ph Immuno armacolooy ELSEVIER Immunopharmacology 28 (1994) 183-192

Expression of naloxone-resistant fl-endorphin binding sites on A20 cells: effects of concanavalin A and dexamethasone

Mahmoud Shaker, Nahid A. Shahabi, Burt M. Sharp* Endocrine-Neuroscience and Neuroimmunomodulation Research Laboratory, Minneapolis Medical Research Foundation, and Departments of

Medicine. Hennepin County Medical Center and University of Minnesota, D-3, 914 South 8th Street, Minneapolis, MN 55415, USA

(Accepted 19 May 1994)

Abstract

fl-Endorphin affects mononuclear cell proliferation, cytokine production and calcium uptake in a naloxone-resistant manner. The presence of naloxone-insensitive binding sites for fl-endorphin have been demonstrated on murine EL4- thymoma cells, transformed human mononuclear cells and normal murine splenocytes. Since murine splenic B cells have been shown to express naloxone-resistant receptors for fl-endorphin in response to the mitogen, concanavalin A (Con A), the A20 B-cell lymphoma line was used to further study regulation of this site by Con A and dexamethasone. Analyses showed two sites: a high-affinity site, Kdl = (8.7 + 2.3) × 10- 11 M and binding capacity (Bmax~) of (2.6 + 2.0) x 103 receptors/cell; and a low-al~ity site, Kd: = (2.2 + 0.8) × 10- 8 M with Bronx2 of (1.5 + 0.8) x 105 receptors/cell. Competi- tion studies showed that N-acetyl-fl-endorphin was approx. 5-fold and fl-endorphin6_31 10-fold less potent than fl-endorphinl_31. Neither fl-endorphinl_27 nor naloxone, morphine or other opioid receptor agonists displaced [125I]fl- endorphin. Con A (20 #g/ml) significantly increased the Bma x (3.5-fold; expressed per cell) and resulted in a loss of the higher-affinity site. However, the increased Bm~ occurred in proportion to the Con-A-induced increase in protein/cell. Dexamethasone (Dex) also increased Bm~,, primarily by increasing (2-3-fold) the number of lower affinity sites. In con- trast to Con A, two binding sites persisted after treatment with Dex, which exerted a minimal effect on protein/cell. Therefore, binding/cell and binding/protein/cell were both significantly enhanced by Dex. The combined effects of Dex and Con A on binding failed to show additivity or synergy. When binding was analyzed per protein/cell, the effect of Con A appeared to dominate; the Dex-enhanced binding/protein/cell was no longer evident in the presence of Dex plus Con A. Thus, Dex and Con A may enhance binding by independent mechanisms.

Key words: fl-Endorphin receptor; Binding kinetics; Concanavalin A; Dexamethasone

1. Introduction

The opiate alkaloids and opioid peptides such as fl-endorphin affect a variety of leukocyte functions, in vivo and in vitro, which are involved in host de-

* Corresponding author.

0162-3109/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 2 - 3 1 0 9 ( 9 4 ) 0 0 0 2 9 - F

fense and immunity (Johnson et al., 1982; Mathews et al., 1983; Sharp et al., 1985; Mandler et al., 1986; Morley et al., 1987; Stead et al., 1987; Payan et al., 1987; Peterson et al., 1987a,b). Some of these can be differentiated according to their sensitivity or resis- tance to the opiate receptor antagonist, naloxone. Mu-like (Ovadia et al., 1989) and delta-like opioid receptors (Carr et al., 1988) have been partially char-

184 M. Shaker et al. / Irnmunopharmacology 28 (1994) 183-192

acterized on murine splenocytes and human periph- eral blood mononuclear cells. In these studies, g-like binding was sensitive to displacement by naloxone or morphine and completely resistant to fl-endorphin. In contrast, the effects of fl-endorphin on mononuclear cell proliferative responses (Gilman et al., 1982; McCain et al., 1982; Puppo et al., 1985; Shahabi et al., 1991), production of cytokines (e.g., interleukin-2; Gilmore and Weiner, 1988) and cal- cium uptake (Himmick and Bidlack, 1987) were re- sistant to inhibition by naloxone.

Several studies have demonstrated the presence ofnaloxone-insensitive binding sites for fl-endorphin on murine EL4-thymoma cells and transformed human mononuclear cells (Hazum et al., 1979; Sch- weigerer et al., 1985). Limited characterization of these binding sites on thymoma cells showed a 76- kDa band after chemical cross-linking (Schweigerer et al., 1985) and Scatchard analysis demonstrated an affinity of 65 nM (Kd). Recent studies in our laboratory have demonstrated the expression of a naloxone-resistant binding site for fl-endorphin on normal lymphoid tissue (Shahabi et al., 1990a). A high-affinity binding site for fi-endorphin was in- duced on normal murine splenocytes during culture, although this was not detectable on freshly obtained normal splenocytes (Shahabi et al., 1990a). Con- canavalin A (Con A), a T-cell mitogen, affected ex- pression of this site in a manner that appeared to be inversely related to its mitogenic effects (Sharp et al., 1991). 48 h after culturing splenocytes, Con A at 7.5 and 10 gg/ml enhanced the specific binding of fl-endorphin but reduced proliferation.

Competition studies have shown that these bind- ing sites are equi-sensitive to displacement by fl-endorphin and N-acetyl-fl-endorphin, and that the C-terminus of fl-endorphin is required for binding (Shahabi et al., 1990a,b). Although these sites were naloxone insensitive, they exhibited properties such as size, salt sensitivity and responsiveness to GTP- 7S which were similar to those observed for fl-endorphin binding to brain opiate receptors (Shahabi et al., 1990b). This site was also rapidly down-regulated by phorbol myristate acetate, an effect which was seen with intact cells but not with membranes (Shahabi and Sharp, 1993). This was due to a reduction in B . . . . and was fully reversible within 24 h.

Several laboratories have reported that the inter- action of fl-endorphin with the naloxone-resistant binding site modulates mitogen-driven mononuclear cell proliferation (Gilman et al., 1982; McCain et al., 1982; Puppo et al., 1985; Shahabi et al., 1991). We have reported that splenocyte proliferation due to low concentrations of phytohemagglutinin (PHA) was inhibited by fi-endorphin at concentrations (10-9-10 -7 M) related to the K d (4.1 x 10 -9 M) for fl-endorphin binding to murine splenocytes (Shahabi etal., 1990a). The inhibitory effect of fl-endorphin was not antagonized by naloxone, and neither des-tyr-fl-endorphin2_ 31 nor fi-endorphinl_27 were effective. N-acetyl-fl-endorphin, which is equi- effective at binding to this site on murine spleno- cytes, did not inhibit PHA-induced proliferation. In- stead, N-acetyl-fi-endorphin reversed the inhibitory effect offl-endorphin on PHA-induced proliferation. Thus, both functional and binding studies show that (i) the C-terminus of fl-endorphin is required for interaction with the naloxone-resistant site and that (ii) a free N-terminus is required for biological ef- fects, but not for binding. The concordance between binding and functional studies indicates that the naloxone-resistant fl-endorphin binding site is a re- ceptor. N-acetyl-fi-endorphin is a naturally occur- ring peptide which may function as an antagonist at this receptor.

To further characterize the naloxone-resistant fl-endorphin receptor and identify factors regulating its expression, the A20 B cell lymphoma line, derived from a spontaneous reticulum cell neoplasm discov- ered in a BALB/cAnN mouse, was investigated. A20 cells have been used to study various properties of B cells (Kim and Dejoy, 1986). Since normal splenic B cells have been shown to express naloxone- resistant receptors for fl-endorphin which were in- creased by Con A (Shahabi et al., data not shown), the A20 cell line was selected to further study the effect of Con A (Sharp et al., 1991) and dexametha- sone. Con A appears to affect normal B-cell differ- entiation, although the mechanism is unknown. It activates B cells to leave G o of the cell cycle, but not to proliferate (Hawrlowicz and Klaus, 1984), and apparently to secrete immunoglobulin (Kim and Dejoy, 1986). Dexamethasone, a potent synthetic glucocorticoid was reported to have an inhibitory effect which favors growth arrest of the murine

M. Shaker et al. / Immunopharmacology 28 (1994) 183-192 185

B-lymphoma cell line WEH-231 (Gold et al., 1991). Other studies have shown that dexamethasone (i) strongly inhibited the proliferation of all mono- nuclear cells fractions including the B cells, after incubation with mitogens in the presence of high concentrations of dexamethasone (Ow et al., 1983) and (ii) significantly inhibited the growth of the Ramos B-cell line at i0 - 7 M (Sasaki et al., 1982).

2. Materials and methods

activity was isolated by filtration through glass filter paper presoaked for at least 30 min at room tem- perature in a solution of K2HPO 4 (50 mM) pH 7.4 containing 0.01~o Triton X-100. Filters were then counted in a gamma counter with 73 ~o efficiency. Competition studies were performed with 0.05 nM [x25I]fl-endorphin and naloxone HC1. Scatchard analysis of the data was performed using the non- linear least squares regression analysis of Ligand by McPherson (McPherson, 1985). All studies were done in triplicate and repeated at least twice.

2.1. Cell culture and preparation for radioligand bind- ing assay

The A20 cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured continuously in suspension in RPMI-1640 (Celox, Hopkins, MN, USA), supple- mented with 10~o heat-inactivated fetal bovine serum (Sigma, St. Louis, MO), 100 U/ml penicillin, 100 #g/ml streptomycin, 2 mM glutamine (Sigma) and 5 x 10-5 M fl-mercaptoethanol. Cell cultures, grown as (1-1.5) × 106 cells/ml, were fed three times per week. They were maintained in a humidified at- mosphere of 5 ~o CO2 at 37 ° C. For binding assays, cells were centrifuged, washed three times with Hanks' buffered saline solution (HBSS; Gibco, Grand Island, NY, USA) containing 0.1 ~o gelatin and 2 mM EDTA, and then resuspended in assay buffer (Tris buffer (50 mM), pH 7.4, containing ba- citracin (50 #g/ml), leupeptin (10/ag/ml), soybean trypsin inhibitor (i0 #g/ml) and benzamidine (1 mM)); all chemicals were from Sigma, unless stated otherwise.

2.3. Proliferation assay

A20 cells were plated in quadruplicate at a den- sity of i x 105 cells/well in 96-well plates. Cells were stimulated with different concentrations of Con A and/or dexamethasone for 48 h. During the last 18 h, cells were pulsed with 0.4/~Ci of [3H]thymidine and harvested onto glass filter paper, using an au- tomated cell harvester (Brandel, Gaithersburg, MD, USA). All studies were repeated at least three times.

2.4. Statistical analysis

Results are expressed as mean + S.E.M. Differ- ences amongst groups were determined by a one- way analysis of variance with Duncan's multiple contrasts, using SYSTAT (Systat, Evanston, IL, USA).

3. Results

3.1. Optimization of the specific binding of [3-endorphin

2.2. Radioligand binding assay

The binding of [ 1251]fl-endorphin (2000 Ci/mmol) was determined by filtration assay as previously de- scribed (Howard et al., 1985; Schweigerer et al., 1985). Briefly, 50-#1 aliquots of a suspension of 0.2 x 106 intact cells were incubated with [ 125I]fl- endorphin in a total volume of 100 #1 of assay buffer. Nonspecific binding was determined in the presence of 4 #M (final concentration) unlabeled fl-endorphin (Peninsula Labs., Belmont, CA, USA). After incu- bation for 60 min at 4 ° C, the receptor-bound radio-

Intact A20 cells were used to define the time course of [125I]fl-endorphin binding at 4, 25 or 37°C (Fig. 1). The fractional specific binding was maximal by 10 min at 37°C, 20 rain at 25°C and 60 min at 4 °C. After that, binding declined-rapidly at 37 and 25 ° C. In contrast, maximal binding was stable at 4°C for more than 200 min. Thus, studies were per- formed at 4 °C for 60 rain.

Fig. 2 shows the effect of cell number on the spe- cific binding of 0.5 n M [ 125I]fl-endorphin. The first data point was obtained with 0.1 x 10 6 cells, and maximum specific binding was achieved with ap-

186 M. Shaker et al. / Immunopharmacology 28 (1994) 183-192

0 . 5

0 . 4

°

0 0 .3 C" m l-- O~ gc l -- Z 0 .2

Z

o.~ ~"~

' ~ ' * o . . . o . . . . . . . . . • • , , . . . . .... ....... .. ...... : ......

30 60 90 120 150 180

TIME [ M I N I

Fig. 1. The effect of time and temperature on [~25I]fl-endorphin specific binding to A20 cells. A20 cells were washed three times with HB S S, cells were resuspended in radioligand binding buffer at 4 × 106 cells/ml; [12Sl]fl-endorphin (0.5 nM) was incubated with 50 ~1 of cell suspension for increasing time intervals at vari- ous temperatures. Non-specific binding (11-23~o at 4°C) was determined using 4 ktM unlabeled fl-endorphin. Each data point is the mean + S.E.M. for 2-3 separate experiments, each done in triplicate.

Z o

M-

I - - Z i,iJ

Z

t ~

1 . 5

~-,w-...= TOTAL

SPECIFIC

0 . 5 . . . . . . . . ~

,o,Oql, o'°'

o ,o ,'5 2o

CELL NUMBER [10 6 ]

Fig. 2. The effect of cell number on ['251]/~-endorphin specific binding. A20 cells were washed three times with HBSS and dif- ferent concentrations of cells were resuspended in radioligand buffer along with [IzsI]/?-endorphin (0.5 nM) for 60 rain at 4°C. Non-specific binding was determined for each point using 4 #M unlabeled fl-endorphin. Each data point is the mean _+ S.E.M. for 2-3 separate experiments, each done in triplicate•

prox. 2.5 × 106 cells/ incubation. Thereafter, 0.2 × 106

cells were used throughout the study (second data point).

3 . 2 . C h a r a c t e r i z a t i o n o f the f l - e n d o r p h i n b i n d i n g s i te on

A 2 0 cel ls

Fig. 3 shows a characteristic saturat ion study in which A20 cells were incubated with concentra t ions of [125I]fl-endorphin from 0.14 n M to 20 nM. Non-

specific binding was linear throughout the dosage range. Analysis using Ligand (McPherson, 1985) showed two sites: a high-affinity binding site of Kd, = 8.1 x 10- xl M and binding capacity (B . . . . ) of 5.4 × 103 receptors/cell; and a low-affinity binding site of Kd2 = 2.9 x 10-8 with a Bmax2 of 0.9 × 105 receptors/cell. The validity of the 2-site analysis was evident from comparisons of the correlation coeffi- cients and residual variances for 2-site vs. 1-site analyses. The correlation coefficient for the best lin- ear fit of all 9 data points was -0 .74 (residual var- iance = 1509), whereas the best fit for points 1,5 and

5,9 generated correlation coefficients of -0 .99 and -0.89, respectively (residual variance for the curvi-

linear fit was 209). This experiment was repeated three separate times and the means + S.E.M. for the K d and Bma x values are shown in Table 1 (control group).

Competi t ion studies using opiate receptor ligands and analogs of fl-endorphinl_3x are shown in Fig. 4. N-Acetyl-fl-endorphin was approx. 2-fold and

Table 1

Analysis of [~25I]/?-endorphin binding to A20 cells treated with Dex or Con A compared to control (n=3-4 experiments/ treatment)

Treatment K~ (Mol) B . . . . . (receptors/cell)

Control (8.7 + 2.3) × 10 1~ 2.6 + 2.0 x 10 3 (2.2+0.8)× 10 -8 1.5+_0.8× 105

Dex 10-7 M (8.7 +0.7)x 10 11 3.2+2.2×103 (3.0+0.01)×10 -8 3.2_+2.3×105

Con A 20 #g/ml (3.3+0.6)× 10 -8 9.3+2.5 x 105

M. Shaker et al. / lmmunopharmacology 28 (1994) 183-192 187

so 9,49['9; TOTAL

' • SPECIFIC , .~ 4 0 . . . . ~ .... N O ~

~r / i - ' - 30- = ~. 4,78[-g, .= 20- . . . . . .......

I 0 "

o 6,46E-84 o so ,oo t~o 2oo 18[-11 3,OI[ ' lO

I z ~ I - B - E N D O R P H I N [ n M ] 1 2 s I - D - E N D O R P H i N B O U N D [I,4]

Fig. 3. Binding isotherm and Scatchard plot of [ ~25I]fl-endorphin binding to A20 cells. A20 cells were washed three times with HBSS, pellets were resuspended in radioligand binding buffer at 4 × 106 cells/ml and increasing concentrations of [ t25I]fl-endorphin were added to 50/~1 of cell suspension. Incubations were performed for 60 min at 4°C. Analysis of the binding data, using the Ligand non-linear curve fitting program (30), indicated the presence of two sites: a high-affinity binding site of Ko] = 8.1 × 10- ~l M and binding capacity (Bmaxl) of" 5.4 × 103 receptors/cell; and a low-affinity binding site of K<, = 2.9 × 10-8 with a Bmax2 = 0.9 × 105 receptors/cell.

fl-endorphin6_3~ 4-fold less potent than fl-endorphin at displacing [125I]fl-endorphin. g i values for fl-endorphin 1-31, N-acetyl-fl-endorphin and ~'

z fl-endorphin6_31 were 1.9, 4.9 and 6.8x 10 -s M, ~ t 2o

respectively. Neither fl-endorphinl_27, which lacks m the C-terminal tetrapeptide, nor naloxone were able

,,t- I O O to displace [ 125I]fl-endorphin. Furthermore, at con- ~- CI)

centrations as high as 10-3 M, morphine, U69,593, DAGO and 2,5 DPDP-enkephalin failed to displace ~ ao [125I]fl-endorphin (data not shown).

3.3. The effects of Con A, dexamethasone (Dex) and combinations

t,D i m g~

z

m

Z A20 cells were treated with Con A (1, 5, 10 and =

Q . 20 pg/ml) or Dex (10- 13-10- 7 M) and binding was assessed 1, 24 or 48 h, thereafter. Differences were =

z only observed after 48 h. Fig. 5A shows that low ,~ concentrations of Con A (1 and 5 #g/ml) failed to ©

I

affect [125I]fl-endorphin binding, whereas 10 and 20 #g/ml significantly increased specific binding per cell by as much as 430~o compared to control. The via- bility of cells after 48 h of treatment with Con A was greater than 90~o. Fig. 5A shows the effect of Con A on thymidine uptake by A20 cells. Con A 1, 2.5 and 5 #g/ml had no effect on thymidine uptake; however, 10 and 20 pg/ml significantly reduced up- take by 18 ~o and 95 ~o, respectively. Thus, Con A 10

60

40

20

B - E N D ( I - 3 1 )

. . . . ,e-.-. N - A C - ~ - E N D ( I - $ 1 )

. . . . g . . . . J i E N O ( 6 - 3 | )

- - - ~ - - ' D - E N D ( I - 2 7 )

. . . . m - . - Nf lLOXONE

,,] /

o o. o - I o -'8 - '6 - '4

L 0 6 [ M ]

Fig. 4. Displacement of [125I]fl-endorphin binding to A20 cells by fl-endorphinl_31, N-acetyl-fl-endorphinl_31, fl-endorpbin 6-31 and ~ 27 and naloxone. Cell pellets were resuspended in radioli- gand binding buffer at 4 x 106 cells/ml; [125I]fl-endorpbin (0.5 nM) was incubated with 50 ttl of cell suspension and increasing concentrations (10 - 13-5 × 105 M) of unlabeled ligand at 4°C for 60 min. Each data point is the mean _+ S.E.M. of 3-5 separate experiments, each done in duplicate.

188 M. Shaker et al. / Immunopharmacology 28 (1994) 183-192

500

400 g, I.= 300 Z

I~ 200

I 0 0 '

3 0 0 A 125 !-9-ENOIIRIPIIINSPEClFIC BIN|ING

10 15 20

CONCRNRVRLIN A [ p g l m l ]

~ 2 0 0 I¢ I-- Z ¢= ¢J

I 0 0

0 0 0 0.0

B

- ,~ .o - ,~ .o -8'.o " -+.~ DEXAMETHASONE tO6 [ H I

Fig. 5. The effect of Con A (panel A) or dexamethasone (Dex; panel B) on thymidine uptake and on the expression of ['251]fl-endorphin binding by A20 cells. A20 cells (1 x 107 in 10 ml of medium) were cultured with varying concentrations of Con A (1-20 yg/ml) or dex- amethasone (10- 13-10 7 M). After 48 h, cells were washed three times with HBSS, pellets were resuspended in radioligand binding buffer at 4 x 106 cells/ml and ['25I]/~-endorphin (0.5 nM) was added to 50/11 of cell suspension. Specific ['25I]fl-endorphin binding was determined after incubating for 60 min at 4°C. Values (averages of 2-3 experiments, performed in triplicate) were expressed as a per- centage of control. Panel A shows that Con A 10 #g/ml and 20 #g/ml significantly increased specific binding/cell compared to control ( p < 0.01). As an index of cell proliferation, thymidine uptake was measured by pulsing ceils with 0.4/~Ci of [3H]thymidine for the last 18 h in culture. Values (averages of 2-3 experiments, performed in quadruplicate) were expressed as a percentage (~o) of control. Panel A shows that a significant reduction in thymidine uptake was induced by Con A 10 and 20 #g/ml (p<0.01 compared to control). Panel B shows that concentrations of 5 x 10- 8 and 10 ? M of Dex significantly increased fl-endorphin binding/cell (p<0.01). In addition, at concentrations of 10- + M and greater, Dex significantly reduced [3H]thymidine uptake (p < 0.01).

yg/ml minimally reduced [3H]thymidine uptake, whereas it substantially increased [ 1251 ]fl-endorphin binding.

The effect of different concentrations of Dex (10 13-10-7 M) on A 20 cells is shown in Fig. 5B. At low concentrations (10-13-10-9 M), there was no change in []25I]fl-endorphin binding. In contrast, concentrations of Dex greater than 5 × 10 8 M significantly increased binding per cell by 240-290~o. Cell viability was greater than 95~o after treatment with all concentrations of Dex. Thy- midine uptake by A20 cells treated with Dex is shown in Fig. 5B. Low concentrations had no effect while higher concentrations (10 -8 M and higher) pro- foundly reduced thymidine uptake (reduced to 56~ o of control at 10 -8 M and 20~ o at 10 -7 M). Thus, Dex 10- 8 M substantially inhibited cell proliferation without enhancing receptor expression which re- quired higher concentrations of Dex.

A20 cells were treated with 10 or 20 yg/ml Con A, 10 - 11 or 10 - 7 M Dex and combinations of Dex plus Con A. Fig. 6 shows that 10-11 M Dex alone did not affect [125I]fl-endorphin binding while 10-7 M Dex alone significantly increased binding per cell by

308~o. 10 and 20 /~g/ml Con A alone significantly increased [125I]fl-endorphin specific binding by 270 and 430~o per cell, respectively. The combination of Con A with Dex was no more effective than either agent alone. Fig. 7 shows that 10- 7 M Dex or Con (10 or 20/~g/ml) alone significantly reduced [3H]thy- midine uptake, but combining these agents showed no evidence of an interaction. Thus, 10-7 M Dex alone (10 -7 M Dex and Con A 0 yg/ml) reduced uptake to 20~o of control; 10 - 7 M Dex and 10 yg/ml Con A tended to further reduce uptake (to 10~o; p>0 .05 compared to 10- 7 M Dex alone), but to an extent similar to that due to 10/~g/ml Con A, alone.

Table 2 compares the effects of Dex (10-7 M), Con A (10 and 20 yg/ml) and a combination of Dex and Con A on specific binding expressed as binding/ cell vs. binding/protein/cell. Protein per cell (column 2) was markedly increased after treatment with Con A or Con A plus Dex, compared to the modest ef- fect of Dex alone. Dex significantly increased binding/cell (column 3; p<0.001) or binding/ protein/cell (column 4; p < 0.01). In contrast, Con A markedly enhanced binding/cell, but this was not evident when binding data were normalized per

M. Shaker et al. / lmmunopharmacology 28 (1994) 183-192 189

Z o

600

500

~ 400"

~ 3 0 0 "

~ 2 0 0 "

Z

IOO"

Z 0

I

[ ] CON A ALONE

" ~ - DEX lO-t lM +CON A

"="Z'-DEX IO-?M + CON A

0 0

C O N C A N A V A L I N A [ l l g / m l ]

" / / / / / / / / / / . I

2 0

Fig. 6. The effect of Con A and dexamethasone on [125I]fl-

endorphin binding to A20 cells. A20 cells (1 × 107 in 10 ml of medium) were cultured with Con A (10 or 20/~g/ml), Dex (10 - 1~ or 10-7 M) or combinations of both. After 48 h, cells were washed three times with HBSS, pellets were resuspended in ra- dioligand binding buffer at 4 x 106 cells/ml and [125I]fl-endorphin (0.5 nM) was added to 50/~1 of cell suspension for 60 min at 4°C. Values (means+ S.E.M.; n= 3-8 experiments/treatment) were expressed as a percentage (~o) of control. 10 7 M Dex alone or Con A (10 and 20/~g/ml) alone significantly increased [125I]fl- endorphin binding/cell (p < 0.001). There was no evidence for an additive or synergistic interaction between Dex and Con A.

120

o I 0 0

Z O

N 8 0

~ 6 0 ,

~ 4 0 ' z

~ 2 0 "

! Z

111111

! / / / / / / / / / / / / / / . / / / / / / / / / / / / / / / / / / / / J / / / / / 5¢~55

( ( ( ( ( ( O

0

[ ] CON A ALONE

' - * " DEX I 0 - I I M + CON A

-.,z-,- flEX I 0 -7M ÷ CON A

1O

c~.~,.iv//i

2 0

CONCANAVALIN A [ l t g / m l l

Fig. 7. The effect of con A and dexamethasone on thymidine uptake into A20 cells. A20 cells were plated in quadruplicate at a density of 1 x 105 cells/well in 96-well plates. Cells were stim- ulated with Con A (10 or 20/tg/ml), Dex (10 11 o r 10 - 7 M) or combinations of both for 48 h, and pulsed with 0.4 #Ci of [3H]thymidine for the last 18 h. Values (means + S.E.M.; n = 3-5 experiments/treatment) were expressed as a percentage of control thymidine uptake. Alone, Dex 10 - 7 M or both concentrations of Con A significantly reduced proliferation (p < 0.00 l). The effects of Dex l0 11 M plus Con A were not different than Con A alone, and Dex 10 - 7 M plus Con A 10 #g/ml was also not different than Dex 10 7 M alone.

cou ld no t be ana lyzed , due to the a p p e a r a n c e o f

pos i t ive coope ra t iv i ty (da ta no t shown) .

pro te in /ce l l . Similar ly, the e n h a n c e m e n t o f b ind ing

by t r e a t m e n t wi th D e x plus C o n A was only ev iden t

w h e n d a t a were ana lyzed as b inding/ce l l . Th is sug-

gests tha t the m e c h a n i s m s w h e r e b y C o n A vs. D e x

e n h a n c e d b ind ing are i ndependen t .

Tab l e 1 s u m m a r i z e s the b ind ing p a r a m e t e r s o f A20 cells t r ea ted wi th 1 0 - 7 M Dex , 20 # g / m l C o n

A or a c o m b i n a t i o n o f b o t h c o m p a r e d to cont ro l .

C o n t r o l and D e x - t r e a t e d cells s h o w e d two b ind ing

sites wi th s imilar affinities, whe rea s C o n A t r e a t m e n t

resu l ted in a loss o f the h igher affinity b ind ing site

and signif icantly i nc rea sed the Bma x o f the lower affinity b ind ing site ( expressed as b ind ing /ce l l ;

p < 0 . 0 1 ) . D e x also t e n d e d to inc rease the Bma x o f

the lower affinity site. B ind ing d a t a o b t a i n e d f r o m cells t r ea ted wi th 10 - 7 M D e x plus 20 # g / m l C o n A

4. D i s c u s s i o n

A na loxone - r e s i s t an t [x25I]f l -endorphin r ecep to r

has been desc r ibed on t r a n s f o r m e d i m m u n e cells,

inc lud ing h u m a n l y m p h o c y t e and m o u s e E L 4 thy-

m A m a cells ( H a z u m et al., 1979; Schwe ige re r et al.,

1985). F u r t h e r s tudies f rom our l abo ra to ry have

c h a r a c t e r i z e d the effects o f f r agmen t s o f f l - e n d o r p h i n

on [125I]f l -endorphin binding, the sensi t ivi ty to

N-ace ty l - f l - endo rph in and the inhibi t ion o f b ind ing

by ca t ions and G T P 7 S ( S h a h a b i et al., 1990a,b). In

the p re sen t s tudy, we o b s e r v e d two b ind ing sites on

con t ro l A 2 0 cells. O n e site had an affinity ( g d = 8 . 7 )< 1 0 l l M ) t h a t w a s considerably greater t han p rev ious ly desc r ibed on n o r m a l mur ine sp leno-

cytes (K o = 4.1 x 1 0 - 9 M ; Shahab i et al., 1990a) and

190 M. Shaker et aL / Immunopharmacology 28 (1994) 183-192

Table 2

fl-Endorphin binding expressed as binding/cell vs. binding/protein/cell"

Treatment Protein/cell Binding/cell Binding/protein/cell

Control 100 100 100 Dex (10- v M) 155 (152, 159) 351 + 22 226.3 + 14.3 Con A (10 #g/ml) 521 (474, 568) 340 (30l, 379) 66.5 (53, 80) Con A (20 #g/ml) 457 (428, 486) 422 (412, 432) 92.5 (96, 89) Dex and Con A (10/~g/ml) 582 (522, 642) 342 (295, 389) 58.8 (57, 61) Dex and Con A (20#g/ml) 427 (395, 460) 326 (313, 320) 76.5 (72, 81)

Values are expressed as percentages of control. Values are means + S.E.M. (n or averages of experiments performed twice (values for individual experiments in triplicate. All treatments with Dex used 10- 7 M.

= 4, separate experiments for Dex or control treatments) are given in parentheses). All experiments were assayed

cell lines (U937 h-monocyte-like cell (K d = 1.2 x 10- 8 M; Shahabi et al., 1990b), EL-4 thymoma (K d = 6.5 x 10- 8 M; Schweigerer et al., 1985b) and IM-9 lymphocyte (Kd= 3 × 10 -9 M; Hazurn et al., 1979)). In contrast, the lower affinity site detected on A20 cells (K d = 2.2 x 10- 8 M) was similar to that reported for U937 and EL-4 thymoma cells, but less than that reported for the IM-9 lym- phocytes.

The structural requirements for binding to the naloxone-resistant [ 125I]fl-endorphin binding site on A20 cells were similar to those required for binding to U937 (Shahabi et al., 1990b) and normal murine splenocytes (Shahabi et al., 1990a). In those studies, fl-endorphinl_27 con sistently failed to displace [ 1251 ]- fl-endorphin, while fl-endorphin6_31 and 28-31 were effective, although less so than fl-endorphinL_31. Thus, the C-terminal peptide is essential for binding, and progressive elongation of the peptide from C to N terminus results in enhanced potency. With A20 cells, N-acetylation resulted in a peptide approx. 2-fold less potent than fl-endorphinl_31, whereas N-acetyl-fl-endorphin was equipotent to fl-endorphin on murine splenocytes and U937 cells.

Con A at concentrations of l0 and 20/~g/ml en- hanced the binding of [ 125I]fl-endorphin to A20 cells and reduced thymidine uptake. Similar observations have been made with normal murine splenocytes stimulated by T-cell mitogens (Sharp et al., 1991). Con A also has been shown to up-regulate the ex- pression offl-endorphin receptors on splenic B cells (Shahabi and Sharp, data not shown). Previous re- ports have shown that soluble Con A did not induce

proliferation by normal B cells (Andersson et al., 1972; Kim and Dejoy, 1986). It also has been re- ported that Con A directly induced immunoglobulin secretion from a normal subset of B cells (Kim and Dejoy, 1986); however, the direct action of this lectin on B cells is highly controversial. Nonetheless, by direct or indirect action, Con A appears to affect events related to B-cell differentiation. It activates B cells to leave Go of the cell cycle, but not to prolif- erate (Hawrlowicz and Klaus, 1984), and apparently to secrete immunoglobulin (Kim and Dejoy, 1986). With the A20 B cell lymphoma line, Con A 20 gg/ ml significantly increased the Bma x by 3.5-fold (ex- pressed per cell) and resulted in a loss of the higher affinity site. It is possible that the assay simply failed to detect this site, in view of the increased number of low-affinity sites detected under these conditions. Table 2 shows that the increased Bma x occurred in proportion to the overall Con-A-induced increase in protein/cell. Thus, binding/protein/cell was not af- fected by Con-A.

The effect of Con A on binding/cell was not pro- portional to the anti-proliferative effect of Con A, since 10 #g/ml Con A reduced proliferation by 18~o (as assessed by [3H]thymidine uptake; Fig. 5A) whereas binding/cell was enhanced by 250Yo (Fig. 5A). In addition, comparison of the effects of 10 ~t/ml Con A to 20/~g/ml Con A showed that 10 #g/ml Con A increased binding/cell by approx. 43 ~o of the effect due to 20 /~g/ml Con A, whereas the anti-proliferative effect of 10 gg/ml Con A was only 20~o of the effect of 20 #g/ml Con A. This suggests that Con-A-enhanced binding/cell did not result di-

M. Shaker et aL / Immunopharmacology 28 (1994) 183-192 191

rectly from the anti-proliferative effect of Con A. Direct cell counts also were performed to verify

the anti-proliferative effect of Con A (data not shown). After 24, 48 and 72 h in the presence of 10 or 20 /~g/ml Con A, cell numbers were stable; the number of cells at these intervals was reduced by approx. 10 or 20-25~o, respectively, compared to the original numbers. Thus, the number of control cells increased 2.0-fold between 48-72 h, whereas the number of Con-A-treated cells did not change. Cell viability was always higher than 90~o at each time interval. These findings strongly suggest that the failure to observe an increase in cell number was due to an anti-proliferative effect of Con A rather than to apoptosis or toxicity. We also found that A20 cells failed to proliferate in serum-free defined medium, and that viability was markedly reduced (unpublished data). This suggests that A20 cell pro- liferation was not autonomous, but required an uni- dentified growth factor(s) present in serum.

Dexamethasone also significantly increased spe- cific binding (Table 2 and Fig. 5B). This appeared to reflect an increased B . . . . primarily due to an ap- prox. 2-fold increase in the number of lower affinity sites (Table 1). In contrast to Con A, two binding sites persisted after treatment with Dex, which ex- erted a minimal affect on protein/cell. Therefore, binding/cell or binding/protein/cell were both signifi- cantly enhanced by Dex. Similar to Con A, Dex was anti-proliferative. Again, a proportional relationship between the anti-proliferative and binding effects of Dex was not evident. For example, 10- 8 M Dex did not affect binding (Fig. 5B), whereas it reduced [3H]thymidine uptake by 40~o of control,

The combined effects of Dex and Con A on bind- ing or proliferation failed to show additivity or syn- ergy (Figs. 6 and 7). When binding was analyzed per protein/cell, the effect of Con A appeared to domi- nate that of Dex; thus, the Dex-enhanced binding/ protein/cell was no longer evident in the presence of Dex plus Con A (Table 2). This suggests that Dex enhanced binding by a different mechanism than Con A, and in combination, the action of Con A was dominant. This idea is further supported by the fact that two binding sites could be distinguished after Dex treatment whereas only one site was evident after Con A (Table 1).

In summary, the B-cell-related A20 cell line ex-

pressed two naloxone-resistant binding sites for fl-endorphin with Kd, --- (8.7 + 2.3) x 10- II M and Kd2 = (2.2 + 0.8) × 10 - 8 M. The Kd2 site was similar to that found on immune cells and reported by sev- eral laboratories (Schweigerer et al., 1985a,b; Sha- habi et al., 1990). In contrast, a naloxone-resistant site for fl-endorphin with an affinity in the range of Ka, has not been reported previously, to our knowl- edge. Dex treatment did not affect this site, whereas it became undetectable after treatment with Con A. Studies of specific binding showed that both Dex and Con A enhanced binding/cell at the Kd2 site. This was due to an increase in Bma × by Dex, and appeared to be due to a similar effect of Con A. However, when analyzed on the basis of binding/ protein/cell, only Dex enhanced binding, since Con A increased protein/cell in proportion to binding/ cell. The effect of Con A on binding appeared to be related to a generalized effect on protein expression/ cell. Thus, Dex and Con A appeared to enhance binding by independent mechanisms; in the pres- ence of both agents, Con A appeared to exert the predominant effect by increasing protein/cell and, secondarily, binding.

Acknowledgement

This work was supported by grants T32-DA- 07239 (M.S.) and DA-04196 (B.M.S.).

References

Andersson J, Sjoberg O, Moiler G. Mitogens as probes for im- munocyte activation and cellular cooperation. Transplant Rev 1972; 11: 131-141.

Carr DJ, Kim C-H, DeCosta B, Jacobson AE, Rice KC, Blalock JE. Evidence for a delta class opioid receptor on cells of the immune system. Cell Immunol 1988; 116: 44.

Gilman SC, Schwartz JM, Milner RJ, Bloom FE, Feldman JD. /3-Endorphin enhances lymphocytes proliferative responses. Proc Natl Acad Sci USA 1982; 79: 4226.

Gilmore W, Weiner LP. /?-Endorphin enhances interleukin-2 (IL-2) production in murine lymphocytes. J Neuroimmunol 1988; 18: 125.

Gold MR, Gajewski TF, DeFranco AL. Regulation of anti- immunoglobulin-induced B lymphoma growth arrest by trans- forming growth factor /31 and dexamethasone. Int Immunol 1991; 3: 1091.

Hawrylowicz CM, Klaus GGB. Activation and proliferation sig-

192 M. Shaker et al. / Immunopharmacology 28 (1994) 183-192

nals in mouse B cells. IV. Concanavalin A stimulates B cells to leave Go, but not to proliferate. Immunology 1984; 53: 703- 708.

Hazum E, Chang KJ, Cuatrecasas P. Specific non-opiate recep- tors for fl-endorphin. Science 1979; 205: 1033.

Himmick LM, Bidlack JM. fl-Endorphin modulation of mitogen- stimulated calcium uptake by rat thymocytes. Life Sci 1987; 41: 1971.

Howard AD, de La Banme S, Gioannini TL, Miller JM, Simon EJ. Covalent labeling of opioid receptors with radioiodinated human fl-endorphin. J Biol Chem 1985; 260: 10833.

Johnson HM, Smith EM, Torres BA, Blalock JE. Regulation of the in vitro antibody response by neuroendocrine hormones. Proc Natl Acad Sci USA 1982; 79: 4171.

Kim KJ, Dejoy SQ. Con A induction of IgG secretion from a B-lymphoid tumor cell line, A20. Immunology 1986; 59:15-21.

Mandler RN, Biddison WE, Mandler R, Serrate SA. fl-endorphin augments the cytotytic activity and interferon production of natural killer cells. J Immunol 1986; 136: 934.

Mathews PM, Froelich CJ, Sibbitt WL, Bankhurst AD. Enhancement of natural cytotoxicity by fl-endorphin. J Immu- nol 1983; 130: 1658.

McCain HW, Lamster IB, Bozzone JM, Gribic JT. fl-Endorphin modulates human immune activity via non-opiate receptor mechanisms. Life Sci 1982; 31: 1619.

McPherson GA. Analysis of radioligand binding experiments: a collection of computer programs for IBM PC. J Pharmacol Methods 1985; 14: 213.

Morley JE, Kay NE, Solomon GF, Plotnikoff NP. Neuropep- tides: conductors of immune orchestra. Life Sci 1987; 41: 527.

Ovadia H, Nitson P, Abrarnsky O. Characterization of opiate binding sites on membranes of rat lymphocytes. J Neuroim- munol 1989; 21: 93.

Ow KT, Abandowitz HM, Sarafty GA, Nash AR, Effects of dihydrotestosterone, 17fl-estradiol and dexamethasone on mi- togen and allogeneic induced lymphoblastogenesis. Endocrinol Exp 1983; 17: 69.

Payan DG, McGillis JP, Renold FK, Mitsuhoshi M, Goetzl EG. Neuropeptide modulation of leukocytes function. Ann NY Acad Sci 1987; 496: 182.

Peterson PK, Sharp BM, Gekker G, Brummitt C, Keane WF. Opioid-mediated suppression of cultured peripheral blood mononuclear cell respiratory burst activity. J Immunol 1987a; 138: 3907.

Peterson PK, Sharp BM, Gekker G, Brummitt C, Keane WF.

Opioid-mediated suppression of interferon production by cul- tured peripheral blood mononuclear cells. J Clin Invest 1987b; 80: 824.

Puppo F, Corsini G, Mangini P, Battaro L, Barreca T. Influence of fl-endorphin on phytohemagglutinin-induced lymphocyte proliferation and on the expression of mononuclear cell surfaces antigens in vitro. Immunopharmacology 1985; 10: 119.

Sasaki R, Mishima Y, Srivastava BI, Minowada J. Effect of dexamethasone on the growth of human lymphoblastoids cell lines. Jpn J Med 1982; 21: 89.

Schweigerer L, Schmidt W, Teschemacher H, Gramsch C. fl-endorphin: surface binding and internalization in thymoma cells. Proc Natl Acad Sci USA 1985a; 82: 5751.

Schweigerer L, Schmidt W, Teschemacher H, Wilhelm S. fl-Endorphin: interaction with specific nonopioid binding sites on EL4 thymoma cells. Neuropeptides 1985b; 6: 445.

Shahabi NA, Linner KM, Sharp BM. Murine splenocytes ex- press a naloxone-insensitive binding site for fl-endorphin. En- docrinology 1990a; 126: 1442.

Shahabi NA, Peterson PK, Sharp BM. fl-Endorphin binding to naloxone-insensitive binding site on a human mononuclear cell line (U937): effects of cations and GTP. Endocrinology 1990b; 126: 3006.

Shahabi NA, Burtness MZ, Sharp BM. N-Acetyl-fi-endorphin ~-31 antagonizes the suppressive effect of fl-endorphinl_3~ on mu- rine splenocyte proliferation via a naloxone-resistant receptor. Biochem Biophys Res Commun 1991; 175: 936.

Shahabi NA and Sharp BM. Activation of protein kinase C rapidly down-regulates naloxone-resistant receptors for fl-endorphin on U937 ceils J Pharmacol Exp Ther 1993; 264: 276.

Sharp BM, Keane WF, Suh HJ, Gekker G, Tsukayama D, Peter- son PK. Opioid peptides rapidly stimulate superoxide produc- tion by human polymorphonuclear leukocytes and peritoneal macrophages. Endocrinology 1985; 117: 793.

Sharp BM, Shahabi NA, Peterson PK, Linner KM. Biochemi- cal characterization of naloxone-resistant receptors for fl-endorphin on a human mononuclear cell line (U937) and murine splenocytes. In: H Friedman, S Spector and TW Klein (eds.), Drugs of Abuse, Immunity, and Immunodeficiency. 1991, p. 215.

Stead RH, Bienenstock J, Stanisz AM. Neuropeptide regulation of mucosal immunity. Immunol Rev 1987; 100: 333.