- Email: [email protected]
Modulation of mitogen-induced spleen cell proliferation and the antibody-forming cell response by beta-endorphin in vivo
Peptides, Vol. 10, pp. 473-479. © Pergamon Press plc, 1989. Printed in the U.S.A.
0196-9781/89 $3.00 + .00
Modulation of Mitogen-Induced Spleen Cell Proliferation and the Antibody-Forming Cell Response by Beta-Endorphin In Vivo A L E X A N D E R W. K U S N E C O V , A L A N J. H U S B A N D , .1 M A U R I C E G. K I N G A N D R O G E R S M I T H *
Department of Psychology and *Faculty of Medicine, University of Newcastle 2308, New South Wales, Australia R e c e i v e d 8 N o v e m b e r 1988
KUSNECOV, A. W., A. J. HUSBAND, M. G. KING AND R. SMITH. Modulation ofmitogen-induced spleen cell proliferation and the antibody-forming cell responseby beta-endorphin in vivo. PEPTIDES 111(2)473--479, 1989.--Experiments were conducted which compared the in vivo effects of beta-endorphin (BEP), gamma-endorphin (-¢EP), methionine-enkephalin (Met-ENK), and acetylated BEP(1-27) on the in vitro proliferative response of rat spleen cells to concanavalin A (ConA). In addition, the influence of BEP administration on the primary and secondary antibody-forming cell (AFC) response to the soluble antigen keyhole-limpet hemocyanin (KLH) was examined. Intravenous administration of BEP enhanced the spleen cell proliferative response to ConA assessed 3 hr after a single bolus infusion. Conversely, infusion with AcBEP(1-27) suppressed the proliferative response, whereas no effects of intravenous ~EP or Met-ENK treatment were observed. The enhancing effect of BEP administration was not detectable 24 hr after a single infusion, but could be maintained over a 44 hr period by multiple infusions. The primary AFC response to KLH was suppressed by a dose of 1 nmole BEP only. On the other hand, the secondary IgG AFC response to KLH was enhanced by 10 pmoles BEP, while the IgM and IgA AFC responses remained unaltered by BEP treatment. The anamnestic in vitro proliferative response of spleen cells cultured with KLH was not altered if BEP was administered at the time of secondary KLH immunization. These results extend previous observations of BEP-induced modulation of in vitro immune function by demonstrating that opioid and nonopioid forms of BEP administered in vivo alter the capacity of spleen cells to proliferate and develop antibody responses to antigen. Beta-endorphin
Spleen cell proliferation
Antibody-forming cell response
MODULATION of the immune system by CNS-related peptides has received considerable attention in recent years (19). The majority of these studies has focussed on the endogenous opioid peptides, and in particular the 31 amino acid peptide betaendorphin (BEP). Beta-endorphin is derived from the larger precursor peptide proopiomelanocortin (POMC), and is found in highest concentrations in the pituitary gland where it is synthesised, stored, and secreted concomitantly with another POMC derivative adrenocorticotropin (ACTH) (2). Although in the brain BEP has been implicated with functions relating to nociception (2), the role of BEP in the periphery is less clearly understood. One hypothesis generated by an increasing number of studies, and addressed in the present paper, suggests that BEP in blood may serve, among other things, to regulate immune function. Much of the evidence for this is based on the results of in vitro experiments in which BEP is incubated with lymphoid cells prior to or during the induction of an immunologic event. Hence, poly-
Concanavalin A
In vivo immune function
morphonuclear cell function, lymphokine production, natural killer cell activity, antibody production, and lymphocyte proliferation have all been shown to be altered in the presence of BEP (6, 15, 16, 21). Given that under conditions of "stress" plasma levels of BEP are significantly elevated (3,18), these in vitro observations suggest that pituitary-derived BEP may modulate immunologic activity in vivo. Moreover, endorphin-like peptides have been shown to be produced by murine and human lymphoid cells (4,13), further suggesting the existence of a close link between the immune system and BEP. Few reports have attempted to examine the effects of BEP on immune function in vivo. Shavit, Lewis, Terman, Gale and Liebeskind (26) reported that rat natural killer cell activity was depressed subsequent to 'opioid' (i.e., inducing naloxone reversible analgesia) electric shock only, with a 'nonopioid' electric shock schedule failing to modify NK activity. A further observation of naloxone reversibility and morphine-induced mimicking of the
tRequests for reprints should be addressed to Dr. A. J. Husband.
473
474
KUSNECOV, HUSBAND, KING AND SMITH
opioid stress effect on NK activity suggested to these authors that the endogenous opioid system was involved (26). Similarly, Kraut and Greenberg (11) observed that mice subjected to a single session of randomly applied tail electric shock had transiently diminished NK cell activity. This effect also was naloxone reversible but could not be reproduced by the intraperitoneal administration of a single dose of morphine or [D-AlaZ-MetS]B-endorphin, a stable analogue of BEP. Recently, we demonstrated that a single intravenous administration of BEP to nonstressed freely moving rats fitted with indwelling jugular catheters enhanced, in a dose-dependent manner, the in vitro mitogen-induced proliferative response of spleen cells harvested 3 hours after peptide administration (12). Interestingly we found that administering naloxone prior to BEP not only reversed the enhancing effect of BEP on proliferation but induced a significant decrement in the response relative to controls. This suggested that there may be different types of receptors on lymphoid cells which may mediate either down- or up-regulatory signals. Given that both opioid and nonopioid receptors have been identified on lymphoid cells (8,14) this finding suggests that BEP may act on nonopioid (not bound by naloxone) receptors that mediate a down-regulatory influence on lymphocyte function. N-acetylation of BEP renders it opioid inactive, while gammaendorphin (',/EP) [BEP(1-17] and Met-enkephalin (Met-ENK) [BEP(I-5)] are substantially less opioid active than BEP in standard preparations of opioid activity (27). In the present report we have extended our previous observations (12) by conducting experiments in which rats were administered -/EP, Met-ENK, and the N-acetylated form of the first 27 amino acids of BEP [AcBEP (1-27)]. In addition, we report experiments investigating further the immunomodulatory properties of BEP by examining 1) the duration of effect on spleen cell proliferation of single and multiple administrations of BEP, and 2) the influence of BEP on the in vivo primary and secondary response of spleen cells to the soluble antigen keyhole-limpet hemocyanin (KLH).
Experimental Procedures Effect of peptide administration on spleen cell proliferation. In all experiments peptide was administered 3--4 days after surgery. All peptides were freshly prepared in sterile PBS from an aliquot of stock stored frozen at - 20°C and administered in a bolus dose (100 fmoles, 10 pmoles, or 1 nmole) in a volume of 0.1 ml. For experiments comparing the effects of BEP, "/EP, Met-ENK and AcBEP(1-27) on spleen cell proliferation to ConA rats were administered one of the 3 doses of each peptide at 0800 hr and sacrificed 3 hr later. Control rats received 0.1 ml PBS. Two experiments examined the duration of effect of single or multiple infusions of BEP on spleen cell proliferation. In the first experiment rats were administered BEP at 0800 hr and sacrificed 24 hr later. In the second experiment rats were allotted to one of 3 groups: PBS, BEP-20, and BEP-44. Rats in the PBS group were given 3 infusions each of 0.1 ml PBS three times daily at 3 hr intervals commencing at 0800 hr on each of two consecutive days, and sacrificed 20 hr after the last treatment. Rats in the BEP-20 group were treated three times with PBS on the first day, and on the second day received three infusions of one of the 3 doses of BEP. Twenty hours later the rats were sacrificed. Rats in the BEP-44 group were administered one of the 3 doses of BEP three times on the first day, beginning at 0800 hr, and treated three times with PBS on the second day. Twenty hours after the last PBS treatment (44 hr after the last BEP treatment) the rats were sacrificed and spleens removed for assay. Effect of BEP on the primary and secondary response to KLH in vivo, KLH was prepared by dissolving in PBS at 4°C overnight,
Indwelling jugular catheters were surgically implanted as described previously (12), with the exception that anaesthesia was induced by light ether immobilization followed by induction of general anaesthesia by slow intravenous (IV) administration of 5% chloral hydrate, prepared in sterile phosphate buffered saline (PBS). Catheter patency was maintained by daily flushing with 10 units of sodium heparin in 0.5 ml sterile PBS. Preliminary experiments showed that this procedure had no discernible immunologic effects which could have contributed to the effects of peptide administration.
following which it was sterile filtered and the protein concentration determined by spectrophotometry. Stock aliquots of KLH were stored at -20°C at a concentration of 1 mg/ml, with fresh aliquots being used each time for immunization. To examine the effect of BEP on the primary response to KLH, rats were immunized with 500 I~g KLH (Calbiochem) in 0.5 ml sterile PBS administered via an indwelling jugular catheter. To examine the effect of BEP on the secondary response to KLH rats were initially primed under ether anaesthesia with 200 I~g KLH in 0.5 ml PBS via a tail vein, and 4-45 weeks later administered a booster dose of 500 ~xg KLH via an indwellingjugular catheter at the same time as peptide administration. In the first of these experiments rats were given either PBS or one of 3 doses of BEP 1 hr before and 1 hr after primary immunization with 500 ~g KLH (Calbiochem) given IV via a jugular catheter. Four days after BEP-KLH administration (previously determined as the peak of the primary antibody-forming cell response) the rats were sacrificed and spleens removed for assay of anti-KLH antibody-forming cells (AFC) using an enzyme-linked immunospot assay (ELISPOT) (5,24). The second experiment examined the effect of BEP on the secondary response to KLH in primed rats. In this experiment, assessment of the number of AFC generated in response to the second KLH exposure included differentiation of cells according to isotype-specificity (IgG, IgM, or IgA). Preliminary experiments showed that the peak of the IgG AFC response begins at around Day 5 following boosting with KLH. Therefore rats were sacrificed 5 days after the BEP-KLH treatment. In addition to the AFC response, spleen cells were also assayed for in vitro anamnestic proliferative response to KLH.
Peptides
Immunologic Assays
Peptides used for IV infusion were human synthetic BEP (1-31) (Sigma), acetylated BEP(I-27) and 3,EP (Peninsula Laboratories), and Met-ENK (Sigma).
All rats were sacrificed by cervical dislocation following light ether anaesthesia and tissues removed immediately and placed in sterile PBS containing 5% Foetal Calf Serum (FCS).
METHOD
Animals Inbred rats of the Australian Albino Wistar strain (Atomic Energy Commission, Lucas Heights) aged 90-120 days at the time of peptide administration were used in all experiments. Prior to surgery rats were group housed ( n = 5 per cage) under a 12:12 hr light:dark schedule (lights on at 0600 hr) with ad lib access to food and water. After surgery rats were housed in individual plastic rat cages under the above light and feeding conditions.
Surgery
BETA-ENDORPHIN AND IMMUNE FUNCTION
Proliferation assays. Lymphocyte proliferation in response to ConA was assessed as previously described (12). For antigeninduced proliferation 1 × 10 6 spleen cells were cultured in triplicate with KLH at 25, 50 or 100 txg/ml final concentration in 96-well round bottom tissue culture plates. The spleen cells were suspended in RPMI-1640 culture medium supplemented with 5% FCS, 100 U/ml penicillin, 100 ixg/ml streptomycin, 20 mM L-glutamine, and 5 x 10 -5 M 2-mercaptoethanol. To confirm the specificity of the proliferative response of spleen cells from KLH-boosted rats, triplicate cultures were established with 100 txg/ml ovalbumin (OVA) (Sigma). In addition, to ensure that the proliferative response observed reflected a memory response, spleen cells from unimmunized rats were cultured with KLH and OVA. Both antigen and mitogen cultures were incubated at 37°C in a 5% CO2 humidified atmosphere for varying periods and either 0.1 txCi or 0.25 txCi/well 3H-thymidine was added for the last 16 hr, at the end of which the cells were harvested and counted as described previously (12). ELISPOT assay. Twenty-four well Costar tissue culture plates were precoated overnight at 4°C with 1.0 ml/well of 10 txg/ml KLH or 10 p~g/ml OVA diluted in 0.1 M borate saline buffer (pH 8.73). The plates were then washed twice with PBS/5% Tween and then twice more with normal PBS. From each rat 5 × l 0 6 spleen cells suspended in PBS supplemented with 5% FCS were then added to duplicate wells in a volume of 300 txl/well. The cells were incubated for 4 hr at 37°C and were then discarded and the plates washed four times with PBS/5% Tween. For assay of the primary response to KLH total anti-KLH Ig-secreting cells were determined by addition of 1.0 ml/well of a 1/1000 dilution of alkaline phosphatase conjugated rabbit anti-rat IgG (heavy and light chain specific) (Bioyeda) followed by incubation overnight at 4°C. For detection of isotype-specific AFC appropriate wells were incubated for 2 hr at 37°C with an optimal dilution of the IgG fraction of purified rabbit anti-rat IgG, IgM and IgA (Miles). This was followed by incubation overnight at 4°C with 1.0 ml/well of a 1/2000 dilution of alkaline phosphatase conjugated goat antirabbit IgG (heavy and light chain specific) (TAGO) in all wells. After overnight incubation with enzyme the plates were washed four times with PBS/5% Tween and substrate-agar solution was then added to each well. The substrate-agar solution was prepared by dissolving at 45°C 10 mg of 5-bromo-4-chloro-3-indolyl phosphate (Sigma) in 8 ml of sterile-filtered 2-amino-2-methyll-propanol (AMP) buffer as prepared by Sedgwick and Holt (24). The substrate solution was then made up to 10 ml by the addition of 2 ml 3% molten agar solution prepared in distilled water. Immediately following this the substrate agar solution was gently mixed and 200 Ixl added to each well. The enzyme-substrate reaction was then allowed to proceed and the number of spots formed counted under a low-powered microscope. Controls for nonspecific immunospot formation were provided by l) using spleen cells from rats not previously immunized with KLH, and 2) incubating all spleen cell samples in wells precoated with OVA. Data Presentation and Analysis The proliferation data are expressed in terms of counts per minute (cpm) and represent the difference between the mean of triplicate experimental wells (containing antigen or mitogen) and the mean of triplicate background wells (containing diluent without antigen or mitogen). For the ELISPOT results the data are expressed in terms of the number of antibody-forming cells (AFC) per 10 x 10 6 cells, and also the number of AFC per spleen. In calculating the former, the difference between the average number of immunospots of duplicate wells for KLH-primed rats and duplicate wells for rats not
475
TABLE 1 PROLIFERATIVE RESPONSE (cpm × 10-3) TO 3.12 ~g/ml ConA AMONG SPLEEN CELLS FROM RATS INFUSED WITH ONE OF SEVERAL DOSES OF EITHER BEP, "yEP, OR MET-ENK 3 HR PRIOR TO ASSAY
Experiment (Peptide Treatment)
Proliferative Response (cpm _+ S.E.)
Experiment (1): BEP PBS (n=6) 39.5 100 fmoles (n=6) 50.2 10 pmoles (n=6) 51.7 1 nmole(n=6) 41.9 Experiment (2): ",/EP PBS (n= 10) 49.2 100 fmoles (n=6) 41.4 10 pmoles (n= 6) 46.5 1 nmole (n=6) 44.7 Experiment (3): Met-ENK PBS (n=7) 49.4 100 fmoles (n=6) 45.3 10 pmoles (n=6) 49.1 1 nmole (n=6) 41.7
Percentage Change From PBS
Statistical Significance
--- 2.5 ± 2.8 _ 7.3 _ 4.2
+27.1% +30.9% + 6.1%
n.s. p<0.05 n.s.
± ± ± ±
0.9 4.0 1.13 3.5
- 15.9% - 5.5% - 9.2%
n.s. n.s. n.s.
± ± ± ±
2.4 2.7 1.7 2.1
- 8.3% 0.0% -15.6%
n.s. n.s.
immunized with KLH was determined and then doubled. To obtain the number of AFC per spleen this value was appropriately adjusted according to the number of splenic leucocytes recovered for each animal. Statistical analysis was performed using one-way analysis of variance (ANOVA) and the Dunnett test for post hoc comparison of group means (10). Additional statistical analyses were conducted using the independent t-test (two-tailed, unless otherwise stated). RESULTS
The proliferative responses to an optimal dose of ConA (3.12 Ixg/ml) of splenic lymphocytes from rats administered BEP, ~/EP, or Met-ENK 3 hr prior to sacrifice are presented in Table 1. A one-way ANOVA of the BEP data revealed a significant main effect of BEP treatment, F(3,20)=3.601, p<0.05. Post hoc comparison of the PBS group with the BEP groups showed that only the increased response of the 10 pmole group was significant [Dunnett critical difference CD(20) = 11.35, p<0.05]. Analysis of the data by ANOVA for animals treated with either Met-ENK or ~/EP did not reveal a significant main effect of either peptide treatment [~/EP: F(3,24) = 1.95, n.s.; Met-ENK: F(3,21)= 2.686, n.s.]. These data therefore reveal modest but statistically significant dose-dependent effects of in vivo administration with BEP, but no observable effects of ~/EP and Met-ENK, on spleen cell proliferation in vitro. Figure 1 presents the results of another experiment in which rats were sacrificed 3 hr after infusion with various doses of the nonopioid active AcBEP(1-27) and the proliferative response of spleen cells to optimal (3.12 Ixg/ml) and suboptimal (0.78 i,tg/ml) doses of ConA examined after 3 or 4 days of culture. One-way ANOVAs conducted on these data indicated a significant effect on both days of culture at the optimal ConA dose [Day 3: F(3,28)= 4.42, p<0.025; Day 4: F(3,28)= 6.11, p<0.0025] but a significant effect was only observed on Day 3 for suboptimal doses [Day 3: F(3,28)=5.39, p<0.005; Day 4: F(3,28)= 1.08, n.s.]. Post hoc comparison of the PBS group with each of the AcBEP(1-27)
476
KUSNECOV, HUSBAND, KING AND SMITH
3.12,ug/ml Con A
0.78,oglml Con A
40
]
Day 3
]
Day 4
1
2 7 x E
30
20
j PBS
100fmoles 10pmoles
1
m
1nmole
PBS
100fmoles lOpmoles
lnmole
Dose o f AcBEP(1-27)
FIG. I. Mean proliferative responses to optimal (3.12 ~g/ml) and suboptimal (0.78 Ixg/ml) concentrations of ConA among spleen cells cultured for 3 and 4 days from rats treated with PBS or various doses of AcBEP(1-27). Vertical bars represent standard error of the mean for n = 8 animals.
groups using the two-tailed Dunnett test (10) revealed that the response of spleen cells from the 100 fmoles group to 3.12 txg/ml ConA was significantly depressed on Day 3 of culture [Critical Difference for Dunnett test, CD(28) = 11.477, p<0.05]. On Day 4 of culture none of the AcBEP(1-27) groups differed significantly from the PBS group using the Dunnett test. However, the proliferative responses of the 100 fmole and 10 pmole groups were significantly greater than the 1 nmole group [1 nmole vs. 100 fmole: t(14) = 4.02, p<0.0025; 1 nmole vs. 10 pmole: t(14) = 3.3, p<0.01]. With respect to the proliferative response of spleen cells from AcBEP(1-27)-treated rats to 0.78 txg/ml ConA, the reduced responses among the 100 fmole and 10 pmole groups on Day 3 of culture were not significantly different from the PBS group using the Dunnett test, On the other hand, a two-tailed t-test comparing the 1 nmole and PBS groups revealed a significant difference, t(14) = 3.316, p<0.01. In addition, the differences between the 1 n mmole group and the 100 fmole and 10 pmole groups, respectively, were found to be significant [1 nmole vs. 100 fmole: t(14)=2.864, p<0.01; 1 nmole vs. 10 pmole: t(14)=5.00, p<0.001]. It is pertinent to note that the depressed proliferative response of spleen cells from the 100 fmole group responding to 3.12 ixg/ml ConA on Day 3 of culture had recovered substantially by Day 4 of culture to exceed that of the PBS group (Fig. i). Similarly, the magnitude of the reduction in the proliferative response of the 1 nmole group to 3.12 txg/ml ConA on Day 4 was two-fold greater than that observed for the PBS group (35.6% vs. 17.0% respectively). These observations suggest that infusion with low and high doses of AcBEP had different effects on the kinetics of the proliferative response of spleen cells to an optimal dose of ConA. Two experiments were conducted to determine the longer term effect of single and multiple in vivo treatments with BEP on lymphocyte proliferation. The results of these experiments are summarized in Fig. 2. It should be noted that in this figure data from the animals in the PBS control groups of both experiments were combined (Experiment 1: n = 7; Experiment 2: n = 10), there being no significant difference between these two control groups, t(15) =0.24, although these data were analyzed separately. In the first experiment rats were given a single infusion of BEP and sacrificed 24 hr later. This experiment did not reveal any effects of BEP at any of the 3 doses used on the proliferative response of spleen cells to an optimal dose of ConA measured on the day of
peak proliferation, F(3,24)=0.302. In the second experiment rats were administered BEP three times at 3 hr intervals and sacrificed 20 or 44 hr after the final infusion. A one-way ANOVA revealed a significant effect of BEP treatment on the proliferative response to ConA, F(4,37)= 5.633, p<0.001. Post hoc comparison of the group means using the two-tailed Dunnett test revealed that only the enhanced proliferative response of spleen cells from rats infused with 100 fmoles and 10 pmoles BEP and sacrificed 44 hr after the last infusion were significantly different from PBS controls [Dunnett CD(37)= 13.41, p<0.05]. The influence of BEP in vivo on the primary AFC response to the soluble protein antigen KLH is shown in Table 2. A one-way ANOVA of the data was found to be significant, F(3,42)= 4.963, p<0.005. Comparison of the group means revealed that only the 1 nmole group was significantly different from the PBS group [Dunnett CD(42) = 81.23, p<0.05]. Table 2 also summarizes the effect of BEP on the secondary AFC response to KLH. The response of all isotypes in the PBS group receiving a second exposure to KLH was significantly greater than the residual group as assessed by a one-tailed t-test
140
x E
[] []
PBS 100 fmoles
[] []
10 pmoles I nmole
120
100
24 hr T i m e of assay a f t e r
20 hr 44 hr last infusion
FIG. 2. Mean proliferative responses to an optimal dose of ConA (3.12 p.g/ml) among spleen cells cultured for 3 days from rats that received a single infusion of BEP and sacrificed 24 hr later, or multiple infusions of BEP and sacrificed 20 or 44 hr after the last infusion. Vertical bars represent standard error of the mean.
BETA-ENDORPHIN AND IMMUNE FUNCTION
477
rats receiving BEP at the time of second exposure to KLH is shown in Fig. 3. The results represented a specific memory response since nonprimed animals did not demonstrate proliferation above background to any of the doses of KLH, and specificity was evident in that all experimental animals demonstrated responses commensurate with background when spleen cells were cultured with OVA. However, for all doses of KLH tested there were no differences between the proliferative responses of PBS and BEP-treated rats on any of the days of culture examined.
TABLE 2 THE MEAN NUMBER OF PRIMARY (EXPERIMENT 1) AND SECONDARY (EXPERIMENT 2) ANTI-KLH AFC (PER 107 CELLS -+ STANDARDERROR OF THE MEAN) IN THE SPLEEN OF RATS TREATED WITH BEP OR PBS AT THE TIME OF IMMUNIZATION Treatment Group (Experiment 1)
Primary AFC Total
Treatment Group (Experiment 2)
Secondary AFC IgG
IgM
168 _ 15
29 ± 12
IgA
DISCUSSION PBS (n = 12)
219.9 --- 17.5
1 nmole BEP ( n - 11)
127.7 ± 19.2
10 pmoles BEP (n= 13)
250.4 -- 26.7
Residual (n 5)
7 ±
1
PBS (n = 6)
271 ± 26 145 ± 33 52 ±
6
1 nmol (n = 6)
335 - 18 165 - 20 70 + 11
Immune function has been shown to be modified by in vitro incubation of human and murine lymphoid cells with BEP and its various derivatives (6). However, the immunologic influence of BEP in vivo has not been extensively studied. As part of a study of the suppressive influence of opioid-mediated analgesic stress on NK cell function, Kraut and Greenberg (11) administered intraperitoneally the proteolytically stable BEP analogue [D-Ala sMetS]-B-endorphin to mice and observed a small, though not statistically significant, increase in poly I:C-induced NK cell activity. More recently, we found that bolus infusion of physiologic doses of BEP via indwelling jugular catheters to conscious rats resulted in a dose-dependent enhancement of the proliferative response of spleen cells to ConA (12). In the present paper we have pursued our investigation of the in vivo effects of BEP on the immune system by way of experiments which address whether 1) opioid and nonopioid active derivatives of BEP exert any influence on lymphocyte proliferation; 2) whether the immunologic changes rendered by BEP administration are of only short duration; and 3) whether the primary and secondary response to soluble antigen can be modified by BEP. Met-enkephalin (Met-ENK) and gamma-endorphin [~/EP: corresponding to BEP(1-17)] are considerably less potent than the complete BEP peptide in standard tests of opioid activity (27). Moreover, total abrogation of opioid activity can be rendered by acetylation of the N-terminal region of BEP (27). In a series of experiments, therefore, we compared the immunomodulatory capabilities of Met-ENK, ~EP, and AcBEP(1-27) when administered in vivo with that of BEP. When single bolus infusions of various doses of BEP were administered via an indwelling jugular
=
100 fmoles 188.4 -- 28.5 BEP (n= 10)
10pmol 389 ± 20 123 -+ 11 52 +- 7 (n = 6) 100fmol 249 - 41 161 - 48 54 +- 9 (n = 6)
[IgG: t(9) = 3.2, p < 0 . 0 1 ; IgM: t(9) = 3.07, p < 0 . 0 1 ; IgA: t(9) = 6.5, p < 0 . 0 0 1 ] . These results indicated that the response of the PBS and BEP groups represented a secondary AFC response. With respect to the IgG isotype, a one-way ANOVA of the data was found to be significant, F ( 4 , 2 4 ) = 7 . 3 8 3 , p < 0 . 0 0 1 . Post hoc comparison of the PBS group with the BEP groups revealed that only the increased response of the 10 pmole group was significant [Dunnett C D ( 2 4 ) = 107.6, p < 0 . 0 5 ] . Although statistical analysis of the IgM and IgA data revealed significant main effects, these were due to the lower response of the residual group, and not attributable to any significant differences between the PBS and BEP groups. The proliferative response to KLH in vitro of spleen cells from
25jug/ml KLH
sOjug/ml KLH
30-
100pg/ml
T
KLH
,,5"::':
,,.,'..!~
o x E
..","t
/,/'."
20
f
10
- PBS . . . . . . . . . Inmole ...... 10pmoles 100fmoles t
3
1
tl
I
S
I
1
3
4 DAY
I
5
I
4
I
5
OF CULTURE
FIG. 3. The mean proliferative responses to various concentrations of KLH among spleen cells cultured for 3, 4, and 5 days from KLH-primed rats administered with PBS or BEP at the time of secondary immunization. For each point vertical bars represent the standard error of the mean for n = 6 rats.
478
catheter to conscious rats a significant enhancement of the proliferative response of splenic lymphocytes to an optimal concentration of ConA was observed among ceils from rats administered 10 pmoles BEP 3 hours prior to assay (see Table 1). This finding is in agreement with the results of our previous report (12). There was no such enhancement of the proliferative response following infusion with Met-ENK or ~EP. In fact, infusion with these peptides resulted in diminished proliferative responses to ConA, but these were not statistically significant. Given that intravenous administration of BEP enhanced the spleen cell proliferative response to ConA, infusion with AcBEP( 127) suppressed the response, and infusion with ~/EP or Met-ENK had no effect, it appears that the enhancing effect of BEP on spleen cell proliferation following in vivo administration requires the C-terminal region of BEP. In the absence of the C-terminal region an inhibitory effect on the proliferative capacity of spleen cells may occur. The observation that AcBEP(1-27) exerted inhibitory effects on spleen cell proliferation where BEP had an enhancing influence indicates further that endogenous peptides with diminished or absent opioid activity may have opposite effects to that of the extremely opioid potent BEP. The foregoing results are consistent with other evidence showing that -yEP in vitro has no effect on lymphocyte proliferation (7). In addition, although there have been no reports of the effect of N-acetylated forms of BEP on lymphocyte proliferation, other parameters of immune function have not been shown to be affected by AcBEP (25). In the present experiments, however, some dose rates of AcBEP(1-27) exerted an inhibitory effect on the capacity of lymphocytes to proliferate in vitro to an optimal dose of ConA, thereby demonstrating a possible immunomodulatory role in vivo. Indeed, acetylated forms of BEP are secreted from the intermediate lobe of the pituitary in response to stress (1), which may serve to oppose the immunomodulatory effects of similarly secreted anterior lobe BEP. Moreover, this functional opposition may also be present at the autocrine level, since murine spleen cells have been shown to synthesize both normal and acetylated forms of BEP (13). The present experiments have also demonstrated that the enhancing effect of BEP in vivo on lymphocyte proliferation can be observed after 2 days when rats are administered BEP three times in a 9 hour period (Fig. 2). Interestingly, there was no detectable effect of multiple BEP treatment on lymphocyte proliferation after 20 hours. Although there is presently no adequate explanation for this delayed effect, the data demonstrate that repeated physiologic increases of BEP in blood can induce immunopotentiating effects which are detectable after a prolonged period. The in vitro plaque-forming cell (PFC) response of murine splenocytes to sheep erythrocytes can be partially inhibited by BEP (9). In the present study it was observed that administering BEP 1 hour before and 1 hour after primary immunization with the soluble antigen KLH resulted in a significant suppression of the splenic AFC response measured 4 days later (Table 2). A high dose of BEP (1 nmole) was required to induce this effect and was not observed with lower physiologic doses. Nonetheless, this observation extends previous in vitro observations (9) that BEP can inhibit in vivo the primary antibody response to antigen. A further experiment examined whether the secondary antibody response to KLH could also be modulated by BEP administration at the time of secondary exposure to antigen. The results showed that only the IgG-secreting AFC were affected by BEP
KUSNECOV, HUSBAND, KING AND SMITH
treatment (Table 2). This is not surprising, since IgG was the major isotype being synthesised in the spleens of animals in the residual group (primed but not boosted), and the secondary antibody response is predominantly of the IgG isotype. In contrast to the influence of BEP on the primary anti-KLH response, the secondary IgG AFC response was potentiated by BEP at a dose of 10 pmoles. This suggests that, unlike the primary response, the secondary antibody response to antigen is resistant to any suppressive influence of high doses of BEP. There was no influence of BEP on the capacity of KLHsensitized spleen cells to proliferate in vitro in response to various concentrations of KLH 5 days after combined KLH/BEP treatment. Whether an effect may have been observed at an earlier time after BEP treatment remains to be determined. Whether the immunologic effects of the various peptide treatments in the present experiments were due to a direct peptidelymphoid cell interaction is not known. Endogenous opioids have been shown to have endocrinologic effects (17) which may mediate the immunologic changes observed in the present report. We have observed previously that BEP has enhancing effects on spleen cell proliferation in vitro at doses similar to those which enhance proliferative capacity in vivo (12). Therefore, it is most probable that the effects of BEP and AcBEP(1-27) on spleen cell proliferation involve a direct interaction with lymphoid cells. However the fact that the effects exerted by BEP varied with dose rate and time is a reflection of the complexity of mechanisms involved in these effects and many further studies will be required before appropriate explanations could be provided. Basal levels of BEP in rat plasma range from 120 to 500 pg/ml (3,22), and subsequent to restraint stress are elevated 6- to 12-fold (3,18). The doses of BEP in our experiments therefore approximated physiologic (100 fmoles=346.5 pg), stress-induced (10 pmoles=34.65 ng), and supraphysiologic (1 nmole=3.465 ~g) plasma levels, It is interesting to note that the enhancing effects of BEP administration on spleen cell proliferation and secondary IgG AFC formation were observed at a dose of 10 pmoles. This suggests that BEP release from the pituitary may serve to facilitate immune function under conditions of stress. On the other hand, the primary response to KLH was suppressed by the supraphysiologic dose of 1 nmole BEP, but was unaffected by 10 pmoles BEP. This indicates that stress-related plasma levels of BEP do not alter the primary response to antigen in vivo at the time of immunization, this being possible only when doses far in excess of those encountered physiologically are administered. The present series of experiments has shown that in vivo administration of some doses of BEP enhances the proliferative response of spleen cells to ConA in vitro and modulates the primary and secondary antibody-secreting cell response to the soluble protein antigen KLH. Moreover, in vivo treatment with Met-ENK or "yEP was without effect on spleen cell proliferation, while AcBEP(1-27) inhibited the proliferative response of spleniclymphocytes to ConA. These observations complement an extensive literature documenting in vitro modulation of immune function by the endogenous opioids. ACKNOWLEDGEMENTS
This work was supported by grants from the National Health and Medical Research Council of Australia. The authors are grateful for the technical assistance of Christine Baker. Thanks also go to Dr. Gerald Pang for his expert advice in establishing the ELISPOT assay.
BETA-ENDORPHIN AND IMMUNE FUNCTION
479
REFERENCES 1. Akil, H.; Shiomi, H.; Mathews, J. Induction of the intermediate pituitary by stress: Synthesis and release of a nonopioid form of B-endorphin. Science 227:424-426; 1985. 2. Akil, H.; Watson, S. J.; Young, E.; Lewis, M. E.; Khachaturian, H.; Walker, J. M. Endogenous opioids: Biology and function. Annu. Rev. Neurosci. 7:223-255; 1984. 3. Berkenbosch, F.; Vermes, I.; Tilders, F. J. H. The B-adrenoceptorblocking drug propanolol prevents secretion of immuuoreactive Bendorphin and et-melanocyte-stimulating hormone in response to certain stress stimuli. Endocrinology 115:1051-1059; 1984. 4. Blalock, J. E.; Bost, K. L.; Smith, E. M. Neuroendocrine peptide hormones and their receptors in the immune system: Production, processing, and action. J. Neuroimmunol. 10:31-40; 1985. 5. Czerkinsky, C. C.; Nilsson, L-A.; Hakan, N.; Ouchterlony, O.; Tarkowski, A. Solid-phase enzyme-linked imunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65:109-121; 1983. 6. Fischer, E. G.; Falke, N. E. B-endorphin modulates immune functions. Psychother. Psychosom. 42:195-204; 1984. 7. Gilman, S. C.; Schwartz, J. M.; Milner, R. J.; Bloom, F. E.; Feldman, J. D. B-endorphin enhances lymphocyte proliferative responses. Proc. Natl. Acad. Sci. USA 79:4226--4230; 1982. 8. Hazum, E.; Chang, K-J.; Cuatrecasas, P. Specific nonopiate receptors for B-endorphin. Science 205:1033-1035; 1979. 9. Johnson, H. M.; Smith, E. M.; Torres, B. A.; Blalock, J. E. Regulation of the in vitro antibody response by neuroendocrine hormones. Proc. Natl. Acad. Sci. USA 79:4171-4174; 1982. 10. Keppel, G. Design and analysis: A researcher's handbook, 2nd ed. Englewood Cliffs, NJ: Prentice-Hall; 1982. 11. Kraut, R. P.; Greenberg, A. H. Effects of endogenous and exogenous opioids on splenic natural killer cell activity. Nat. Immun. Cell Growth Regul. 5:28-40; 1986. 12. Kusnecov, A.W.; Husband, A. J.; King, M. G.; Pang, G.; Smith, R. In vivo effects of B-endorphin on lymphocyte proliferation and interleukin-2 production. Brain Behav. Immun. 1:88-97; 1987. 13. Lolait, S. J.; Clements, J. A.; Markwick, A. J.; Cheng, C.; McNally, M.; Smith, A. I.; Funder, J. Pro-opiomelanocortin messenger ribonucleic acid and posttranslational processing of beta endorphin in spleen macrophages. J. Clin. Invest. 77:1776-1779; 1986. 14. Lopker, A.; Abood, L. G.; Hoss, W.; Lionetti, F. J. Stereoselective muscarinic acetylcholine and opiate receptors in human phagocytic leukocytes. Biochem. Pharmacol. 29:1361-1365; 1980.
15. Lori Brown, S.; Van Epps, D. E. Opioid peptides modulate production of interferon gamma by human mononuclear cells. Cell. Immunol. 103:19-26; 1986. 16. Loft Brown, S.; Van Epps, D. E. Suppression of T lymphocyte chemotactic factor production by the opioid peptides B-endorphin and met-enkephalin. J. Immunol. 134:3384-3390; 1985. 17. Millan, M. J.; Herz, A. The endocrinology of the opioids. Int. Rev. Neurobiol. 26:1-83; 1985. 18. Mueller, G. Beta-endorphin immunoreactivity in rat plasma: Variations in response to different physical stimuli. Life Sci. 29:16691674; 1981. 19. Payan, D. G.; McGillis, J. P.; Goetzl, E. J. Neuroimmunology. Adv. Immunol. 39:299-323; 1986. 20. Peterson, P. K.; Sharp, B.; Gekker, G.; Brummitt, C.; Keane, W. F. Opioid-mediated suppression of cultured peripheral blood mononuclear cell respiratory burst activity. J. Immunol. 138:3907-3912; 1987. 21. Peterson, P. K.; Sharp, B.; Gekker, G.; Brummitt, C.; Keane, W. F. Opioid-mediated suppression of interferon-gamma production by cultured peripheral blood mononuclear cells. J. Clin. Invest. 80: 824-831; 1987. 22. Pettibone, D. J.; Mueller, G. P. Differential effects of adrenergic agents on plasma levels of immunoreactive B-endorphin and alphamelanotropin in rats. Proc. Soc. Exp. Biol. 176:168-174; 1984. 23. Puppo, F.; Corsini, G.; Mangini, P.; Bottaro, L.; Barreca, T. Influence of B-endorphin on phytohemagglutinin-induced lymphocyte proliferation and on the expression of mononuclear cell surface antigens in vitro. Immunopharmacology 10:119-125; 1985. 24. Sedgwick, J. D.; Holt, P. G. A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting ceils. J. lmmunol. Methods 57:301-309; 1983. 25. Sharp, B. M.; Tsukayama, D. T.; Gekker, G.; Keane, W. F.; Peterson, P. K. B-endorphin stimulates human polymorphonuclear leukocyte superoxide production via a stereoselective opiate receptor. J. Pharmacol. Exp. Ther. 242:579-582; 1987. 26. Shavit, Y.; Lewis, J. W.; Terman, G. W.; Gale, R. P.; Liebeskind, J. C. Opioid peptides mediate the suppressive effect of stress on natural killer cell cytotoxicity. Science 223:188-190; 1984. 27. Taylor, J. W.; Kaiser, E. T. The structural characterization of B-endorphin and related peptide hormones and neurotransmitters. Pharmacol. Rev. 38:291-319; 1986.
0196-9781/89 $3.00 + .00
Modulation of Mitogen-Induced Spleen Cell Proliferation and the Antibody-Forming Cell Response by Beta-Endorphin In Vivo A L E X A N D E R W. K U S N E C O V , A L A N J. H U S B A N D , .1 M A U R I C E G. K I N G A N D R O G E R S M I T H *
Department of Psychology and *Faculty of Medicine, University of Newcastle 2308, New South Wales, Australia R e c e i v e d 8 N o v e m b e r 1988
KUSNECOV, A. W., A. J. HUSBAND, M. G. KING AND R. SMITH. Modulation ofmitogen-induced spleen cell proliferation and the antibody-forming cell responseby beta-endorphin in vivo. PEPTIDES 111(2)473--479, 1989.--Experiments were conducted which compared the in vivo effects of beta-endorphin (BEP), gamma-endorphin (-¢EP), methionine-enkephalin (Met-ENK), and acetylated BEP(1-27) on the in vitro proliferative response of rat spleen cells to concanavalin A (ConA). In addition, the influence of BEP administration on the primary and secondary antibody-forming cell (AFC) response to the soluble antigen keyhole-limpet hemocyanin (KLH) was examined. Intravenous administration of BEP enhanced the spleen cell proliferative response to ConA assessed 3 hr after a single bolus infusion. Conversely, infusion with AcBEP(1-27) suppressed the proliferative response, whereas no effects of intravenous ~EP or Met-ENK treatment were observed. The enhancing effect of BEP administration was not detectable 24 hr after a single infusion, but could be maintained over a 44 hr period by multiple infusions. The primary AFC response to KLH was suppressed by a dose of 1 nmole BEP only. On the other hand, the secondary IgG AFC response to KLH was enhanced by 10 pmoles BEP, while the IgM and IgA AFC responses remained unaltered by BEP treatment. The anamnestic in vitro proliferative response of spleen cells cultured with KLH was not altered if BEP was administered at the time of secondary KLH immunization. These results extend previous observations of BEP-induced modulation of in vitro immune function by demonstrating that opioid and nonopioid forms of BEP administered in vivo alter the capacity of spleen cells to proliferate and develop antibody responses to antigen. Beta-endorphin
Spleen cell proliferation
Antibody-forming cell response
MODULATION of the immune system by CNS-related peptides has received considerable attention in recent years (19). The majority of these studies has focussed on the endogenous opioid peptides, and in particular the 31 amino acid peptide betaendorphin (BEP). Beta-endorphin is derived from the larger precursor peptide proopiomelanocortin (POMC), and is found in highest concentrations in the pituitary gland where it is synthesised, stored, and secreted concomitantly with another POMC derivative adrenocorticotropin (ACTH) (2). Although in the brain BEP has been implicated with functions relating to nociception (2), the role of BEP in the periphery is less clearly understood. One hypothesis generated by an increasing number of studies, and addressed in the present paper, suggests that BEP in blood may serve, among other things, to regulate immune function. Much of the evidence for this is based on the results of in vitro experiments in which BEP is incubated with lymphoid cells prior to or during the induction of an immunologic event. Hence, poly-
Concanavalin A
In vivo immune function
morphonuclear cell function, lymphokine production, natural killer cell activity, antibody production, and lymphocyte proliferation have all been shown to be altered in the presence of BEP (6, 15, 16, 21). Given that under conditions of "stress" plasma levels of BEP are significantly elevated (3,18), these in vitro observations suggest that pituitary-derived BEP may modulate immunologic activity in vivo. Moreover, endorphin-like peptides have been shown to be produced by murine and human lymphoid cells (4,13), further suggesting the existence of a close link between the immune system and BEP. Few reports have attempted to examine the effects of BEP on immune function in vivo. Shavit, Lewis, Terman, Gale and Liebeskind (26) reported that rat natural killer cell activity was depressed subsequent to 'opioid' (i.e., inducing naloxone reversible analgesia) electric shock only, with a 'nonopioid' electric shock schedule failing to modify NK activity. A further observation of naloxone reversibility and morphine-induced mimicking of the
tRequests for reprints should be addressed to Dr. A. J. Husband.
473
474
KUSNECOV, HUSBAND, KING AND SMITH
opioid stress effect on NK activity suggested to these authors that the endogenous opioid system was involved (26). Similarly, Kraut and Greenberg (11) observed that mice subjected to a single session of randomly applied tail electric shock had transiently diminished NK cell activity. This effect also was naloxone reversible but could not be reproduced by the intraperitoneal administration of a single dose of morphine or [D-AlaZ-MetS]B-endorphin, a stable analogue of BEP. Recently, we demonstrated that a single intravenous administration of BEP to nonstressed freely moving rats fitted with indwelling jugular catheters enhanced, in a dose-dependent manner, the in vitro mitogen-induced proliferative response of spleen cells harvested 3 hours after peptide administration (12). Interestingly we found that administering naloxone prior to BEP not only reversed the enhancing effect of BEP on proliferation but induced a significant decrement in the response relative to controls. This suggested that there may be different types of receptors on lymphoid cells which may mediate either down- or up-regulatory signals. Given that both opioid and nonopioid receptors have been identified on lymphoid cells (8,14) this finding suggests that BEP may act on nonopioid (not bound by naloxone) receptors that mediate a down-regulatory influence on lymphocyte function. N-acetylation of BEP renders it opioid inactive, while gammaendorphin (',/EP) [BEP(1-17] and Met-enkephalin (Met-ENK) [BEP(I-5)] are substantially less opioid active than BEP in standard preparations of opioid activity (27). In the present report we have extended our previous observations (12) by conducting experiments in which rats were administered -/EP, Met-ENK, and the N-acetylated form of the first 27 amino acids of BEP [AcBEP (1-27)]. In addition, we report experiments investigating further the immunomodulatory properties of BEP by examining 1) the duration of effect on spleen cell proliferation of single and multiple administrations of BEP, and 2) the influence of BEP on the in vivo primary and secondary response of spleen cells to the soluble antigen keyhole-limpet hemocyanin (KLH).
Experimental Procedures Effect of peptide administration on spleen cell proliferation. In all experiments peptide was administered 3--4 days after surgery. All peptides were freshly prepared in sterile PBS from an aliquot of stock stored frozen at - 20°C and administered in a bolus dose (100 fmoles, 10 pmoles, or 1 nmole) in a volume of 0.1 ml. For experiments comparing the effects of BEP, "/EP, Met-ENK and AcBEP(1-27) on spleen cell proliferation to ConA rats were administered one of the 3 doses of each peptide at 0800 hr and sacrificed 3 hr later. Control rats received 0.1 ml PBS. Two experiments examined the duration of effect of single or multiple infusions of BEP on spleen cell proliferation. In the first experiment rats were administered BEP at 0800 hr and sacrificed 24 hr later. In the second experiment rats were allotted to one of 3 groups: PBS, BEP-20, and BEP-44. Rats in the PBS group were given 3 infusions each of 0.1 ml PBS three times daily at 3 hr intervals commencing at 0800 hr on each of two consecutive days, and sacrificed 20 hr after the last treatment. Rats in the BEP-20 group were treated three times with PBS on the first day, and on the second day received three infusions of one of the 3 doses of BEP. Twenty hours later the rats were sacrificed. Rats in the BEP-44 group were administered one of the 3 doses of BEP three times on the first day, beginning at 0800 hr, and treated three times with PBS on the second day. Twenty hours after the last PBS treatment (44 hr after the last BEP treatment) the rats were sacrificed and spleens removed for assay. Effect of BEP on the primary and secondary response to KLH in vivo, KLH was prepared by dissolving in PBS at 4°C overnight,
Indwelling jugular catheters were surgically implanted as described previously (12), with the exception that anaesthesia was induced by light ether immobilization followed by induction of general anaesthesia by slow intravenous (IV) administration of 5% chloral hydrate, prepared in sterile phosphate buffered saline (PBS). Catheter patency was maintained by daily flushing with 10 units of sodium heparin in 0.5 ml sterile PBS. Preliminary experiments showed that this procedure had no discernible immunologic effects which could have contributed to the effects of peptide administration.
following which it was sterile filtered and the protein concentration determined by spectrophotometry. Stock aliquots of KLH were stored at -20°C at a concentration of 1 mg/ml, with fresh aliquots being used each time for immunization. To examine the effect of BEP on the primary response to KLH, rats were immunized with 500 I~g KLH (Calbiochem) in 0.5 ml sterile PBS administered via an indwelling jugular catheter. To examine the effect of BEP on the secondary response to KLH rats were initially primed under ether anaesthesia with 200 I~g KLH in 0.5 ml PBS via a tail vein, and 4-45 weeks later administered a booster dose of 500 ~xg KLH via an indwellingjugular catheter at the same time as peptide administration. In the first of these experiments rats were given either PBS or one of 3 doses of BEP 1 hr before and 1 hr after primary immunization with 500 ~g KLH (Calbiochem) given IV via a jugular catheter. Four days after BEP-KLH administration (previously determined as the peak of the primary antibody-forming cell response) the rats were sacrificed and spleens removed for assay of anti-KLH antibody-forming cells (AFC) using an enzyme-linked immunospot assay (ELISPOT) (5,24). The second experiment examined the effect of BEP on the secondary response to KLH in primed rats. In this experiment, assessment of the number of AFC generated in response to the second KLH exposure included differentiation of cells according to isotype-specificity (IgG, IgM, or IgA). Preliminary experiments showed that the peak of the IgG AFC response begins at around Day 5 following boosting with KLH. Therefore rats were sacrificed 5 days after the BEP-KLH treatment. In addition to the AFC response, spleen cells were also assayed for in vitro anamnestic proliferative response to KLH.
Peptides
Immunologic Assays
Peptides used for IV infusion were human synthetic BEP (1-31) (Sigma), acetylated BEP(I-27) and 3,EP (Peninsula Laboratories), and Met-ENK (Sigma).
All rats were sacrificed by cervical dislocation following light ether anaesthesia and tissues removed immediately and placed in sterile PBS containing 5% Foetal Calf Serum (FCS).
METHOD
Animals Inbred rats of the Australian Albino Wistar strain (Atomic Energy Commission, Lucas Heights) aged 90-120 days at the time of peptide administration were used in all experiments. Prior to surgery rats were group housed ( n = 5 per cage) under a 12:12 hr light:dark schedule (lights on at 0600 hr) with ad lib access to food and water. After surgery rats were housed in individual plastic rat cages under the above light and feeding conditions.
Surgery
BETA-ENDORPHIN AND IMMUNE FUNCTION
Proliferation assays. Lymphocyte proliferation in response to ConA was assessed as previously described (12). For antigeninduced proliferation 1 × 10 6 spleen cells were cultured in triplicate with KLH at 25, 50 or 100 txg/ml final concentration in 96-well round bottom tissue culture plates. The spleen cells were suspended in RPMI-1640 culture medium supplemented with 5% FCS, 100 U/ml penicillin, 100 ixg/ml streptomycin, 20 mM L-glutamine, and 5 x 10 -5 M 2-mercaptoethanol. To confirm the specificity of the proliferative response of spleen cells from KLH-boosted rats, triplicate cultures were established with 100 txg/ml ovalbumin (OVA) (Sigma). In addition, to ensure that the proliferative response observed reflected a memory response, spleen cells from unimmunized rats were cultured with KLH and OVA. Both antigen and mitogen cultures were incubated at 37°C in a 5% CO2 humidified atmosphere for varying periods and either 0.1 txCi or 0.25 txCi/well 3H-thymidine was added for the last 16 hr, at the end of which the cells were harvested and counted as described previously (12). ELISPOT assay. Twenty-four well Costar tissue culture plates were precoated overnight at 4°C with 1.0 ml/well of 10 txg/ml KLH or 10 p~g/ml OVA diluted in 0.1 M borate saline buffer (pH 8.73). The plates were then washed twice with PBS/5% Tween and then twice more with normal PBS. From each rat 5 × l 0 6 spleen cells suspended in PBS supplemented with 5% FCS were then added to duplicate wells in a volume of 300 txl/well. The cells were incubated for 4 hr at 37°C and were then discarded and the plates washed four times with PBS/5% Tween. For assay of the primary response to KLH total anti-KLH Ig-secreting cells were determined by addition of 1.0 ml/well of a 1/1000 dilution of alkaline phosphatase conjugated rabbit anti-rat IgG (heavy and light chain specific) (Bioyeda) followed by incubation overnight at 4°C. For detection of isotype-specific AFC appropriate wells were incubated for 2 hr at 37°C with an optimal dilution of the IgG fraction of purified rabbit anti-rat IgG, IgM and IgA (Miles). This was followed by incubation overnight at 4°C with 1.0 ml/well of a 1/2000 dilution of alkaline phosphatase conjugated goat antirabbit IgG (heavy and light chain specific) (TAGO) in all wells. After overnight incubation with enzyme the plates were washed four times with PBS/5% Tween and substrate-agar solution was then added to each well. The substrate-agar solution was prepared by dissolving at 45°C 10 mg of 5-bromo-4-chloro-3-indolyl phosphate (Sigma) in 8 ml of sterile-filtered 2-amino-2-methyll-propanol (AMP) buffer as prepared by Sedgwick and Holt (24). The substrate solution was then made up to 10 ml by the addition of 2 ml 3% molten agar solution prepared in distilled water. Immediately following this the substrate agar solution was gently mixed and 200 Ixl added to each well. The enzyme-substrate reaction was then allowed to proceed and the number of spots formed counted under a low-powered microscope. Controls for nonspecific immunospot formation were provided by l) using spleen cells from rats not previously immunized with KLH, and 2) incubating all spleen cell samples in wells precoated with OVA. Data Presentation and Analysis The proliferation data are expressed in terms of counts per minute (cpm) and represent the difference between the mean of triplicate experimental wells (containing antigen or mitogen) and the mean of triplicate background wells (containing diluent without antigen or mitogen). For the ELISPOT results the data are expressed in terms of the number of antibody-forming cells (AFC) per 10 x 10 6 cells, and also the number of AFC per spleen. In calculating the former, the difference between the average number of immunospots of duplicate wells for KLH-primed rats and duplicate wells for rats not
475
TABLE 1 PROLIFERATIVE RESPONSE (cpm × 10-3) TO 3.12 ~g/ml ConA AMONG SPLEEN CELLS FROM RATS INFUSED WITH ONE OF SEVERAL DOSES OF EITHER BEP, "yEP, OR MET-ENK 3 HR PRIOR TO ASSAY
Experiment (Peptide Treatment)
Proliferative Response (cpm _+ S.E.)
Experiment (1): BEP PBS (n=6) 39.5 100 fmoles (n=6) 50.2 10 pmoles (n=6) 51.7 1 nmole(n=6) 41.9 Experiment (2): ",/EP PBS (n= 10) 49.2 100 fmoles (n=6) 41.4 10 pmoles (n= 6) 46.5 1 nmole (n=6) 44.7 Experiment (3): Met-ENK PBS (n=7) 49.4 100 fmoles (n=6) 45.3 10 pmoles (n=6) 49.1 1 nmole (n=6) 41.7
Percentage Change From PBS
Statistical Significance
--- 2.5 ± 2.8 _ 7.3 _ 4.2
+27.1% +30.9% + 6.1%
n.s. p<0.05 n.s.
± ± ± ±
0.9 4.0 1.13 3.5
- 15.9% - 5.5% - 9.2%
n.s. n.s. n.s.
± ± ± ±
2.4 2.7 1.7 2.1
- 8.3% 0.0% -15.6%
n.s. n.s.
immunized with KLH was determined and then doubled. To obtain the number of AFC per spleen this value was appropriately adjusted according to the number of splenic leucocytes recovered for each animal. Statistical analysis was performed using one-way analysis of variance (ANOVA) and the Dunnett test for post hoc comparison of group means (10). Additional statistical analyses were conducted using the independent t-test (two-tailed, unless otherwise stated). RESULTS
The proliferative responses to an optimal dose of ConA (3.12 Ixg/ml) of splenic lymphocytes from rats administered BEP, ~/EP, or Met-ENK 3 hr prior to sacrifice are presented in Table 1. A one-way ANOVA of the BEP data revealed a significant main effect of BEP treatment, F(3,20)=3.601, p<0.05. Post hoc comparison of the PBS group with the BEP groups showed that only the increased response of the 10 pmole group was significant [Dunnett critical difference CD(20) = 11.35, p<0.05]. Analysis of the data by ANOVA for animals treated with either Met-ENK or ~/EP did not reveal a significant main effect of either peptide treatment [~/EP: F(3,24) = 1.95, n.s.; Met-ENK: F(3,21)= 2.686, n.s.]. These data therefore reveal modest but statistically significant dose-dependent effects of in vivo administration with BEP, but no observable effects of ~/EP and Met-ENK, on spleen cell proliferation in vitro. Figure 1 presents the results of another experiment in which rats were sacrificed 3 hr after infusion with various doses of the nonopioid active AcBEP(1-27) and the proliferative response of spleen cells to optimal (3.12 Ixg/ml) and suboptimal (0.78 i,tg/ml) doses of ConA examined after 3 or 4 days of culture. One-way ANOVAs conducted on these data indicated a significant effect on both days of culture at the optimal ConA dose [Day 3: F(3,28)= 4.42, p<0.025; Day 4: F(3,28)= 6.11, p<0.0025] but a significant effect was only observed on Day 3 for suboptimal doses [Day 3: F(3,28)=5.39, p<0.005; Day 4: F(3,28)= 1.08, n.s.]. Post hoc comparison of the PBS group with each of the AcBEP(1-27)
476
KUSNECOV, HUSBAND, KING AND SMITH
3.12,ug/ml Con A
0.78,oglml Con A
40
]
Day 3
]
Day 4
1
2 7 x E
30
20
j PBS
100fmoles 10pmoles
1
m
1nmole
PBS
100fmoles lOpmoles
lnmole
Dose o f AcBEP(1-27)
FIG. I. Mean proliferative responses to optimal (3.12 ~g/ml) and suboptimal (0.78 Ixg/ml) concentrations of ConA among spleen cells cultured for 3 and 4 days from rats treated with PBS or various doses of AcBEP(1-27). Vertical bars represent standard error of the mean for n = 8 animals.
groups using the two-tailed Dunnett test (10) revealed that the response of spleen cells from the 100 fmoles group to 3.12 txg/ml ConA was significantly depressed on Day 3 of culture [Critical Difference for Dunnett test, CD(28) = 11.477, p<0.05]. On Day 4 of culture none of the AcBEP(1-27) groups differed significantly from the PBS group using the Dunnett test. However, the proliferative responses of the 100 fmole and 10 pmole groups were significantly greater than the 1 nmole group [1 nmole vs. 100 fmole: t(14) = 4.02, p<0.0025; 1 nmole vs. 10 pmole: t(14) = 3.3, p<0.01]. With respect to the proliferative response of spleen cells from AcBEP(1-27)-treated rats to 0.78 txg/ml ConA, the reduced responses among the 100 fmole and 10 pmole groups on Day 3 of culture were not significantly different from the PBS group using the Dunnett test, On the other hand, a two-tailed t-test comparing the 1 nmole and PBS groups revealed a significant difference, t(14) = 3.316, p<0.01. In addition, the differences between the 1 n mmole group and the 100 fmole and 10 pmole groups, respectively, were found to be significant [1 nmole vs. 100 fmole: t(14)=2.864, p<0.01; 1 nmole vs. 10 pmole: t(14)=5.00, p<0.001]. It is pertinent to note that the depressed proliferative response of spleen cells from the 100 fmole group responding to 3.12 ixg/ml ConA on Day 3 of culture had recovered substantially by Day 4 of culture to exceed that of the PBS group (Fig. i). Similarly, the magnitude of the reduction in the proliferative response of the 1 nmole group to 3.12 txg/ml ConA on Day 4 was two-fold greater than that observed for the PBS group (35.6% vs. 17.0% respectively). These observations suggest that infusion with low and high doses of AcBEP had different effects on the kinetics of the proliferative response of spleen cells to an optimal dose of ConA. Two experiments were conducted to determine the longer term effect of single and multiple in vivo treatments with BEP on lymphocyte proliferation. The results of these experiments are summarized in Fig. 2. It should be noted that in this figure data from the animals in the PBS control groups of both experiments were combined (Experiment 1: n = 7; Experiment 2: n = 10), there being no significant difference between these two control groups, t(15) =0.24, although these data were analyzed separately. In the first experiment rats were given a single infusion of BEP and sacrificed 24 hr later. This experiment did not reveal any effects of BEP at any of the 3 doses used on the proliferative response of spleen cells to an optimal dose of ConA measured on the day of
peak proliferation, F(3,24)=0.302. In the second experiment rats were administered BEP three times at 3 hr intervals and sacrificed 20 or 44 hr after the final infusion. A one-way ANOVA revealed a significant effect of BEP treatment on the proliferative response to ConA, F(4,37)= 5.633, p<0.001. Post hoc comparison of the group means using the two-tailed Dunnett test revealed that only the enhanced proliferative response of spleen cells from rats infused with 100 fmoles and 10 pmoles BEP and sacrificed 44 hr after the last infusion were significantly different from PBS controls [Dunnett CD(37)= 13.41, p<0.05]. The influence of BEP in vivo on the primary AFC response to the soluble protein antigen KLH is shown in Table 2. A one-way ANOVA of the data was found to be significant, F(3,42)= 4.963, p<0.005. Comparison of the group means revealed that only the 1 nmole group was significantly different from the PBS group [Dunnett CD(42) = 81.23, p<0.05]. Table 2 also summarizes the effect of BEP on the secondary AFC response to KLH. The response of all isotypes in the PBS group receiving a second exposure to KLH was significantly greater than the residual group as assessed by a one-tailed t-test
140
x E
[] []
PBS 100 fmoles
[] []
10 pmoles I nmole
120
100
24 hr T i m e of assay a f t e r
20 hr 44 hr last infusion
FIG. 2. Mean proliferative responses to an optimal dose of ConA (3.12 p.g/ml) among spleen cells cultured for 3 days from rats that received a single infusion of BEP and sacrificed 24 hr later, or multiple infusions of BEP and sacrificed 20 or 44 hr after the last infusion. Vertical bars represent standard error of the mean.
BETA-ENDORPHIN AND IMMUNE FUNCTION
477
rats receiving BEP at the time of second exposure to KLH is shown in Fig. 3. The results represented a specific memory response since nonprimed animals did not demonstrate proliferation above background to any of the doses of KLH, and specificity was evident in that all experimental animals demonstrated responses commensurate with background when spleen cells were cultured with OVA. However, for all doses of KLH tested there were no differences between the proliferative responses of PBS and BEP-treated rats on any of the days of culture examined.
TABLE 2 THE MEAN NUMBER OF PRIMARY (EXPERIMENT 1) AND SECONDARY (EXPERIMENT 2) ANTI-KLH AFC (PER 107 CELLS -+ STANDARDERROR OF THE MEAN) IN THE SPLEEN OF RATS TREATED WITH BEP OR PBS AT THE TIME OF IMMUNIZATION Treatment Group (Experiment 1)
Primary AFC Total
Treatment Group (Experiment 2)
Secondary AFC IgG
IgM
168 _ 15
29 ± 12
IgA
DISCUSSION PBS (n = 12)
219.9 --- 17.5
1 nmole BEP ( n - 11)
127.7 ± 19.2
10 pmoles BEP (n= 13)
250.4 -- 26.7
Residual (n 5)
7 ±
1
PBS (n = 6)
271 ± 26 145 ± 33 52 ±
6
1 nmol (n = 6)
335 - 18 165 - 20 70 + 11
Immune function has been shown to be modified by in vitro incubation of human and murine lymphoid cells with BEP and its various derivatives (6). However, the immunologic influence of BEP in vivo has not been extensively studied. As part of a study of the suppressive influence of opioid-mediated analgesic stress on NK cell function, Kraut and Greenberg (11) administered intraperitoneally the proteolytically stable BEP analogue [D-Ala sMetS]-B-endorphin to mice and observed a small, though not statistically significant, increase in poly I:C-induced NK cell activity. More recently, we found that bolus infusion of physiologic doses of BEP via indwelling jugular catheters to conscious rats resulted in a dose-dependent enhancement of the proliferative response of spleen cells to ConA (12). In the present paper we have pursued our investigation of the in vivo effects of BEP on the immune system by way of experiments which address whether 1) opioid and nonopioid active derivatives of BEP exert any influence on lymphocyte proliferation; 2) whether the immunologic changes rendered by BEP administration are of only short duration; and 3) whether the primary and secondary response to soluble antigen can be modified by BEP. Met-enkephalin (Met-ENK) and gamma-endorphin [~/EP: corresponding to BEP(1-17)] are considerably less potent than the complete BEP peptide in standard tests of opioid activity (27). Moreover, total abrogation of opioid activity can be rendered by acetylation of the N-terminal region of BEP (27). In a series of experiments, therefore, we compared the immunomodulatory capabilities of Met-ENK, ~EP, and AcBEP(1-27) when administered in vivo with that of BEP. When single bolus infusions of various doses of BEP were administered via an indwelling jugular
=
100 fmoles 188.4 -- 28.5 BEP (n= 10)
10pmol 389 ± 20 123 -+ 11 52 +- 7 (n = 6) 100fmol 249 - 41 161 - 48 54 +- 9 (n = 6)
[IgG: t(9) = 3.2, p < 0 . 0 1 ; IgM: t(9) = 3.07, p < 0 . 0 1 ; IgA: t(9) = 6.5, p < 0 . 0 0 1 ] . These results indicated that the response of the PBS and BEP groups represented a secondary AFC response. With respect to the IgG isotype, a one-way ANOVA of the data was found to be significant, F ( 4 , 2 4 ) = 7 . 3 8 3 , p < 0 . 0 0 1 . Post hoc comparison of the PBS group with the BEP groups revealed that only the increased response of the 10 pmole group was significant [Dunnett C D ( 2 4 ) = 107.6, p < 0 . 0 5 ] . Although statistical analysis of the IgM and IgA data revealed significant main effects, these were due to the lower response of the residual group, and not attributable to any significant differences between the PBS and BEP groups. The proliferative response to KLH in vitro of spleen cells from
25jug/ml KLH
sOjug/ml KLH
30-
100pg/ml
T
KLH
,,5"::':
,,.,'..!~
o x E
..","t
/,/'."
20
f
10
- PBS . . . . . . . . . Inmole ...... 10pmoles 100fmoles t
3
1
tl
I
S
I
1
3
4 DAY
I
5
I
4
I
5
OF CULTURE
FIG. 3. The mean proliferative responses to various concentrations of KLH among spleen cells cultured for 3, 4, and 5 days from KLH-primed rats administered with PBS or BEP at the time of secondary immunization. For each point vertical bars represent the standard error of the mean for n = 6 rats.
478
catheter to conscious rats a significant enhancement of the proliferative response of splenic lymphocytes to an optimal concentration of ConA was observed among ceils from rats administered 10 pmoles BEP 3 hours prior to assay (see Table 1). This finding is in agreement with the results of our previous report (12). There was no such enhancement of the proliferative response following infusion with Met-ENK or ~EP. In fact, infusion with these peptides resulted in diminished proliferative responses to ConA, but these were not statistically significant. Given that intravenous administration of BEP enhanced the spleen cell proliferative response to ConA, infusion with AcBEP( 127) suppressed the response, and infusion with ~/EP or Met-ENK had no effect, it appears that the enhancing effect of BEP on spleen cell proliferation following in vivo administration requires the C-terminal region of BEP. In the absence of the C-terminal region an inhibitory effect on the proliferative capacity of spleen cells may occur. The observation that AcBEP(1-27) exerted inhibitory effects on spleen cell proliferation where BEP had an enhancing influence indicates further that endogenous peptides with diminished or absent opioid activity may have opposite effects to that of the extremely opioid potent BEP. The foregoing results are consistent with other evidence showing that -yEP in vitro has no effect on lymphocyte proliferation (7). In addition, although there have been no reports of the effect of N-acetylated forms of BEP on lymphocyte proliferation, other parameters of immune function have not been shown to be affected by AcBEP (25). In the present experiments, however, some dose rates of AcBEP(1-27) exerted an inhibitory effect on the capacity of lymphocytes to proliferate in vitro to an optimal dose of ConA, thereby demonstrating a possible immunomodulatory role in vivo. Indeed, acetylated forms of BEP are secreted from the intermediate lobe of the pituitary in response to stress (1), which may serve to oppose the immunomodulatory effects of similarly secreted anterior lobe BEP. Moreover, this functional opposition may also be present at the autocrine level, since murine spleen cells have been shown to synthesize both normal and acetylated forms of BEP (13). The present experiments have also demonstrated that the enhancing effect of BEP in vivo on lymphocyte proliferation can be observed after 2 days when rats are administered BEP three times in a 9 hour period (Fig. 2). Interestingly, there was no detectable effect of multiple BEP treatment on lymphocyte proliferation after 20 hours. Although there is presently no adequate explanation for this delayed effect, the data demonstrate that repeated physiologic increases of BEP in blood can induce immunopotentiating effects which are detectable after a prolonged period. The in vitro plaque-forming cell (PFC) response of murine splenocytes to sheep erythrocytes can be partially inhibited by BEP (9). In the present study it was observed that administering BEP 1 hour before and 1 hour after primary immunization with the soluble antigen KLH resulted in a significant suppression of the splenic AFC response measured 4 days later (Table 2). A high dose of BEP (1 nmole) was required to induce this effect and was not observed with lower physiologic doses. Nonetheless, this observation extends previous in vitro observations (9) that BEP can inhibit in vivo the primary antibody response to antigen. A further experiment examined whether the secondary antibody response to KLH could also be modulated by BEP administration at the time of secondary exposure to antigen. The results showed that only the IgG-secreting AFC were affected by BEP
KUSNECOV, HUSBAND, KING AND SMITH
treatment (Table 2). This is not surprising, since IgG was the major isotype being synthesised in the spleens of animals in the residual group (primed but not boosted), and the secondary antibody response is predominantly of the IgG isotype. In contrast to the influence of BEP on the primary anti-KLH response, the secondary IgG AFC response was potentiated by BEP at a dose of 10 pmoles. This suggests that, unlike the primary response, the secondary antibody response to antigen is resistant to any suppressive influence of high doses of BEP. There was no influence of BEP on the capacity of KLHsensitized spleen cells to proliferate in vitro in response to various concentrations of KLH 5 days after combined KLH/BEP treatment. Whether an effect may have been observed at an earlier time after BEP treatment remains to be determined. Whether the immunologic effects of the various peptide treatments in the present experiments were due to a direct peptidelymphoid cell interaction is not known. Endogenous opioids have been shown to have endocrinologic effects (17) which may mediate the immunologic changes observed in the present report. We have observed previously that BEP has enhancing effects on spleen cell proliferation in vitro at doses similar to those which enhance proliferative capacity in vivo (12). Therefore, it is most probable that the effects of BEP and AcBEP(1-27) on spleen cell proliferation involve a direct interaction with lymphoid cells. However the fact that the effects exerted by BEP varied with dose rate and time is a reflection of the complexity of mechanisms involved in these effects and many further studies will be required before appropriate explanations could be provided. Basal levels of BEP in rat plasma range from 120 to 500 pg/ml (3,22), and subsequent to restraint stress are elevated 6- to 12-fold (3,18). The doses of BEP in our experiments therefore approximated physiologic (100 fmoles=346.5 pg), stress-induced (10 pmoles=34.65 ng), and supraphysiologic (1 nmole=3.465 ~g) plasma levels, It is interesting to note that the enhancing effects of BEP administration on spleen cell proliferation and secondary IgG AFC formation were observed at a dose of 10 pmoles. This suggests that BEP release from the pituitary may serve to facilitate immune function under conditions of stress. On the other hand, the primary response to KLH was suppressed by the supraphysiologic dose of 1 nmole BEP, but was unaffected by 10 pmoles BEP. This indicates that stress-related plasma levels of BEP do not alter the primary response to antigen in vivo at the time of immunization, this being possible only when doses far in excess of those encountered physiologically are administered. The present series of experiments has shown that in vivo administration of some doses of BEP enhances the proliferative response of spleen cells to ConA in vitro and modulates the primary and secondary antibody-secreting cell response to the soluble protein antigen KLH. Moreover, in vivo treatment with Met-ENK or "yEP was without effect on spleen cell proliferation, while AcBEP(1-27) inhibited the proliferative response of spleniclymphocytes to ConA. These observations complement an extensive literature documenting in vitro modulation of immune function by the endogenous opioids. ACKNOWLEDGEMENTS
This work was supported by grants from the National Health and Medical Research Council of Australia. The authors are grateful for the technical assistance of Christine Baker. Thanks also go to Dr. Gerald Pang for his expert advice in establishing the ELISPOT assay.
BETA-ENDORPHIN AND IMMUNE FUNCTION
479
REFERENCES 1. Akil, H.; Shiomi, H.; Mathews, J. Induction of the intermediate pituitary by stress: Synthesis and release of a nonopioid form of B-endorphin. Science 227:424-426; 1985. 2. Akil, H.; Watson, S. J.; Young, E.; Lewis, M. E.; Khachaturian, H.; Walker, J. M. Endogenous opioids: Biology and function. Annu. Rev. Neurosci. 7:223-255; 1984. 3. Berkenbosch, F.; Vermes, I.; Tilders, F. J. H. The B-adrenoceptorblocking drug propanolol prevents secretion of immuuoreactive Bendorphin and et-melanocyte-stimulating hormone in response to certain stress stimuli. Endocrinology 115:1051-1059; 1984. 4. Blalock, J. E.; Bost, K. L.; Smith, E. M. Neuroendocrine peptide hormones and their receptors in the immune system: Production, processing, and action. J. Neuroimmunol. 10:31-40; 1985. 5. Czerkinsky, C. C.; Nilsson, L-A.; Hakan, N.; Ouchterlony, O.; Tarkowski, A. Solid-phase enzyme-linked imunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65:109-121; 1983. 6. Fischer, E. G.; Falke, N. E. B-endorphin modulates immune functions. Psychother. Psychosom. 42:195-204; 1984. 7. Gilman, S. C.; Schwartz, J. M.; Milner, R. J.; Bloom, F. E.; Feldman, J. D. B-endorphin enhances lymphocyte proliferative responses. Proc. Natl. Acad. Sci. USA 79:4226--4230; 1982. 8. Hazum, E.; Chang, K-J.; Cuatrecasas, P. Specific nonopiate receptors for B-endorphin. Science 205:1033-1035; 1979. 9. Johnson, H. M.; Smith, E. M.; Torres, B. A.; Blalock, J. E. Regulation of the in vitro antibody response by neuroendocrine hormones. Proc. Natl. Acad. Sci. USA 79:4171-4174; 1982. 10. Keppel, G. Design and analysis: A researcher's handbook, 2nd ed. Englewood Cliffs, NJ: Prentice-Hall; 1982. 11. Kraut, R. P.; Greenberg, A. H. Effects of endogenous and exogenous opioids on splenic natural killer cell activity. Nat. Immun. Cell Growth Regul. 5:28-40; 1986. 12. Kusnecov, A.W.; Husband, A. J.; King, M. G.; Pang, G.; Smith, R. In vivo effects of B-endorphin on lymphocyte proliferation and interleukin-2 production. Brain Behav. Immun. 1:88-97; 1987. 13. Lolait, S. J.; Clements, J. A.; Markwick, A. J.; Cheng, C.; McNally, M.; Smith, A. I.; Funder, J. Pro-opiomelanocortin messenger ribonucleic acid and posttranslational processing of beta endorphin in spleen macrophages. J. Clin. Invest. 77:1776-1779; 1986. 14. Lopker, A.; Abood, L. G.; Hoss, W.; Lionetti, F. J. Stereoselective muscarinic acetylcholine and opiate receptors in human phagocytic leukocytes. Biochem. Pharmacol. 29:1361-1365; 1980.
15. Lori Brown, S.; Van Epps, D. E. Opioid peptides modulate production of interferon gamma by human mononuclear cells. Cell. Immunol. 103:19-26; 1986. 16. Loft Brown, S.; Van Epps, D. E. Suppression of T lymphocyte chemotactic factor production by the opioid peptides B-endorphin and met-enkephalin. J. Immunol. 134:3384-3390; 1985. 17. Millan, M. J.; Herz, A. The endocrinology of the opioids. Int. Rev. Neurobiol. 26:1-83; 1985. 18. Mueller, G. Beta-endorphin immunoreactivity in rat plasma: Variations in response to different physical stimuli. Life Sci. 29:16691674; 1981. 19. Payan, D. G.; McGillis, J. P.; Goetzl, E. J. Neuroimmunology. Adv. Immunol. 39:299-323; 1986. 20. Peterson, P. K.; Sharp, B.; Gekker, G.; Brummitt, C.; Keane, W. F. Opioid-mediated suppression of cultured peripheral blood mononuclear cell respiratory burst activity. J. Immunol. 138:3907-3912; 1987. 21. Peterson, P. K.; Sharp, B.; Gekker, G.; Brummitt, C.; Keane, W. F. Opioid-mediated suppression of interferon-gamma production by cultured peripheral blood mononuclear cells. J. Clin. Invest. 80: 824-831; 1987. 22. Pettibone, D. J.; Mueller, G. P. Differential effects of adrenergic agents on plasma levels of immunoreactive B-endorphin and alphamelanotropin in rats. Proc. Soc. Exp. Biol. 176:168-174; 1984. 23. Puppo, F.; Corsini, G.; Mangini, P.; Bottaro, L.; Barreca, T. Influence of B-endorphin on phytohemagglutinin-induced lymphocyte proliferation and on the expression of mononuclear cell surface antigens in vitro. Immunopharmacology 10:119-125; 1985. 24. Sedgwick, J. D.; Holt, P. G. A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting ceils. J. lmmunol. Methods 57:301-309; 1983. 25. Sharp, B. M.; Tsukayama, D. T.; Gekker, G.; Keane, W. F.; Peterson, P. K. B-endorphin stimulates human polymorphonuclear leukocyte superoxide production via a stereoselective opiate receptor. J. Pharmacol. Exp. Ther. 242:579-582; 1987. 26. Shavit, Y.; Lewis, J. W.; Terman, G. W.; Gale, R. P.; Liebeskind, J. C. Opioid peptides mediate the suppressive effect of stress on natural killer cell cytotoxicity. Science 223:188-190; 1984. 27. Taylor, J. W.; Kaiser, E. T. The structural characterization of B-endorphin and related peptide hormones and neurotransmitters. Pharmacol. Rev. 38:291-319; 1986.