Regulation of β-endorphin receptor expression in mouse spleen cells with con A and rIL-2

0192-0561/92 $5.00 + .00 Pergamon Press Ltd. ©1992 International Society for Immunopharmacology.

Int. J. lmmunopharmac., Vol. 14, No. 5, pp. 809-819, 1992. Printed in Great Britain.

REGULATION OF/3-ENDORPHIN RECEPTOR EXPRESSION IN MOUSE SPLEEN CELLS WITH CON A A N D rlL-2 LING JIA,* HIDEK1 HARA,* TOSHIKAZU OKOCHI t and SHIGERU NEGORO *~ *Osaka University Medical School, Department of Medicine III, 1-5-50 Fukushima, Fukushima-ku, Osaka 553; fOsaka University, Faculty of Health and Sport Sciences, Toyonaka, Osaka 560, Japan

(Received 17 July 1991 and in final form 30 December 1991)

-The expression of the/3-endorphin receptor on both activated and unstimulated mouse spleen cells was studied. Results showed that unstimulated cells have only one type of/3-endorphin receptor with a specific low affinity (Kd = 1.034 _+ 0.0237 x 10 -7 M, 25,000 sites/cell). After Con A stimulation, cells express two types of receptors, one with a low affinity (Kd = 1.034 __+0.024 x 10 -7 M, 320,000 sites/cell) and the other with a high affinity (Kd = 1.052_ 0.033 x 10 9 M, 49,000 sites/cell). The kinetic experiments during 4 days after Con A activation indicated that the receptor of high affinity emerged from 24 to 72 h, while the low affinity one increased in number after stimulation. The receptor numbers of both high and low affinity ones reached a maximum peak at 72 h, then began to decline. The addition of exogenous rlL-2 depressed the Con A-induced increment of the receptor numbers of both the high and low affinity ones, but enhanced the proliferative response of the cells. It is suggested that the degree of the expression of the receptors does not simply depend on the mitogenic degree of the cells. In addition, our experiment demonstrated that splenocytes cultured in medium with or without Con A or Con A + rlL-2 for 96 h did not secrete any detectable amount of/3-endorphin with use of the RIA assay, which is sensitive enough to detect the much lower levels of/3-endorphin than that necessary for biological effects. We suggest that the expression of the high affinity/3-endorphin receptor on the activated T-lymphocytes may have to precede the production of IL-2 to potentiate the T-cell proliferative response. The mechanisms and modes of interaction between the neuroendocrine system and the immune system were discussed. Abstract

The interaction between the immune and the neuroendocrine systems has been verified by numerous data obtained from humans and animals (Brown & Blalock, 1990; Besedovsky & Sorkin, 1977; Dantzer & Kelley, 1989; Dion & Blalock, 1988). The interactions have been thought to have important roles in stress response. Blalock (1984) has mentioned that the immune system may function as a sensory organ that recognizes noncognltive stimuli such as bacteria, viruses and other kind of antigens. O f the large array o f neuroendocrine transmitters considered to be involved in the regulation of immune responsiveness, the glucocorticoid h o r m o n e has been the prototype and has received much attention. The blood levels o f the h o r m o n e are known to increase not only under various kinds of stress but also after immunizations (Besedovsky & Sorkin, 1977; Khansari, Murgo & Faith, 1990; Shek & Sabiston, 1983). The h o r m o n e suppresses the

production of IL-1 and IL-2 which have many important roles in immune and inflammation responses (Khansari et al., 1990). The limbic system of the CNS, thalamus, hypothalamus and hypophysis controls the production and release of the hormone from the adrenal gland via the adrenocorticotrophic h o r m o n e (ACTH). /~-Endorphin and A C T H originate from the same prohormone, proopiomelanocortin (POMC) (Nakanish et al., 1979), and are closely related in their tissue-specific processing and coordinate release (Zakarian & Smyth, 1982; Mains, Epper, Glembotski & Dotes, 1983; Dores, Akii & Watson, 1984)./3-Endorphin is a 31 amino acid peptide which induces analgesic effects and behavioral changes in experimental animals (Loh, Tseng, Wei & Li, 1976; Li, 1977; Akil, Watson, Young, Lewis, Khachaturian & Walker, 1984). It was originally isolated from the m a m m a l i a n pituitary (Li, 1982). U p o n stress,/3-endorphin is also

*Author to whom correspondence should be addressed. 809

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L. JIA et al.

apparently released from the pituitary into the blood (Rossier, French, River, Ling, Guillemin & Bloom, 1977). /3-Endorphin in the circulation has been reported to modulate monocyte chemotaxis (Van Epps & Saland, 1984) and superoxide generation (Peterson, Sharp, Gekker, Brummitt & Keane, 1987a), inhibit lymphocyte ),-interferon secretion (Peterson, Sharp, Gekker, Brummitt & Keane, 1987b), enhance natural killer cell activity (Mandler, Biddison, Mandler & Serrate, 1986; Mathews, Froelich, Sibbitt & Bankhurst, 1983) and inhibit the antibody response to sheep erythrocytes (Jonson, Smith, Torres & Blalock, 1982)./3-Endorphin also has been shown to influence the proliferative response and interleukin-2 production by mouse spleen cells (Gilmore & Weiner, 1989; Gilman, Schwartz, Milner, Bloom & Feldman, 1982; Gilmore & Weiner, 1988), the proliferative response of human peripheral blood mononuclear cells (Fontana, Fattorossi, D'Amelio, Migliorati & Perricone, 1987; Puppo, Corsini, Mangini, Bottaro & Barreca, 1985; McCain, Lamster, Bozzone & Grbic, 1982) and calcium uptake by rat thymocytes (Hemmick & Bidlack, 1987). Recently, Zakarian, Eleazar & Silvers (1989) reported that /3-endorphin production in the rat pituitary increased to 15-fold during graft rejection. These reports suggest that fl-endorphin has important relevance to the immune response. It is widely accepted that the biological effect of a hormone on a target cell not only depends on the concentration of the hormone, but also on the extent and nature of receptor expression. Indeed, the presence of fl-endorphin binding sites has been reported on fresh intact human lymphocytes, several transformed human mononuclears (Hazum, Chang & Cuatrecasas, 1979; Shahabi, Peterson & Sharp, 1990a), murine EL4-thymoma (Schweigerer, Schmidt, Teschemacher & Wilhelm, 1985a), and cultured murine splenocytes (Shahabi, Linner & Sharp, 1990b). However, the binding sites and affinity are different from report to report. fl-Endorphin has been reported to regulate the functions of activated lymphocytes, but not resting, unstimulated lymphocytes in the above reports. Identifying /3-endorphin receptor expression during immune response may be critical to the understanding of the process of immunoregulation by fl-endorphin. Therefore, it is interesting to examine whether the fl-endorphin receptor on the activated lymphocytes is any different from that on the resting, unstimulated ones. However, to our knowledge, little is known about the characteristic difference between the fl-endorphin receptors on

fresh unactivated lymphocytes and activated ones. This prompted us to investigate the character of the receptor on fresh unactivated and activated lymphocytes. We also examined the regulatory mechanism of receptor expression in relation to proliferative activity of the cells and the effect of exogenous interleukin-2.

EXPERIMENTAL PROCEDURES

Animals. Balb/c mice aged 8 weeks were purchased from Japan SLC (Shizuoka). All of the mice used in this study were female. Mice were housed in our laboratories until use and were used at around 10 weeks of age for the spleen cell preparation. The weight of mouse ranged around 1 9 - 23 g. Medium and reagents. RPMI 1640 medium was obtained from Flow Laboratories, Inc. (McLean, VA) and FCS from Irvine Scientific (lot 300070206). RPMI 1640 medium was supplemented with 5 × 10 5M 2-mercaptoethanol, 10o70 FCS, penicillin (100 U/ml), streptomycin (100 #g/ml), and gentamycin (80 #g/ml). Concanavalin A (Con A product No., c5275) and /3-endorphin (human synthetic product No., E6261) were purchased from Sigma. The binding buffer for the /3-endorphin receptor assay consists of 25 mM Tris, 5 mM KC1, 120 mM NaCI, 1.1 mM EDTA, 1.4 mM MgSO4 and 10 mM glucose (pH 7.6). The buffer also contains 0.5% BSA, 0.1 mg/ml bacitracin (Sigma). Hank's balanced salt solution (Hank's) was obtained from Nakarai Tesque (Japan). Nine volumes of 0.85°70 NH4C1 and one volume of 170 mM Tris (pH 7.65) solutions were mixed immediately before use and used as mouse RBC lysis buffer (ACT). [~2~I] RIA kits for /3-endorphin (human) and [3H]thymidine (743.7 GBq/mmol) were purchased from Dupont (U.S.A.). (3-[~2~I]iodotyrosy127) fl-endorphin was obtained from Amersham (72 TBq/mmol). Preparation o f the cell suspension. Ten mice were killed by cervical dislocation and the spleens were aseptically removed and placed in complete medium. The spleens were teased to gain single cell suspensions and red blood cells were lysed by incubating with lysis buffer (ACT) with shaking in a water bath at 37°C for about 7 min. After three washes (1600 rev/min 5 min at 4°C) with Hank's, the cells were resuspended in complete medium. The cell number was counted by a hemocytometer and viability was determined by the dye exclusion method with trypan blue.

/3-endorphin Receptor

Spleen cell culture. Sixty milliliters of spleen cell suspensions (I × 106 cell/ml) were placed in flask (FALCON 3024, from B e c t o n - Dickinson). The cell suspensions were cultured with Con A (2.5/ag/ml) for cell stimulation in the presence or absence of 200 U/ml rIL-2 when necessary and cultured at 37°C in a CO2 incubator. To prevent cultured spleen cells from overcrowding, the cell suspensions were divided equally into two flasks and supplemented with 30 ml of fresh complete medium to each flask at 48 h and the cell cultures were continued. The cultured spleen cells were harvested at the indicated time point and dead cells were removed by centrifugation on F i c o l l - Paque (d = 1.077) (Pharmacia, U.S.A.). The cell number and viability were determined before being used in a binding assay. Cell viabilities were 91 _ 3%. Competitive inhibition radioligand binding assay. Cells were suspended at the cell density from 35 to 45 × 106 viable cells/ml in the binding buffer. By using this binding buffer, the specific binding became 15% higher than that obtained by using Hank's (data not shown). Cell suspensions (fresh intact cells 9 × 106, Con A-stimulated cells 7 x 106) were incubated in triplicate with a fixed concentration of (3-['25I]iodotyrosy127) /J-endorphin (5 × 10-~0 M to l0 -9 M, depending on the assay)for 2 h in the presence of serial dilutions of unlabeled fl-endorphin (cold/3-endorphin, l0 -5 to l0 10 M) in a final volume of 200/A in 4 ml polypropylence tubes placed in the ice bath. In our preliminary experiments, the equivalent or similar value of competitive binding assays was attained during ice-cold incubation from 1.5 h to overnight. The binding was saturable since the cell bound (3-[~25I]iodotyrosy~27)fl-endorphin was displaced by an excess of unlabeled /~-endorphin. Then we performed our experiments with a 2 h incubation period as the saturable equilibrium condition. Binding increased as function of spleen cell concentration within the range from 15 × 106 to 60 × 106cells/ml (data not shown). The cell suspension was layered over a 400/al cushion of H a n k ' s - F i c o l l - P a q u e solution (d = 1.05) in 1.5 ml Eppendorf tubes and was centrifuged at 14,000 rev/min for 60 s at 4°C in a Hitachi microfuge. This procedure effectively separated the cell bound ligand from free unbound ligand with complete cell recovery at the bottom of tube. Supernatants were aspirated and the portion of tube with the cell pellet was cut apart with scissors and counted in a ),-emission counter (752-JCD). Nonspecific binding represents the cell bound radioactivity that can be obtained in the presence of

811

10 -5 M unlabeled/3-endorphin. Specific binding was calculated by subtraction of the nonspecific binding from the cell bound radioactivity in the presence of indicated cold or unlabeled/3-endorphin. Scatchard analysis (Scatchard, 1949). The analysis was based on rearrangement of data from the Competitive Inhibition Radioligand Binding Assay. Briefly, the number of the bound fl-endorphin (B) was calculated by multiplying bound activity (counts/min)/total activity (counts/min) by the total number of /3-endorphin (cold /3-endorphin plus iodinated /3-endorphin). The amounts of free /3-endorphin (F) were calculated as the total number of /3-endorphin subtracted from the number of bound/3-endorphin. The curve of the Scatchard plot was best fitted to these data by the nonlinear least squares regression analysis using the computer program Cricket Graph on Macintosh II (Apple). The equilibrium constant and the number of /3-endorphin receptors per spleen cell were obtained by the method of curve-fitting as described by Berson & Yalow (1964). Cellproliferation assay. Spleen cells were cultured with [3H]thymidine (0.5/aCi/well) for the last 6 h before cell harvest. The cells were harvested on a Printed Filtermat A (L.K.B. Wallac, Finland) by a 1295-001 cell Harvester (L.K.B. Wallac) and the radioactivity of the harvested cells was counted in a 1205 Betarplate (liquid scintilation counter; L.K.B. Wallac). Radioimmunoassay for fl-endorphin. As we could not obtain the anti-mouse endorphin antibody, the concentration of/3-endorphin in culture supernatants nonstimulated, Con A-stimulated and Con A + rlL-2-stimulated groups was measured by using a commercially available fl-endorphin [~25I] RIA Kit (human). Mouse fl-endorphin differs by only two amino acids from the human fl-endorphin (Li, 1986). The antibody used in this experiment also has been shown to react with the mouse/3-endorphin and its precursor fl-lipotropin (Isseroff et al., 1989). The assay was carried out according to the procedure designated by the supplier (DuPont, U.S.A.).

RESULTS

Competitive inhibition radioligand binding assay of fresh intact mouse spleen cell Intact fresh mouse spleen cells were prepared and suspended to the cell density at 45 × 106 viable cells/ml. The amount of specific binding of (3-['25I]iodotyrosy127) /3-endorphin to the cells was

812

L. JIA et al.

determined by the use of the competitive inhibition radioligand binding assay as described in Experimental Procedures. As shown in Fig. 1, we could find a monophasic inhibition curve and estimate the dissociation constant (Kd) from the concentration of cold unlabeled /3-endorphin required to reduce the binding activity of '25I-/3-endorphin on the cells to a level of the 50% of the m a x i m u m binding activity. Thus, the intact fresh spleen cells were shown to have the specific /3-endorphin receptors whose Kd and Ka were 1.034 ± 0.0237 × 10 -7 and 9.68 -+ 0.219 × 108 M - ' , respectively. These values of Kd and Ka were comparable with those of the low affinity/J-endorphin receptors on human lymphocytes.

120

100,

80.

40.

20,

O~ -10

-9

-8

-7

-6

-5

Concontralion of cold 13-endorphin (log M)

Competitive inhibition radioligand binding assay o f the Con A-stimulated spleen cells Spleen cells were stimulated and cultured with 2.5/~g/ml o f Con A for 72 h. Viable cells were recovered and suspended to the cell density at 35 × 106 viable cells/ml. The/3-endorphin receptors on these cells were assessed by the competitive inhibition radioligand binding assay. The representative results are shown in Fig. 2. The inhibition curve exhibited biphasic shape which suggested the existence o f two kinds of different affinity sites on the cells. The high affinity binding site began to reach a plateau at around 2 × 10 -~ M. The lower one began to reach a plateau at around 10 -6 M. The dissociation constants (Kd) or affinity constants (Ka) calculated from the above data are Kd = 1.052_+0.033 x 10 9 M or Ka = 9.51_+ 0.295 × 108 M -t for the former, and Kd = 1.034+_0.024 x 10 7 M or Ka = 9.68_+0.219 × 108 M -~ for the latter, respectively. The former binding site could be called the high affinity fl-endorphin receptor and the latter the low affinity one.

Scatchard analys& o f the data f r o m the competitive inhibition radioligand binding assay of fresh intact and Con A-stimulated mouse spleen cells To determine the character and the number of the receptors on the fresh intact and Con A-activated spleen cells, the data presented above were rearranged to a Scatchard plot as described in Experimental Procedures. As shown in Fig. 3, the linearity of the rearranged plots of the data obtained from the fresh intact spleen cells indicated the presence of fl-endorphin receptors o f one kind of affinity, while the curvilinearity of those obtained from Con A-stimulated spleen cells indicated the presence of the receptors with two different kinds of

Fig. 1. The competitive inhibition radioligand binding assay to fresh intact mouse spleen cells. Each of the cell suspensions (45 × 106 cells/ml) was incubated at 0°C for 2 h with a fixed concentration (10 -9 M) of hot (3-['25I]iodotyrosy127)/3-endorphin and various concentrations (10 -~° to 10 -5 M) of cold unlabeled/3-endorphin. The binding activity obtained in the presence of 10 5M /3endorphin was considered nonspecific binding. The specific binding activity (B~) was determined by subtracting the nonspecific binding from the binding activity in the presence of various concentrations of cold unlabeled /3-endorphin. The maximum specific binding activity (B0) was obtained as the binding activity at the lowest concentration 10 t°M of cold /3-endorphin. The data were plotted as the percentage of inhibition of the specific binding activity to the maximum binding activity ( I - B / B o ) × 100. The affinity of the receptors was estimated from the ICs0of the cold ligand.

affinity. The number of/3-endorphin receptors per spleen cell was estimated as described in Experimental Procedures. The numbers of the receptors with low affinity on fresh intact and Con A-stimulated spleen cells are 25,000 and 320,000 sites/cell, respectively. The number of those with a high affinity on activated spleen cells was 49,000 sites/cell. Intact fresh spleen cells had no detectable high affinity receptors.

Time course o f the affinity and the number o f fl-endorphin receptors expressed after Con A stimulation The O-endorphin receptors expressed on the spleen cells were assessed at 0, 24, 48, 72 and 96 h after the

813

~-endorphin Receptor 0.04-

120.

• O

72B/F freshB/F

100.

0.03 80,

60,

0.02

40. 0.01

20'

0 -10

0.00

i -9

-8

-7

-6

-5

C o n A s t i m u l a t i o n by m e a n s o f the competitive i n h i b i t i o n r a d i o l i g a n d b i n d i n g assay as described above. T h e affinity o f the specific /J-endorphin receptor was estimated f r o m the results described above. As s h o w n in T a b l e 1, the dissociation c o n s t a n t s o f the low a f f i n i t y / 3 - e n d o r p h i n receptor s h o w e d n o significant changes after the C o n A s t i m u l a t i o n o f the cells. H o w e v e r , a n e w / 3 - e n d o r p h i n receptor with a high affinity emerged o n the spleen cells at 24 h a n d b e g a n to d i s a p p e a r at 96 h after the stimulation. T h e results were also analysed by a S c a t c h a r d plot to d e t e r m i n e the n u m b e r o f / 3 - e n d o r p h i n receptors o n the cells. T h e kinetics o f n u m b e r o f low a n d high affinity receptors are s h o w n in Fig. 4. Simultaneously, kinetics o f the proliferative r e s p o n s e o f the cells are also s h o w n in Fig. 4. The n u m b e r s o f b o t h receptors with high a n d low affinities reached a m a x i m u m peak at 72 h. T h e n u m b e r s at the m a x i m u m peak are 320,000 sites/cell for low affinity

200~

3OO00O

Sites/coil

Concentration of cold [~-endorphln (log M)

Fig, 2. The competitive inhibition radioligand binding assay of Con A-stimulated mouse spleen cells. Each of the cell suspensions (35 × l0 s cells/ml) was incubated at 0°C for 2 h with a fixed concentration (10 -9 M) of hot (3-['251] iodotyrosy127)/3-endorphin and various concentrations (10 -~° to 10 -5 M) of cold unlabeled /3-endorphin. The binding activity obtained in the presence of 10-SM fl-endorphin was considered nonspecific binding. The specific binding activity (B~) was determined by subtracting the nonspecific binding activity from the binding activity in the presence of various concentrations of cold unlabeled fl-endorphin. The maximum specific binding activity (Bo) was obtained as the binding activity at the lowest concentration 10-~°M of cold fl-endorphin. The data were plotted as the percentage of inhibition of the specific binding activity to the maximum binding activity (1-Bx/B0) × 100. The affinities of the two kinds of receptors were estimated from the IC,0 of the cold ligand.

i

1OOOO0

Fig. 3. Scatchard analysis of data obtained from the competitive inhibition radioligand binding assay of fresh unstimulated and Con A-stimulated spleen cells. Data from the competitive inhibition radioligand binding assay of fresh unstimulated (O) and Con A-stimulated cells (O) were rearranged into a Scatchard plot as described in Experimental Procedures. The ordinate represents the number of binding sites per cell vs the number of free fl-endorphins. The abscissa represents the number of bound sites per cell.

Table 1. Time course of dissociation constants of low affinity (Kdl) and high affinity (Kd2)/3-endorphin receptors on the spleen cells after Con A stimulation Culture time

Low affinity (Kdl M)

High affinity (Kd2 M)

Freshly 24 h 48 h 72h 96 h Mean _+ S.E.

1.08 × 10 -7 1.08 x 10 -7 1.17 x 10 -7 1.00 × 10 7 9.8 × 10 -s 1.06 __ 0.03 × 10 -7

None* 3.8 × 10 -9 1.10 × 10 -9 1.04 X 10 9 None*

Mouse spleen cells were cultured with 2.5/ag/ml Con A from 0 - 9 6 h as described in Experimental Procedures. Cultured cells were harvested at each indicated time point and viable cells were adjusted to the cell density of 35 × 106 cells/ml. The affinities of the receptors were determined as described in Fig. 2. *Expressed none. *Expressed in low number.

receptors a n d 49,000 sites/cell for high affinity receptors, respectively. T h e proliferative response also reached a m a x i m u m peak at 72 h.

814

L. J|A et al.

4O

10

Table 2. Effects o f the addition of exogenous rlL-2 on the expression of fl-endorphin receptors on Con A-activated mouse spleen cells

8-

3o

c ,20

o

10 i

_g

10'

o

Proliferation ( x 10 -3 c o u n t s / m i n )

Con A +rlL-2

186.73 _+ 4.47

Con A

77.52 _+ 6.67

i

|

--

Group A

Kd(M)

Sites/cell

1.02 × 10 7 22 × 104 1.00 x 10 -9 1.87 × 104 1.08 x 1.14 ×

32 x 104 4.9 × l&

1 0 -7 10 -9

2-

~o

O"

o~

20

40

60

80

100

0

Culturl time (hr)

Fig. 4. The kinetics of the expressed n u m b e r of fl-endorphin receptors and the proliferative response of mouse spleen cells after C o n A stimulation. The n u m b e r s of low ( • --) and high ( - • -) affinity fl-endorphin receptors on the m o u s e spleen cells cultured for 0 - 9 6 h after Con A stimulation. At the time indicated, receptor numbers were determined by Scatchard analysis as described in Fig. 3. The proliferative responses of the cells were determined by [3H]TdR uptake ( (3--) as described in Experimental Procedures.

10000 -

Mouse spleen cells cultures were started at a cell density of l06 cells/ml with 2.5/~g/ml of Con A or with 2.5/~g/ml C o n A plus 200 U / m l rIL-2 for stimulation. After 72 h culture, cells were harvested. One aliquot of the cells was placed in microtiter wells and cultured with [3H] thymidine (0.5/JCi/well) for 6 h. Values are means _+ S.E. of four wells. The other aliquot of the cells was used for the competitive inhibition radioligand binding assay. The dissociation constants (Kd) were determined as shown in Fig. 2. The n u m b e r s of/3-endorphin receptors on the cells were determined with use of Scatchard analysis of the data.

100.

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00OO

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Concentration of [[3-endorphlnadded(pg/mt) in the samples to be measured 1"u -10

10-9

1(~"8

lt) -7

1(; .6

1O'~-S

1() "4

Concentration of c o l d [~.endorphln (M)

Fig. 5. The effect of the addition of exogenous IL-2 on the binding of C o n A-stimulated mice spleen cells to [~2~I]/3-endorphin. Spleen cells cultures were started at a cell density of 106 cells/ml with 2.5 # g / m l C o n A in the presence ( - - • ) or absence ( ©--) of 200 U / m l rlL-2. After 72 h culture, cells were harvested and used for the competitive inhibition radioligand binding assay. The concentration of [~2~l]fl-endorphin used in this experiment was 5 × 10 -~° M. Specific binding activity was determined as described in Experimental Procedures. Mean binding values _+ S.E. of triplicates are shown and the data were obtained with cells from five mice. Very similar data have been obtained in several experiments.

Fig. 6. The sensitivity and standard curve of the fl-endorphin ['25I] RIA Kit. According to the procedure designated by the supplier (DuPont, U.S.A.), we added 100/al of various concentrations of/3-endorphin to the tube containing 100 t~l (about 9300 c o u n t s / m i n ) []zsI]fl-endorphin and 100 tal of anti-fl-endorphin antibody solutions. After incubation for 20 h at 4°C, free antigen was removed by centrifugation after the addition 0.5 ml of a charcoal suspension. The radioactivity of supernatant containing i m m u n e complexes was measured. (%B) The percent b o u n d for each standard and sample were determined by dividing the average net c o u n t s / m i n of the supernatants by the average net c o u n t s / m i n of total counts tubes. (%B/Bo) The normalized percent b o u n d was calculated as follows: %B/Bo = [%B of standard or s a m p l e / % B of " 0 " standard] x 100.

3-endorphin Receptor Table 3. The concentration of /J-endorphin in culture supernatants Sample

Culture time (h)

Standard

°7oB/Bo

/3-endorphin value

95.6

5 pg

Medium

24 48 72 96

101 100.8 100.5 99.3

<5 <5 <5 <5

pg pg pg pg

Con A

24 48 72 96

98.3 98.9 98.7 99.7

<5 <5 <5 <5

pg pg pg pg

rlL-2 + Con A

24 48 72 96

100 100.1 100 98.2

<5 <5 <5 <5

pg pg pg pg

The spleen cells (106 cells/ml) were cultured in medium with various combinations of Con A (2.5/ag/ml) and rlL-2 (200 U/ml) for 24-96 h. The culture supernatant at indicated times after the start of culture was measured for the concentration of/3-endorphin with use of the 3-endorphin [t2~|] RIA Kit.

The down regulation o f 3-endorphin receptor expression on activated cells by exogenous rlL-2 From the similar kinetic patterns of the expression of the receptor numbers and proliferative response as shown in Fig. 4, we could imagine that the increment of/3-endorphin receptor numbers depended on the degree of proliferative response of spleen cells induced by the mitogenic stimulation. We next examined whether this was the case. It is well known that major T-cell proliferation is mediated by IL-2 after Con A activation. If the receptor expression on activated cells is regulated by the degree of proliferation induced by IL-2, the addition of exogenous rlL-2 will lead to enhancement of receptor expression on the activated cell. In fact, there was a 2.5-fold increment of proliferative response of the cells by the addition of exogenous rlL-2. However, as shown in Fig. 5, the addition of exogenous rlL-2 leads to the reduction of binding capability on activated cells as compared with that without rIL-2 addition. The data shown in Fig. 5 were analysed by a Scatchard plot and the results are summarized in Table 2. The results indicated that the addition of rlL-2 reduced the number of both high and low type receptors on the cells but did not induce any change in the affinities of either type of receptor.

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fl-Endorphin levels in the culture supernatant Finally, we measured the amount of fl-endorphin levels in culture supernatants of the spleen cells at 24, 48, 72 and 96 h after stimulation with various combinations of Con A (2.5/~g/ml) and rIL-2 (200 U/ml). As shown in Fig. 6, the radioimmunoassay system used could detect the concentration of fl-endorphin or less than 5 pg/ml (1.44 x 10-" M) which was much lower than that necessary for the biological effect to be observed. The results shown in Table 3 indicate that the culture supernatants obtained at any time point after the stimulation contained less than 5 pg/ml (1.44 × 10-" M) of /3-endorphin.

DISCUSSION In this paper we clearly demonstrated that intact unstimulated mouse spleen cells possessed a 3-endorphin receptor of a specific low affinity (Kd = 1.034 _+ 0.024 x l0 -7 M, 25,000 sites/cell) (Figs 1 and 3). Hazum et al. (1979) reported a similar receptor on fresh intact human lymphocytes. Interestingly, Shahabi et aL (1990b) could not detect any 3-endorphin receptor on fresh intact murine splenocytes. The discrepancy between the data may be due to the differences in cell number and the separating method used. Shahabi et al. used fewer cells and separated the cell-bound from the unbound ligand by washing the cells repeatedly during which dissociation of the ligand from a low affinity receptor might have occurred. We also found that after Con A activation mouse spleen cells expressed two different kinds of receptors (Figs 2 and 3). The low affinity receptor showed a Kd of 1.034_+0.024 x 10 -TM and 320,000 sites/cell, and the high affinity receptor had a Kd of 1.052 _+ 0.033 x 10 -9 M and 49,000 sites/ cell, respectively. /3-endorphin receptors have been detected on transformed human lymphocytes RPMI 6237 (Hazum et al., 1979), monocytes U937 (Shahabi et al., 1990a) and mouse EL4-thymoma cells (Schweigerer et al., 1985a) and murine splenocytes cultured (Shahabi et al., 1990b). RPMI 6237 was reported to have two kinds of receptors for which the Kd were 3 x 10 -9 M and 10 -7 M , respectively. The number of binding sites was not determined. EL4-thymoma cells were shown to have two kinds of receptors for which the Kd were 6.5 × 10 -8 and 2.2 x I0 6 M, respectively. The number of binding sites per cell was 19,000 for the former and 260,000 for the latter, respectively. Shahabi et al. reported

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that murine spleen cells cultured for 96 h expressed a /3-endorphin receptor for which the K d and number were 4.1 × 10 9 M and 2600 sites/cell, respectively. Although they did not mention the process of receptor expression, the receptor may be derived from the small number of cells which were activated by the autologous mixed leukocyte reaction. Similar results were also observed in our experiment (data not shown). When spleen ceils were cultured in only complete medium, the specific binding sites for /3-endorphin on the cells tended to increase during 120 h culture, but the number of binding sites was lower than that of Con A-stimulated cells. To our knowledge, our paper is the first report which shows two different kinds of/3-endorphin binding sites on Con A-activated spleen cells. The character of the /3-endorphin receptors on murine and human mononuclear cells has been reported to be nonopiate in nature. Our preliminary results with use of naloxone (data not shown) also confirmed these previous reports. Hasum et al. (1979) have reported that cultured human lymphocytes (RPMI 6237) exhibited specific binding to /3-endorphin. The binding was inhibited by low concentrations of/3-endorphin but was not affected by opiate agonists and antagonists, or by enkephalin analogs. The carboxy-terminal region of /3-endorphin is essential for this binding activity, since a-endorphin was not active. They also suggested the presence of low affinity sites by an inhibition binding assay. Schweigerer et al. (1985a) have shown the presence of specific nonopioid binding sites on murine EL4 thymoma ceils. Shahabi et al. (1990a) have reported the expression of a naloxone-insensitive binding site for/3-endorphin on murine splenocytes. Recently, Sharp, Shahabi, Peterson & Linner (1991) have shown the biochemical character of this naloxone-resistant site. A large number of reports demonstrated that the /3-endorphin may regulate various immune functions. It is interesting to note that all the reports have shown that /3-endorphin only has effects on the immunocompetent cells under an activated state, but not when the cells are unstimulated. Fontana et al. (1987) have reported that the physiological concentration of /3-endorphin (10 ,2_ 10-,3 M) showed modulatory effects on the Con A-induced human lymphocyte proliferative response. A physiological concentration of/3-endorphin has also been reported to enhance the proliferative response of murine spleen cells to T-cell mitogens such as Con A and phytohemagglutinin (Gilmore & Weiner, 1989; Gilman et al., 1982). The concentration of fl-endorphin in plasma under a physiological

condition was reported to be around 1 0 - 100 pg/ml (De Carolis et al., 1984). A higher concentration of /3-endorphin was attained under various stressful situations (Colt, Wardlaw & Frantz, 1981; Contos, Rust, Hollt, Mahr, Kromer & Teschemacher, 1979). Rossier et al. (1977) have reported that the concentration of circulating/3-endorphin reached as high as 10 ng/ml under foot shock. Our study also suggests that the circulating /3-endorphin has important physiological roles upon activated lymphocytes through the high affinity/3-endorphin receptors. The experiments of the kinetic studies indicated that the receptor of high affinity was expressed from 24 to 72 h and the number of the low affinity receptors was enhanced after Con A stimulation (Table 1). The number of both types of receptors reached the peak at 72 h. Proliferative activity also reached a peak at 72 h (Fig. 4). Then, we examined whether the proliferative activity of the cell has any effects upon /3-endorphin receptor expression. The activation of the cell by Con A is a complex process including at least three stages: (1) release of a soluble factor from monocytes/macrophages; (2) the release of IL-2 by a particular T-cell subset under the influence of the monokine; and (3) binding of the IL-2 to the IL-2 receptors on the cells resulting in a proliferative response (Smith, 1980). To determine whether the enhancement of /3-endorphin receptor expression is enhanced by the proliferative response caused by IL-2, exogenous rlL-2 was added to the spleen cell culture. The results showed that the proliferative response was enhanced, however, the number of fl-endorphin receptors on activated spleen cells was rather decreased by the addition of rlL-2 (Fig. 5 and Table 2). The results indicate that the degree of fl-endorphin receptor expression on the ceils does not simply correlate with the degree of proliferation of the cells. It is interesting to note that Sharp et al. (1991) reported recently that expression of/3-endorphin binding sites on mouse spleen cells was enhanced in proportion to the concentration of Con A in an in vitro culture but the proliferative response was rather suppressed at higher concentrations of Con A. In the present experiments, we used the concentration of Con A which induced the optimum proliferative response of the cells. A higher concentration of Con A might have induced a suppressor mechanism (cell) for cell proliferation. In the present experiments, we examined /3-endorphin receptor expression and the proliferative response of the unfractionated splenocytes as a whole which contained various kinds and a series of T-cells, B-cells and monocytes/macrophages. It has been

/3-endorphin Receptor reported that macrophages did not have/3-endorphin receptors (Hazum et al., 1979). Although /3-endorphin has been shown to increase the proliferative response of spleen cells to the T-cell mitogens Con A and PHA, the hormone has no effect on the proliferative response of the cells to a mixture of lipopolysaccharide and dextran sulfate which are both specific B-cell mitogens (Gilman et al., 1982). Moreover, the majority of cells which responded to Con A stimulation was T-cells. Thus, we suspect that most of the cell populations which expressed /3-endorphin receptors might be T-cells. The more precise analyses on the cell subsets are under experimentation. It has been reported by several investigators that cells other than neural cells were able to secrete /3-endorphin. Corticotropin-releasing factor (CFR), Newcastle disease virus and LPS were reported to induce ACTHI_39 and /3-endorphin from leukocytes (Kavelaars, Ballieux & Heijnen, 1989; Westley, Kleiss, Kelley, Wong & Yuen, 1986; HarbourMcMenanin, Smith & Blalock, 1985). Mouse spleen macrophages were shown to contain immunoreactive ACTH and immunoreactive /3-endorphin (Lolait, Lim, Toh & Funder, 1984; Lolait, Clements, Markwick, Cheng, McNally, Smith & Funder, 1986). Phytohemagglutinin (PHA)-stimulated human T-lymphocytes were shown to express pro-opiomelanocortin (POMC) mRNA. The P O M C mRNA was also reported to be expressed in cloned cytotoxic murine T-cells by IL-2 stimulation (Farrar, Hill, Annick & Vinocour, 1987). Thus, in our present experiment, the addition of exogenous rlL-2 would have induced activated splenocytes to secrete /3endorphin. In addition, it has been mentioned that after the binding of/3-endorphin to the receptor, the receptor- ligand complex was internalized to induce a down regulation of receptor expression (Schweigerer, Schmidt, Teschemacher & Wilhelm, 1985b). To test this mechanism could explain our results, we measured the level of/3-endorphin in Con A-stinmlated cell culture supernatants in the presence or absence of rlL-2. However, our results indicated that there was no detectable amount of /3-endorphin in the culture supernatant by using a RIA which is sensitive enough to detect

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1.44 × 10 -1. M (5 pg/ml) of/3-endorphin (Table 3). In the present study, we did not examine the possible cross-reactivity of IL-2 and /3-endorphin to each receptor. Although the mechanism to suppress /3-endorphin receptor expression yet has to be determined, our results clearly show the interrelation between the IL-2 production and /3-endorphin receptor expression. Gilman et al. (1982) have reported that/3-endorphin but not a-endorphin and (Ala2, Met 5) enkephalin was able to enhance the proliferative response of mouse spleen lymphocytes to Con A and PHA. Gilmore & Weiner (1988) have shown that/3-endorphin enhanced the production of IL-2 from mitogen-stimulated, unfractionated mouse spleen cells as well as a cloned T-cell line. These results indicate that/3-endorphin may enhance the proliferative response of the cell through the enhancement of IL-2 production. As IL-2 suppresses or down regulates/3-endorphin receptor expression, we predict that the binding of/3-endorphin to the high affinity receptor on activated cells should have to precede the production of IL-2 to exhibit the efficient help to the proliferative response of the cells. Although the physiological role of/3-endorphin in the immune response has yet to be determined, it is interesting to note that/3-endorphin and ACTH are produced from same precursor of POMC by hypophysis (Nakanish et al., 1979). ACTH is well known as a stimulator of the adrenal gland to produce and release glucocorticoid which suppresses IL-2 production by lymphocytes. We suggest that the most important physiological role of fl-endorphin in immune response might be the enhancement of IL-2 production by lymphocytes. Once enough IL-2 was produced, IL-2 suppresses /3-endorphin receptor expression halting the overproduction of IL-2 in collaboration with glucocorticoid. To examine the more precise regulatory mechanisms of receptor expression and post-receptor mechanisms during the immune response, further investigations are in progress. Acknowledgements - - The authors wish to thank Prof. T.

Kishimoto for his excellent advice and comment on this manuscript, Dr S. Kasayama for useful discussion and Dr A. Ogata for his kind help with operation of the computer.

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