Proc. Nat!. Acad. Sci. USA Vol. 88, pp. 360-364, January 1991 Neurobiology

Immunomodulatory activity of jm- and K-selective opioid agonists (neuroimmunology/antlbody formatlon/immunoregulation) DENNIS D. TAUB*, TOBY K. EISENSTEIN*, ELLEN B. GELLERt, MARTIN W. ADLERt, AND THOMAS J. ROGERS*t Departments of *Microbiology and Immunology, and tPharmacology, Temple University School of Medicine, Philadelphia, PA 19140

Communicated by Hans W. Kosterlitz, October 17, 1990 (received for review May 18, 1990)

important to determine whether different receptor types are involved in immunoregulation. In the present study, we have found that the exogenous IL-selective agonists morphine and Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGE) and the highly K-selective agonists U50,488H and U-69,593 are all able to inhibit antibody production in vitro. Each inhibitory activity is repressed by antagonists specific for the appropriate opioid receptors. These results suggest that this opioid-mediated regulation occurs by way of the classical IL and K receptor-ligand pathways. The stereospecificity of this regulation was demonstrated using enantiomers of U50,488H, in which the (-) isomeric form of the K agonist possessed significantly greater immunomodulatory activity. Furthermore, only K agonists suppressed antibody responses in a p.-deficient mouse strain, whereas ,u and K agonists were effective in the parent -unresponsive strain. In addition, pretreatment studies with these opiate compounds suggest that murine splenocytes possess surface receptor-like structures for opioids. These results support the hypothesis that regulatory pathways exist between the immune and central nervous systems and that the endogenous opioid system is part of that regulation.

Opioids and opioid peptides have been shown ABSTRACT by numerous laboratories to modulate various parameters of the immune response, but little attention has been given to the type of opioid receptor that might be involved. This study focuses on the in vitro influences of morphine and DAMGE (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol), p-selective agonists, and U50,488H and U69,593, Kc-selective agonists, on the generation of antibody to sheep erythrocytes in vitro. It was found that the p and K opioid agonists were able to inhibit the capacity of murine lymphoid cells to generate antibody at concentrations as low as 10-10 M. The effects were almost completely blocked by pretreatment with naloxone or naltrexone, opioid-specific antagonists. Only the K-agonist activity was abrogated by pretreatment with norbinaltorphimine, a K-specific antagonist. The stereospecificity of the K effect was demonstrated using isomers of U50,488H, with the (-) form possessing scantly greater immunomodulatory activity. Additional studies, using a p receptor-deficient mouse strain, demonstrated that only the K agonists were capable of suppressing antibody responses, whereas p- and K-selective agonists suppressed the parent p-responsive strain. Our results clearly indicate that pa and K opioid receptors are involved in regulation of lymphoid cell production of antibodies.

MATERIALS AND METHODS Mice. BALB/cAnSKH mice were obtained from the breeding facility at the Skin and Cancer Hospital, Temple University School of Medicine, Philadelphia. C57BL/6By and CxBK/By mice were purchased from The Jackson Laboratory. Mice received an i.p. injection of 0.2 ml of a 10%o suspension of sheep erythrocytes (SRBCs) and were used after 2 wk as a source of immune splenocytes. Reagents. The following drugs were used in this study: morphine sulfate (National Institute on Drug Abuse, Rockville, MD), DAMGE, and [D-Pen2'5]enkephalin (Peninsula Laboratories), racemic and isomeric forms of trans-3,4-

Opioids have been found by numerous laboratories to exert profound effects on immune function. Clinical evidence suggests that opiate addicts suffer increased susceptibility to a number of infections (1-3). In addition, phagocytic cell activity and the proliferation capacity of peripheral blood lymphocytes appear to be impaired in these individuals (4-8). The administration of opioid compounds to rodents results in increased susceptibility to bacterial and fungal infections (9). These animals also exhibit reduced natural killer cell activity and fail to manifest a normal delayed-type hypersensitivity skin test reaction (10-12). Recent studies have shown that mice implanted with morphine pellets exhibit reduced proliferative responses to Con A or endotoxin (13). We have also found that these mice are unable to generate normal levels of antibody (14). These findings, the description of opiate-like receptors on murine (15) and human (16) lymphocytes, and the discovery that lymphoid cells are able to produce immunoreactive f3-endorphin (17-19) support the hypothesis that regulatory pathways exist between the immune system and the central nervous system. Despite the findings cited above, little attention has been given to the opioid receptor type involved, and it has been assumed that the ,u receptor is the receptor of importance. Since the endogenous opioid system involves at least three receptor types, each mediating different physiological and biochemical processes and each responding differently to ligands from three families of precursor molecules, it is

dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyllbenzeneacetamide methanesulfonate [U50,488H], and (5a,7a,8f3)(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-(4,5)dec-8yl]benzeneacetamide [U69,593] (generously donated by

Upjohn), naloxone (Sigma), naltrexone (Sigma), and norbinaltorphimine [nor-BNI] (Research Biochemicals, Natick, MA) at concentrations between 10-12 and 10-5 M. The drugs were dissolved in minimal essential medium (MEM) prior to addition to splenocytes. Mishell-Dutton Cultures. Antibody-forming cells were generated in vitro using a Mishell-Dutton microculture system (20, 21) as modified previously (22). Briefly, SRBC-immune cells were cultured with various molar concentrations of morphine or U50,488H in flat-bottomed 96-well microtiter plates at a final cell density of 2 x 107 cells per ml in a tissue Abbreviations: SRBC, sheep erythrocyte; DAMGE, Tyr-D-Ala-GlyN-Me-Phe-Gly-ol; nor-BNI, norbinaltorphimine; PFC, plaqueforming cell. jTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 360

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Proc. Natl. Acad. Sci. USA 88 (1991)

culture medium (TCM) consisting of MEM supplemented with 1 mM nonessential amino acids, 1 mM sodium pyruvate, 50 ,ug of gentamicin per ml, 2 mM L-glutamine, 10%o fetal calf serum, and 0.05 mM 2-mercaptoethanol. Each well received 50 Atl of the cell suspension and 50 1ul of a 0.1% suspension of SRBCs in TCM. A culture represents a row of eight wells, and cells were pooled accordingly at the time of the plaqueforming cell (PFC) assay, 5 days after the initiation of the culture. Twenty-four hours after the initiation ofthe cultures, the cells were fed 0.05 ml of a nutritional cocktail consisting of MEM supplemented with a final concentration of 3 mM nonessential amino acids, 3 mM essential amino acids, 6 mg of dextrose per ml, 6 mM L-glutamine, 0.67% sodium bicarbonate, 33% fetal calf serum, and cytosine, adenosine, uridine, and guanosine at 41 Ag/ml. In designated experiments, immune splenocytes were pretreated with various concentrations of the opioid antagonist naloxone, naltrexone, or nor-BNI for 2 hr at 37°C prior to addition of the opioid agonist. Detection of Secondary in Vitro Anti-SRBC Response. The anti-SRBC PFC response in Mishell-Dutton cultures was determined by a hemolytic plaque assay (23). Triplicate determinations of the PFC responses were performed for each group and the mean value per group was determined. Only the direct (IgM) PFC response was measured. Results are expressed as PFC per culture and include the standard error of the mean for each group.

RESULTS Effect of j, and Receptor Agonists on the Generation of Antibody. In this study, graded molar concentrations of morphine, DAMGE, U50,488H, or U69,593 were added with antigen (SRBCs) to immune splenocytes at the initiation of a 5-day Mishell-Dutton culture. The results (Fig. 1) demonstrate that all of these opioid compounds are capable of inhibiting antibody production, with the K agonists being more effective than the ,u agonists. Morphine, when added at the initiation of the culture, suppressed the in vitro antibody response at concentrations as low as 10- M. DAMGE also inhibited antibody formation, but only at 10-6 M. The K agonist U50,488H suppressed antibody formation at concentrations as low as 10-10 M, whereas another K agonist, U69,593, was active at 10-8 M. The results presented here are representative of the results from several experiments. Typ0-0

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ical suppression for morphine at 1 ,M ranges from 30% to 60%. Typical suppression for U50,488H ranges from 75% to 90%. Additional experiments were carried out with the selective receptor agonist [D-Pen2'5]enkephalin (data not shown). This opioid agonist, at concentrations ranging from 10-12 to 1o-5 M, exhibited no detectable effect on the antibody response. The various drug treatments did not significantly alter cell viability at any of the concentrations tested (Table 1), since quite similar numbers of cells were recovered from these opiate-treated cultures and from untreated control cultures. These results suggest that the inhibitory effect of the opioid compounds cannot be attributed simply to cytotoxicity. Inhibition of Morphine- and U50,488H-Induced Immunosuppression by Opioid Antagonists. This experiment was designed to determine whether morphine and U50,488H mediate their inhibitory effects by way of opioid receptors. Immune splenocytes were pretreated with various molar concentrations of the opioid antagonist naloxone for 2 hr at 37°C. The agonists were then added to the cultures at concentrations that had previously demonstrated significant suppressive activity. The results show (Fig. 2) that the inhibitory effects of morphine and U50,488H were blocked by naloxone. Statistically significant (P < 0.05) effects were observed at concentrations of naloxone as low as 10-10 M. Cultures that received naloxone alone demonstrated essentially no inhibitory activity. Similar studies were performed using naltrexone, another opioid antagonist, and the results were similar to those observed with naloxone (data not shown). The involvement of K receptors was further demonstrated using pretreatment with nor-BNI, a highly selective K antagonist. The results in Fig. 3 demonstrate that nor-BNI blocked U50,488H-induced, but not morphine-induced, suppression of antibody formation. The antagonistic effect on U50,488H activity was detected in concentrations of nor-BNI as low as 10-9 M (P < 0.05), with complete antagonism at 10-8 M. nor-BNI alone exerted no inhibitory activity on the antiSRBC response. These studies demonstrate that the inhibitory activity mediated by the K agonist U50,488H occurs by way of K-Opioid receptors. Typical opioid receptors show a striking degree of stereospecificity for (-) isomers of their specific ligands. In an effort to examine the specificity of the interactions between these opioid compounds and the immune target cells, a pair of isomers of U50,488H was analyzed for suppressive activity. The results (data not shown) demonstrate that the (-) isomer is -100 times more suppressive than the (+) isomer. The studies described above indicate that immunoregulatory cells within a murine splenocyte population possess opioid-like receptors. To further explore the drug-cell interTable 1. Effect of opioid agonists on the viability and cell numbers of immune murine splenocytes after 5 days in culture Morphine U50,488H % viablet Cell no.* Conc., M Cell no.* % viablet 10-5 2.2 x 106 89 2.3 x 106 83 10-6 1.8 x 106 74 3.4 x 106 76 10-7 2.5 x 106 83 85 2.2 x 106 10-8 2.1 x 106 91 2.2 x 106 89 10-9 2.0 x 106 78 3.1 x 106 74 2.2 x 106 lo-lo 81 2.4x 106 86 2.8 x 106 lo-11 82 2.5 x 106 74 2.0 x 106 lo-12 72 2.0x 106 85 *Cell numbers were determined 5 days after the initiation of the Mishell-Dutton cultures. Cell numbers recovered from control cultures were 2.5 x 106 cells per ml with a viability of 81%. tViable cells were determined using a trypan blue exclusion analysis.

Neurobiology: Taub et al.

362

Proc. Natl. Acad. Sci. USA 88 (1991)

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action, experiments were carried out to determine whether opioid agonists could be suppressive if splenocytes were exposed to them only briefly at the initiation of culture. Accordingly, immune splenocytes were incubated with various concentrations of morphine or U50,488H at 370C for 2 hr. These agonist-treated cells were then extensively washed and placed in culture with antigen for 5 days. This short-term treatment of cells with either morphine or U50,488H resulted in significant inhibition of the antibody response (Fig. 4). The immunomodulatory effect was observed at concentrations as low as 10-8 M for U50,488H and 10-7 M for morphine and suggests that cells within a resting murine splenocyte population bear surface opioid-like receptors. Effect of p and K Agonists on Antibody Formation in It Receptor-Deficient Mice. The CxBK/By mouse strain, known to be deficient in ,u opioid receptors (24, 25), was used to

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FIG. 6. Inhibition of K agonist-mediated suppression of A receptor-deficient mice (Upper) or their normal parental strain (Lower) by nor-BNI. CxBk/By and C57BL/6By immune splenocytes were pretreated for 2 hr at 370C with various concentrations of nor-BNI. U69,593 (10-8 M) and U50,488H (10-8 M) were then added (day 0) to the 5-day Mishell-Dutton culture. The agonist controls of U69,453 (o)- and U50,488H (A)-treated splenocytes were cultured in the absence of nor-BNI. The control responses for the CxBk/By and C57BL/6By mice were 6480 348 and 3200 368 PFC per culture, respectively. ±

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further substantiated using the K-selective antagonist norBNI. The results given in Fig. 6 demonstrate that the inhibitory activity of U50,488H and U69,593 was blocked by nor-BNI as low as 10-8 M in the CxBK/By and C57BL/6By mice. In all experiments, nor-BNI alone was not inhibitory (data not shown).

DISCUSSION The existence of bidirectional communication between cells of the immune system and the nervous system has been' established by numerous laboratories. Recent studies indicate that immune cells recognize and respond to endogenous opioids by modulation of their cytotoxic activity, proliferative responses to various mitogens, interleukin release, immunoglobulin production, and surface antigen expression. We have shown here that and K agonists modulate the antibody response in vitro, whereas the 8 agonist does not. The immunomodulatory activity appears to occur through interactions with receptors on splenocytes that are similar to those present in the brain. This was demonstrated using agonist and antagonist competition studies in two different mouse strains and in mice with a selective defect in the pu opioid receptor. The activity of the various opioid agonists was blocked by naloxone and naltrexone, indicating the presence of classically defined opioid receptors. In addition, the activity of the K-selective (but not p.-selective) agonists was abrogated by nor-BNI, a K-specific antagonist. The agonist and antagonist competition studies suggest the following: (i) opioid receptors similar to those found on nervous tissue are present on murine splenocytes; (it) agonists and

363

K agonists mediate their effects by way of different receptors; (iii) binding of an agonist to the K opioid receptor, and to a lesser extent to the ,u receptor, on splenocyte populations results in an immunosuppressive event. Further evidence for the specificity of the interactions between opioids and immune cells was demonstrated using the CxBK/By mouse strain. Moskowitz and Goodman (24) and Reith et al. (26) show that this inbred strain is deficient in ,. opioid receptors, whereas the 8 and K opioid receptor populations are unaltered. They demonstrated a diminished response to morphine (24, 27), but the response to the 8 agonist [D-Pen2,L-Pen5]enkephalin remained unaltered (24, 25). This mouse strain provides an excellent opportunity to examine the effects of p. and K agonists on immune function. Our results with the CxBK/By and the parental C57BL/6By mouse strains indicate that the activities of p. agonists occur through classical ,p opioid receptors, since these compounds had little to no activity on immune cells from the p. opioid receptor-deficient strain. Additionally, both strains appeared to be equally responsive to the K-specific agonists, suggesting that the K receptor function is independent of the p. receptor function. The inhibitory effects of K opioid agonists on CxBK/By splenocytes were specifically abrogated by norBNI. These results, along with the pretreatment studies (Fig. 4), suggest that murine splenocytes bear classically defined opioid receptors and that this can mediate an immunomodulatory event following ligand binding. Of particular significance is the finding that the K opioid system is involved and that the expected stereospecificity exists. Although the K type of opioid receptor has been implicated in various central nervous system processes, including analgesia and thermoregulation (28, 29), the present report indicates its importance in immune function. Several laboratories (30-33) have recently demonstrated the existence of at least two K receptor subtypes present in brain tissue. In the present study, we have found that the inhibitory activity of K-selective agonists U50,488H and U69,593 was specifically abrogated by nor-BNI, suggesting that a receptor of the K1 subtype (30) is involved in these regulatory activities. However, the clear difference in the potency of U50,488H (K1 and K2 selective) and U69,593 (K1 selective) on antibody formation suggests the possibility that both (or multiple) K receptor subtypes may exist on lymphoid cells. The relationship of the K receptor system to the other opioid receptor systems and a study of the K receptor subtypes that are involved promise to be a fruitful avenue of investigation. The studies presented here provide further evidence for a link between the immune system and the central nervous system. Although the role of opioids in a normal immune response remains to be defined, the presence of functional receptors on these peripheral cells suggests a role for coordinated regulation of immune and neural function. Coordinated regulation is consistent with the observation that opiate drug abusers commonly suffer recurrent infections and appear to be immunocompromised (2, 3, 34). Further, the implications of these observations are interesting to consider in light of reports that lymphoid cells produce 8-endorphin (17-19). The receptors acted on by the exogenous opioids used in the present studies may be part of an as yet unrecognized autoregulatory pathway in the immune system mediated by the endogenous opioid peptides. We thank Mr. Gregory Harvey for excellent editorial assistance. This work was supported in part by Grants DA00376 from the National Institute on Drug Abuse and A123828 and A115613 from the National Institutes of Health. 1. Hussey, H. H. & Katz, S. (1950) Am. J. Med. 9, 186-193. 2. Louria, D. B., Hensle, T. & Rose, J. (1967) Ann. Intern. Med. 67, 1-22.

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