β-Endorphin's modulation of lymphocyte proliferation is dose, donor, and time dependent

BRAIN,

BEHAVIOR,

AND

IMMUNITY

4, 232-242 (1990)

P-Endorphin’s Modulation of Lymphocyte Proliferation Dose, Donor, and Time Dependent DAVID

B. MILLAR,’

CHRISTOPHER J. HOUGH,~ DENISE JOSEPH E. GOOTENBERG~

L. MAZOROW,’

Is

AND

Laboratory of Biochemical Genetics, National Institute of Mental Health Neuroscience Center St. Elizabeths, Washington, D.C. 20032; and Immunobiology and Transplantation Department, Naval Medical Research Institute, Bethesda, Maryland 20814

at

We find that 8-endorphin (Bend) can have, positive, negative, or neutral dosedependent effects on the mitogen-stimulated proliferation of human peripheral blood lymphocytes. The distribution of positive, negative, or neutral responses was nonrandom. In studies carried out over a year, we show that an individual’s mitogen-stimulated lymphocyte proliferative response to Bend can change with time. We show that the inhibition induced by cortisol can be, in part, relieved by Bend. On the basis of our results and those of others in the field, we put forward a model that can qualitatively account for many of the observations we and other investigators have made. 0 1990 Academic

Press, Inc.

INTRODUCTION

The concept of an autonomous immune system uncommunicative with and uninfluenced by the central nervous system is no longer tenable, (Blalock, 1984). The present consensus suggests that there is bidirectional, functional communication between the central nervous system (CNS) and the immune system. Information exchange between the two occurs by means of messengers and receptors used in common (Blalock, McMenamin, & Smith, 1985; Besedovsky, de1 Rey, & Sorkin, 1985; Payan & Goetzl, 1985; Pert, Ruff, Weber, & Herkenheim, 1985). With regard to prospective messengers, a number of neuropeptides have exhibited modulating activity on various components of the immune system. The most frequently studied are probably the enkephalins and the endogenous opioid, P-endorphin (Bend). Bend is a stress-responsive polypeptide (Guillemin, Vargo, Rossier, Minick, Ling, Rivier, Vale, & Bloom, 1977; Rossier, French, Rivier, Ling, Guillemin, & Bloom, 1977; Dubois, Pickar, Cohen, Roth, Macnamara, & Bunney , 1981; DeSouza & Van Loon, 1985) comprising 31 amino acids derived from cleavage of the prohormone pro-opiomelanocortin (Roberts & Herbert, 1977; Dores, Akil, & Watson, 1984). Within the CNS, it is clear that Bend is a potent neuromodulator, e.g., intracerebrospinal injection of Bend has profound ’ Present address: Laboratory of Biochemical Genetics, National Institute of Mental Health Neuroscience Center at St. Elizabeths, Washington, DC 20032. ’ Present address: Department of Environmental Medicine, Naval Medical Research Institute, Bethesda, MD 20814. 3 Present address: Department of Pediatric Oncology, Lombardi Center, Georgetown University School of Medicine, Washington, DC 20007. 232 0889-1591/90 $3.00 Copyright 6 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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consequences including long-lasting rigidity similar to catatonia (Bloom, Segal, Ling, & Guilleman, 1976); periaqueductal injection also results in catalepsy as well as deep sedation (Jacquet & Marks, 1976). Its actions on some elements of the immune system, however, are less well defined and results from seemingly similar experiments are contradictory. This is strikingly the case for lymphocyte proliferation in the presence of Bend in which suppression, enhancement, or both have been reported (McCain, Lamster, Bozzone, & Grbic, 1982; Gilman, Schwartz, Miller, Bloom, & Feldman, 1982; Nordland & Mutt, 1986; Kusnecov, Husband, King, Pang, & Smith, 1987; Fontana, Fattorossi, D’Amelio, Miglioratti, & Perricone, 1987; Heijnen, Croiset, Zijlstra, & Ballieux, 1987). While methodology and dose differences can explain some of the apparent contradictions, the existence within studies of nonresponders (Gilman et al., 1982; Fontana et al., 1987) as well as responders suggests the problem to be more complex than initially appreciated. Such an opinion is buttressed by the observations of Oleson and Johnson (1988) who showed that human natural killer cells exhibited differential response toward Bend in a manner reflecting their degree of cytolytic activity. Similarly, Brummitt, Sharp, Gekker, Keane, and Peterson (1988) demonstrated that Bend’s influence on interferon-y (IFN-y) production by human peripheral blood mononuclear cells was donor, time, and culture medium dependent. In this report, we present results which show that the direction of Bend’s modulation of human peripheral blood lymphocyte (PBL) proliferation is not necessarily a constant with a given donor but can vary with time as well as dose. Finally, we present a hypothesis that can qualitatively account for many of the reported effects of Bend on lymphocyte proliferation. METHODS

Peripheral blood lymphocytes: Venous blood samples were taken from laboratory personnel in apparent good health who had given informed consent; the age range was from 21 to 55. All donors maintained that they were medication and opiate free. Blood was drawn in heparin (Calbiochem)-containing syringes, as close to 8:30 AM as possible and never varied more than 30 min from this time. After bulk removal of red blood cells by 1 h, room temperature gravity sedimentation in dextran T500 (Pharmacia, equal volumes of blood and 2% dextran in normal saline), the supernatant was diluted 1: 1 in Hanks’ (GIBCO)/lS mM Hepes (Pharmacia; Hanks’/Hepes) and lymphocytes were isolated using Litton Bionetics lymphocyte separation medium (LSM) following the manufacturers instructions. The cells were then washed twice by centrifugation (8OOg for 10 min, 20°C) in RPM1 1640, 10% heat-inactivated GIBCO fetal calf serum, 0.2 mM glutamine, 50 units/ml penicillin G, and 50 p,g streptomycin sulfate and resuspended in this medium (growth medium) to a density of 1 X lo6 cells/ml. Viability was determined by trypan blue exclusion and was never less than 95%. Mitogen-stimulated blastogenesis was assumed to be represented by uptake of tritium-labeled thymidine (New England Nuclear; sp act, 5 Ci/mmol) into DNA which was measured as follows: 1 x lo5 cells were placed in each of 96-well sterile flat bottom plates and Bend was added to give a desired final concentration-typically 3, 30, or 300 a-when the final volume (200 ~1) was achieved. Phytohemagglutinin (PHA;

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Boehringer-Mannheim) was 10 pg/ml. Mitogen and opiates as well as other test substances were routinely diluted into growth medium from frozen stocks stored at - 40°C. Incubation took place for 48 h at 37°C in a 5% COZ atmosphere. At this point, 0.5 to 1 t.&i of tritiated thymidine was added and a further incubation of 18 h was carried out. Cells were harvested by suction onto glass fiber paper (Whatman) and washed extensively with water to remove unreacted label. The paper was allowed to dry and spots containing label were punched out into scintillation vials and counted on a Beckman LS 2500 using Aquasol (New England Nuclear). Each assay was done at least in quadruplicate. Experiments in which the effect of Bend modulation of cortisol inhibition of mitogen-stimulated proliferation were carried out as described except that Bend was added just prior to cortisol. The levels of cortisol used were determined by trial experiments containing up to 50 kg/ml cortisol. Concentrations of cortisol greater than 25 pg/ml were nearly lethal to most cells. It was found that concentrations of 0 to 5 l&ml were most useful. RESULTS

1. Variation

in PBL Proliferative

Response to Bend between Individuals

Donor PBLS mitogen-stimulated proliferation the proliferation ratio (PR) and is defined as PR =

response to Bend is expressed as

CPM thymidine incorporated with Bend present CPM thymidine incorporated minus Bend (control)

*

Further, when used in this paper, the word proliferation shall be taken to mean mitogen-stimulated proliferation. Positive modulation is a PR greater than 1.0 (stimulation) and negative modulation (inhibition) is a PR of less than 1.0. Figure 1 shows the extreme differences found in individual dose-response char1.61

I

30

J

Bend, nM

FIG. 1. Dose response for PBL proliferation to 300 nA4 Bend for three donors. Vertical bars are standard deviations. The triangles, squares, and diamonds delineate the three different donors.

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IS VARIABLE

acteristics for three different donors determined at identical Bend concentrations. The phenomenon of positive, negative, and neutral PBL proliferation responses to Bend has been reported previously by Heijnen et al. (1987) but the reversal of negative responses by higher concentrations of Bend is, we believe, now reported for the first time. The Bend-modulated production of IFN-), by cultured peripheral blood mononuclear cells, however, displays heterogeneity among donors and is also time dependent (Brummitt et al., 1988). 2. The Variability with Time

of Bend’s Modulation of the Proliferative

Response

A point not previously addressed in the literature is whether Bend’s modulation of a donor’s proliferative response is invariable with time. The question is important since the answer potentially can shed light on the mechanism through which Bend modulates the proliferation response. Figure 2 presents proliferation assays on one donor taken over 10 weeks. Measured at three different times, this donor shows both positive, negative, and almost neutral modulation of proliferation responses by Bend. The possibility exists that this donor might be aberrant and not representative of the normal population. We addressed this question by means of experiments conducted over time on a fixed donor population. Figure 3 shows the results of Bend modulation of mitogen-stimulated PBL proliferation assays on four donors conducted over the space of 1 year. Donors A and C are female; B and D are male. The results presented here were obtained at a Bend concentration of 300 m&f but data were gathered (not shown) at 30 and 3 niV as well and exhibited a similar fluctuation. These results confirm that time variability in the Bend-modulated

1

1.4 i

0.6 A 0

I 3

I 30

I 300

Bend, nM

2. Time variability in PBL proliferation dose-response curves to 3, 30, and 300 nM Bend for one donor. Assays performed over a period of 10 weeks. Bars indicate standard deviations. The circles, diamonds, triangles, and squares represent the results obtained for this donor at the four different time periods. FIG.

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1.5

. . f -. -.

1.0 E

.

-ii I

-c : -9i

A . T .

.

.

0.5

FIG. 3. Variation in PBL proliferative response among four donors to 300 ti Bend over a period of 1 year. Upper and lower horizontal bars represent standard deviations; middle bar is the mean of the measurements.

proliferation proliferative

response can exist within donors and that time-variable, nonlinear response profiles to Bend among donors are not unusual.

3. Frequency Distribution of Positive, Negative, and Neutral Proliferation Responses to Bend

We wanted to determine whether or not there was a strong bias toward one response to Bend as this information is of importance in trying to understand the mechanism by which Bend modulates proliferation. To estimate the frequency distribution of positive, negative, and neutral responses to Bend by mitogenstimulated PBL, we expanded the donor population to 14 and increased the number of proliferation assays to 37. Table 1 shows the results. For these calculations, a neutral response is defined as one with a PR of 1.OO, a positive response as 1.Ol or greater, and a negative response as a PR of 0.99 or less. Clearly, the neutral response is in the minority, while positive and negative responses are in the plurality. Averaging the positive responses across the three Bend concentrations

Distribution

TABLE 1 Frequencies in Percentage of Neutral, Positive, and Negative Mitogen-Stimulated Proliferative Responses to Bend” Bend (ru%Ij

Neutral Positive Negative a N = 37 proliferation

3

30

300

11 49 40

8.0 57 35

11 43 46

assays for each concentration

of Bend.

PBL

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gives a frequency of 50%. This is close to the 53% positive response observed by Heijnen et al. (1987) with a donor population of 17 and to those of Gilman et al. (1982) who observed a 55% fraction of positive responders to Bend in mouse spleenocytes (18 donors) at concentrations of Bend in the general region we have used. For our data, there does not appear to be a very strong bias for or against any response except against neutral responses. Our results, however, are in sharp contrast to the work of McCain et al. (1982) who reported only inhibition of proliferation at 100 nM Bend and to those of Fontana et al. (1987) who using extremely low concentrations of Bend (lo- I2 M) reported 83% positive responders (40 donors) and 17% nonresponders (our neutral response). 4. Inhibition of Mitogen-Stimulated PBL Proliferation Bend inhibits cortisol suppression of PBL proliferation. We next investigated those elements that might be inhibitors of proliferation and to the possibility that Bend might modulate this inhibition. This is a significant question since some of the profiles seen in Figs. 1 and 2 might be the result of Bend-modulated inhibition of proliferation. Corticosteroid abrogation of immunocompetence is well established (Gillis, Crabtree, & Smith, 1979); Sapolsky & Donnely, 1985). The fact that Bend and adrenocorticotropin are secreted simultaneously by the pituitary (Guilleman et al., 1977) and that steroids bind at sigma receptors (Tsung-Ping, London, & Jaffe, 1988) suggested to us that differential effects between steroids and Bend might occur with PBLs. Therefore, we tested whether stress-induced circulating corticosteroids interacting with PBLs might be responsible for the highly variable proliferative responses to Bend (Dantzer & Kelley, 1989). Table 2 shows the proliferative consequences of including cortisol and/or Bend in the incubation medium. The proliferative response of all donors is muted by cortisol to varying degrees, but particularly that of donor A whose response is practically obliterated, However, the inhibition by cortisol in 21 of 24 assays is progressively relieved by increasing concentrations of Bend-even donor A’s feeble proliferative response is enhanced by Bend. Certainly, there is no evidence (with the one exception of donor C at 5 pg/ml of cortisol) of any depression of proliferative response by Bend. These results show that all levels of cortisol that we used are inhibitory to mitogen-stimulated PBL proliferation and that Bend generally acts to partially reverse this inhibition. Our data cannot, however, eliminate the possibility that a cortisol resistant, Bend-responsive subpopulation is responsible for the apparent reversal. It should be noted that Holaday, Bernton, & Bryant (1989) have reported that chronic morphine administration in mice results in corticosteroid-mediated immunosuppression that could be prevented by simultaneously dosing with naloxone. Our results suggest that the complex variations in PBL proliferative response to Bend that we observe may in part result from differing levels of steroids in our donors. DISCUSSION

There is little doubt that recent work on Bend and its action on the immune system has but barely sketched the features of what will likely prove to be a highly

238

MILLAR Bend Modulation

Donor A

B

C

Eb

Cortisol (cLg/ml) 0.0 1.25 2.50 5.00 0.0 1.25 2.50 5.00 0.0 1.25 2.50 5.00 0.0 1.25 2.50 5.00

ET AL.

TABLE 2 of Cortisol Inhibition of PBL Proliferation Expressed as PR” Bend (nkf) 0

3

1.0

0.017 2 1.0 0.35 2 0.34 2 0.30 2 1.0 0.25 i 0.14 f 0.15 k 1.0

,002 .052 .033 .041 .051 .020 .020

-

0.21 f .018

1.13 f 0.043 k 1.24 2 0.54 f 0.39 f 0.43 +1.011 k 0.21 k 0.21 f 0.11 f 2.65 2

-

.08 .002 .12 .056 .070 .051 .019 .035 .020 .040 .021

0.38 2 .18

30

300

1.16 f .09 0.047 + .005 1.47 * .16 0.54 + .077 0.39 k .068 0.44 2 .14 1.16 f .14 0.28 + ,007 0.20 rfr .029 0.13 k .026 3.26 f .32 0.31 f .027

1.09 -+ .07 0.044 * .015 1.41 k .19 0.71 * .O% 0.61 t .12 0.57 2 .036 1.14 + .03 0.30 f .016 0.32 * .006 0.099 k .029 3.46 f .38 0.47 2 .24

n Each value is the average of four assays. b Male donor.

complex portrait. Indeed, the molecular consequence of Bend’s impact on lymphocytes are just starting to be outlined, (Hough, Halperin, Mazorow, Yeandle, & Millar, 1990; Mazorow, Hough, & Millar, 1989). In at least some cases, Bendmodulated inhibition of lymphocyte proliferation is now beginning to be understood as a multicellular process involving interaction of Bend with monocytes resulting in the elaboration of monocyte metabolites (Peterson, Sharp, Gekker, Brummitt & Keane, 1987; Brummitt et al., 1988) which inhibit proliferation at an early stage (Vercammen & Ceuppens, 1987; Bidlack & Hemmick, 1989). But in our experiments no preincubation with Bend was performed and we observed donor-dependent inhibition or stimulation of proliferation or no response at all. The observation that Bend was partially able to reverse cortisol inhibition of lymphocyte proliferation leaves little doubt that there is also a positive modulatory effect of Bend which is active even in cells exposed to the powerful immunosuppressive agent used here. But this observation does not explain all the findings of Gilman et al. (1982), Fontana et al. (1987), Brummitt et al. (1988), Heijnen et al. (1987), and our own. These studies have in common that they are all characterized by departures from simple behavior in the fact that individuals either vary in direction of modulation with time or dose or do not respond at all to Bend. In this regard, it is important to remember that the cell populations investigators use in these studies derive from subjects who may be exposed to a wide variety of immunoactive agents (Brummitt et al, 1988; Dantzer & Kelley, 1989; Weiss, Sundar, Becker, & Cierpial, 1989). These agents do not necessarily wash out after cell harvesting and might prime the system for modulation in a definite direction depending upon the protocol details. A potent example of the

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latter is Bend incubation vis a vis mitogen exposure (Dubois et al., 1981; Goodwin, Bromberg, Staszak, Kaszubowski, Messner, & Neal, 1981; McCain et al., 1982; Peterson et al., 1987). Indeed, the work of Goodwin et al. (1981) suggests this aspect of the problem to be even more complex. These authors demonstrate that stress increases the sensitivity of lymphocytes to exogenous PGE, action and it is by no means certain that this is due to increased concentrations of cortisol or changes in suppressor cell populations. With these caveats in mind one can sketch out a simple outline of the elements that we propose govern the response of mitogen-stimulated PBL to Bend. 1. The direction of Bend modulation of proliferation results from the net influence of positive modulation sites (lymphocytes) and negative modulation sites (monocytes, Goodwin, Bankhurst, & Pressner, 1977; Peterson et al., 1987, lymphocytes) which we feel are both targets of Bend. The profile of the lymphocyte proliferation dose-response curve to Bend therefore can take on a variety of shapes depending on whichever modulation effect is dominant (this work, Fig. 1). 2. This net influence can, in part, arise from the ratio of monocytes to lymphocytes and their state of activation which can vary from experiment to experiment (Peterson et al., 1987). Thus, the lymphocyte separation medium and the lymphocyte isolation protocol used may well influence the response to Bend because of the number of monocytes contained in the PBL preparation. 3. Madden, Donahoe, Zwemer-Collins, Shafer, & Falek (1987) have shown that purified lymphocytes from different individuals bind naloxone with as much as a fivefold difference in capacity with accompanying changes in cooperativity. Brummitt et al. (1988) have demonstrated large differences in production rates of IFN-+y among individuals challenged by Bend. These results, and those shown in Fig. 2, appear to reflect the following concept: Different opiate binding abilities combined with the relative expressive vigor of negative and positive modulation sites will contribute to the outcome of exposure to Bend. 4. Stress-elaborated elements such as PGE, and cortisol will modulate the degree of reactivity of the cell system to Bend as has been shown by Goodwin et al. (1981), Goodwin and Ceuppens (1983), Holaday et al. (1989), and this work (Table 2). 5. McCain et al. (1982) and Peterson et al. (1987) have shown that substantial preincubation of Bend with the lymphocyte preparation is required for inhibition of lymphocyte function. These findings are consistent with the idea: The time exposure protocol of Bend vs mitogen can strongly guide the degree and/or the direction of modulation conferred by Bend because of activation of different, kinetically irreversible steps early in the cell cycle. The path taken first-activation or inhibition-can result in resistance to the opposite path achieving maximum effect. 6. The extent of lymphocyte stimulation by mitogen can effect the modulation by Bend (Gilman et al., 1982; Fontana et al., 1987; Gilmore & Weiner, 1988).

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This arises because a lymphocyte fully stimulated by mitogen may have no proliferative reserve available for Bend activation. Choice of mitogen may be also critical since Bend has been reported to modulate a subset of T cells that are responsive to concanavalin A (Con A) but not to PHA (Hemmick & Bidlack, 1987). What are the neuroimmunomodulatory consequences of postulates 1 through 6? It is fully possible with these postulates to have a range of proliferative responses to Bend going from positive to neutral to negative. The model also allows for changes in an individual’s proliferation dose-response profile when measured at different calendar times. This degree of freedom arises since functions 1 through 4 could be time dependent and 1 through 6 can be either antagonistic or interactive or combinations of both allowing a richness of immunomodulatory response to Bend. While this portion of the model adequately explains in a qualitative sense simple PBL reactions to Bend, it falls short of dealing with the complex curves seen in Figs. 1 and 2 and those of Heijnen et al. (1987). Oleson and Johnson (1988) also observed bell shaped and skewed effects of opioids on natural killer cell activity and attributed the results to two receptors with differing opioid affinities and biological activities. Foris, Medgyesi, and Hauck (1986) suggest a similar concept for the action of Met-enkephalin on macrophage functions. We favor a similar explanation to be operative in our studies because the PR profiles seen in Figs. 1 and 2 are consistent with the hypothesis of two receptors with different afftnities for Bend and opposing functions with respect to proliferation. We therefore added postulate 7 to the model: 7. With respect to positive and negative modulation there are, at least, two different Bend receptors with different affinities and different biological activities toward proliferation (Oleson & Johnson, 1988; Foris et al., 1986). According to this postulate, the results of Fontana et al. (1987) could be explained by the presence of a high affinity, positive modulation site for Bend that is fully operative at very low concentrations of Bend. Additionally, the results of Figs. 1 and 2 suggest that receptor number could be a time variable quantity. It should be noted that it might be difficult to distinguish between the above hypothesis and one in which one receptor is coupled to two different effecters and coupling is modified by the metabolic state of the cell. With the addition of postulate 7, we believe that the model can qualitatively account for many of the proliferative responses to Bend so far reported. The fact that we and others do not always see bell or curvilinear responses may be attributed to variations in the opioid affinity and/or quantity of the types of receptors among subjects (Madden et al., 1987) as well as the purity of the cell preparation and concentration of Bend and type of mitogen (Gilmore & Weiner, 1988; Hemmick & Bidlack, 1987) employed in the assay. The proposal we present here has the virtue that it lists concepts which can aid in experimental design and can be tested in a logical manner. In this context, and in view of the cellular complexity of a standard PBL preparation, it might be well

IMMUNOMODULATION

advised to use a purified T-cell approach in opioidflymphocyte (1987) and Madden et al. (1987). a more inclusive understanding

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population as a starting point. The utility of this studies has been demonstrated by Peterson et al. Such investigations may provide a framework for of the immunomodulatory properties of Bend. REFERENCES

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