of 5 : 1. Interestingly at the higher stimulator/responder ratio overall proliferation in the A M L R decreased, presumably due to the effect of suppressor macrophages. We routinely harvested cells at approximately 7 days, a time when the A M L R maximally appears to proliferate. To determine if B-END (10 -8 M) modified the kinetics of the augmented A M L R , we performed a series of four time-course experiments; the optimal stimulator/responder ratio of 1 : 1 was utilized for these studies. As shown in Fig. 1, no appreciable difference in proliferation occurred on days 1 and 3. However, on day 4 and thereafter wells containing the opiate peptide demonstrated augmented A M L R proliferation over control cultures.

t~

TABL E 3 E F F E C T S OF D I F F E R E N T A U T O L O G O U S S T I M U L A T O R C E L L C O N C E N T R A T I O N S O N END O R P H I N - M E D I A T E D E N H A N C E M E N T OF A M L R b

Density of stimulator cells

B-END a

cpm

1 × 10 s lxl05 2×105 2 x 105 5 xlO s 5 x 10 5

+ + +

19.6+4.1 34.3+8.9 14.0+5.1 22.8 + 5.9 12.8_+7.1 15.4 ± 8.7

a ( + ) , B-END added, ( - ) mock culture. b Values are mean cpm X 10 3 ± SD.

As shown in Table 3 as increasing numbers of macrophage containing stimulator cells were added to the culture, the proliferative response of mock-treated cultures progressively declined. The attenuated proliferation was probably due to the suppressive effects of monocytes present in the stimulator cell fraction (Smolen et al. 1981). Moreover, one of the major soluble factors that mediate this suppression is PGE 2. It has been shown that B-END can modulate the effect of prostaglandins on mast cell function (Yamasaki et al. 1982). Since prostaglandins are considered to be produced by monocyte macrophages and suppress T cell proliferation in part by directly modulating interleukin-2 (IL-2) secretion and IL-2 receptor acquisition (Tilden and Balch 1983), the effect of B-END on PGE 2 added to AMLR cultures was examined. In addition other investigators (Rappaport and Dodge 1982) have shown the down-regulation of T cell proliferation was chiefly mediated by monocytes that released PGE 2. Table 4 shows that B-END alone did augment the AMLR in a group of responsive volunteers (n = 3) whereas the addition of PGE 2 significantly depressed blastogenesis. Moreover, the PGE2-induced suppression was not due to a decreased secretion of the T cell growth factor IL-2. However, when

50 4o

70_ 3o N

20 IO 0

m

0

I

2

3

4

5

6

7

Day

Fig. 1. Influence of B-END on kinetics of A M L R proliferation. Cells were processed as described in Materials and Methods. Cultures contained medium (O) or B-END (10 -8 M (11)). Standard deviations did not exceed the mean values by more than ± 0.3 (cpm × 10-3).

TABLE 4 EFFECT OF STIMULATOR CELL DEPLETED OF MONOCYTES ON AMLR: MODULATION BY PGE 2 AND fl-END Supernatants were harvested on day 4 and quantitated in the CTLL-20 assay as described in Materials and Methods. Treatment fl-END

PGE 2

Concentration 0 10 -8 10 -1° 10-12 10 -9 10 -8 10 -7 10 - 6

PGE 2 + fl-END

10 - 9 + 10 -8 10-8+10 -8 10-7+10 -8 10 - 6 + 1 0 - 8

AMLR a 14.4± 9.8 41.3±11.2 36.8± 9.1 41.4 + 13.4 8.4+ 5.8 7.3± 9.4 7.8± 6.4 4.4± 5.8 48.4± 7.8 * * 44.85:6.4 ** 39.9± 4.8 * 34.3± 7.4

AMLR-generated IL-2 units ( X 1 0 - 1 )

b

18.4_+8.8 13.4±4.1 16.8±9.4 14.4 ± 7.4 19.8+6.2 18.7±2.2 14.6±7.8 18.9± 2.0 16.4±6.4 15.8±4.3 14.9±7.2 17.3±4.5

a Values expressed as mean cpm+SD×10 -3 (n = 3). Values compared by the Student t-test against AMLR performed in the absence of modulating factors (i.e., PGE2, fl-END). * * P < 0.0l; * P < 0.05. b Spontaneous release of isolated responder cells was < 0.1 units.

f l - E N D ( 1 0 - 8 M ) was a d d e d to the cultures, the o p i a t e p e p t i d e clearly a m e l i o r a t e d the suppressive effects o f P G E 2.

Effect of fl-END on lymphoreactivity with monoclonal antibodies R e c e n t l y it was r e p o r t e d ( P u p p o et al. 1985) that w h e n m o n o n u c l e a r cells were p r e - i n c u b a t e d w i t h f l - E N D a p r o f o u n d d e c r e a s e in O K T 3 + a n d O K T 4 + cells was a p p a r e n t . If this d a t a is correct a n d the d e c r e a s e d n u m b e r of p h e n o t y p i c a l l y d e f i n e d T4 cells correlates with d e c r e a s e d h e l p e r f u n c t i o n t h e n p e r h a p s an a t t e n u a t i o n a n d n o t a u g m e n t a t i o n of the A M L R w o u l d b e a p p a r e n t . I n this regard, in a series o f five e x p e r i m e n t s n o n a d h e r e n t m o n o n u c l e a r cells were e x p o s e d to f l - E N D (10 - 8 M ) for 24 h after which l y m p h o r e a c t i v i t y with a p a n e l of m o n o c l o n a l a n t i b o d i e s was assessed b y flow c y t o m e t r y . S u r p r i s i n g l y O K T 3 ÷ cells a n d O K T 4 ÷ cells d e c r e a s e d b y a p p r o x i m a t e l y 50% whereas a 50% i n c r e a s e in O K M 1 ÷ cells was o b s e r v e d . D u r i n g the p e r f o r m a n c e of this s t u d y we n o t e d significant loss o f cells f o l l o w i n g filtration t h r o u g h the n y l o n mesh, a step r e q u i r e d to r e m o v e c l u m p e d cells w h i c h c o u l d p o t e n t i a l l y ' c l o g ' the lines of the flow c y t o m e t e r . W e s u s p e c t e d t h a t f l - E N D c a u s e d a n o n s p e c i f i c e n h a n c e m e n t of a d h e s i v e n e s s of T cells w h i c h r e s u l t e d in their a g g r e g a t i o n a n d r e m o v a l b y the n y l o n mesh. Since this c o u l d c o n t r i b u t e to the o b s e r v e d p h e n o t y p i c a l t e r a t i o n o b s e r v e d following i n c u b a t i o n with f l - E N D , we p r o c e e d e d to re-assess m o n o c l o n a l l y m p h o r e a c t i v i t y b y direct visualization. A s shown in T a b l e 5, n o p h e n o t y p i c a l t e r a t i o n s were o b s e r v e d f o l l o w i n g i n c u b a t i o n w i t h f l - E N D . It should b e n o t e d that d e s p i t e a t t e m p t s to m i n i m i z e cell c l u m p i n g b y

8

TABLE 5 PHENOTYPIC CHARACTERIZATIONOF MONONUCLEAR CELLS TREATED WITH fl-END USING MONOCLONALANTIBODIES Treatment

Methodof analysis

Immunofluorescent-positivecells (%) OKTll O K T 3 O K T 4 OKT8

0 10 8

Flow cytometric b

85.5+5.1 79+4.7 81 +5.7 44+6.8

0 10-8

Direct vision c

84 +7.4 85 +_4.9

a

49+6.1 30+4.0

80+6.9 44+6.1 77+_4.7 49+9.6

OKM1 a

ig+

24 + 5 . 1 23+7.8 17.8+4.7 47±6.0

8.9+6.0 8.1-t-2.3

27 +7.9 21+_6.1 24 +-3.1 23_+4.8

7.4+_2.8 6.8+_3.1

PBMC were incubated for 24 h in the concentration of designated r-END. b Flow cytometrywas performed as described in Materials and Methods. c Direct visualization was performed as described in Materials and Methods. d 9% of OKM1+ cells were esterase positive. a

repeated Pasteur pipetting, many of the mononuclear cells were observed in aggregates of 3-5 cells which were predominantly O K T 3 + / O K T 4 +.

Discussion

In this report we have extended the immunomodulating influence of r - E N D to the AMLR. When M L R studies were generated no augmentation was noted. These observed differences are difficult to reconcile but perhaps can be attributed to the maximal proliferation that occurs during an M L R which can not be further augmented by r - E N D whereas the A M L R represents a suboptimal stimulatory culture system which can be augmented by r - E N D . How r - E N D augments the A M L R remains conjectural. Pre-incubation of the B cells with r - E N D and adding them to the A M L R did not produce augmentation. It has been reported that when r - E N D (concentration range 1-1000 nM) was added to B cells in conjunction with either B cell growth factor or recombinant interleukin-2 (IL-2), B cells became activated and underwent proliferation. Proliferation was maximal on day 3 (Alvarez-Mon et al. 1985). In this regard, activated B cells have been shown to be more potent stimulators of the A M L R than resting B cells (Crow and Kunkel 1985). These combined observations may explain in part why augmentation of the A M L R begins to occur during the fourth day of culture. In addition, investigators using an antigen-induced T cell proliferation system have observed that r - E N D enhances IL-2 receptor expression (Kleinheinz and Kilroy 1985). Thus, the responder T cells in the A M L R may be more receptive to IL-2 produced in this system and thereby enhance proliferation. Since P G E 2 is known to decrease IL-2 receptor acquisition (Tilden and Balch 1982), it is possible that r - E N D antagonizes the effect of PGE 2 at the level of the responder cell. Experiments are under way to determine if P G E 2 decreases the number of high-affinity IL-2 receptors on IL-2stimulated cells. Thereafter r - E N D will be added to determine if the opiate peptide reverses the effect of PGE2.

A l t h o u g h specific o p i a t e r e c e p t o r s have b e e n d e m o n s t r a t e d o n h u m a n m o n o n u c l e a r cells ( M e h r i s h i a n d Mills 1983), we s h o w e d that the o p i a t e a n t a g o n i s t N A L o n l y p a r t i a l l y decreases a u g m e n t a t i o n of the A M L R . T h u s it a p p e a r s that f l - E N D b i n d s to l y m p h o i d cell n o n o p i a t e r e c e p t o r s ( H a z u m et al. 1975) a n d t h e r e b y m e d i a t e s e n h a n c e m e n t via this receptor. H o w e v e r , given the v a r i e t y o f cells p r e s e n t in the A M L R the p a r t i a l i n h i b i t i o n b y N A L m a y reflect the fact that s o m e cells m a y have classical o p i a t e r e c e p t o r s whereas o t h e r cells in the A M L R cultures m a y possess nonclassical o p i a t e receptors. F i n a l l y we p r e s e n t d a t a that c o n t r a d i c t s that of o t h e r i n v e s t i g a t o r s w h o d e m o n s t r a t e d a p r o f o u n d decrease in the p e r c e n t a g e of T3 + a n d T4 + cells, w h e n the m o n o n u c l e a r cells were p r e - i n c u b a t e d with f l - E N D ( P u p p o et al. 1985). W e were u n a b l e to c o r r o b o r a t e their observations. T h e a p p a r e n t differences are n o t r e a d i l y e x p l a i n a b l e except p e r h a p s that the c o n t r a d i c t o r y results are a t t r i b u t a b l e to m e t h o d ologic differences. I n s u m m a r y , we p r e s e n t evidence that f l - E N D is a b l e to a u g m e n t the A M L R . This o b s e r v a t i o n further e x p a n d s the i m m u n o m o d u l a t o r y p o t e n t i a l of the o p i a t e peptide fl-END.

Acknowledgement I w o u l d like to t h a n k G e n e v i e v e L a P i n s k a as always for h e r excellent secretarial assistance.

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