Immunosuppression by -Opioid Antagonist Naltrindole: - and Triple / / -Opioid Receptor Knockout Mice Reveal a Nonopioid Activity

The -opioid antagonist naltrindole has been shown to inhibit graft rejection in vivo and suppress allogeneic mixed lymphocyte reaction (MLR) in vitro, similarly to cyclosporin A. We investigated whether this action is mediated by -opioid receptors using both genetic and pharmacological tools. Naltrindole and two related compounds, 7-benzylidene-7-dehydronaltrexone and naltriben, inhibited MLR performed with lymphocytes from wild-type and -opioid receptor knockout mice, with comparable potency. Furthermore, these compounds suppressed the proliferation of spleen cells from triple / / -opioid receptor-deficient animals as well. Finally, the highly -selective, but structurally distinct, antagonist N,N-dimethyl-Dmt-Tic-OH and the general opioid antagonist naltrexone were inactive in the MLR assay. In conclusion, we demonstrate for the first time that the immunosuppressive activity of naltrindole and close derivatives is not mediated by any of the three cloned opioid receptors. Therefore, the postulated inhibitory activity of naltrindole in the graft rejection process is mediated by a target, which remains to be discovered. Morphine and related opiate agonists are first line medication for moderate-to-severe pain (Mather and Smith, 1999). Opiate compounds also exhibit many other pharmacological activities (Vaccarino and Kastin, 2000), including the alteration of immune responses and there is evidence that the endogenous opioid system plays an important role in regulating immunity (Eisenstein and Hilburger, 1998). Opiate drugs and endogenous opioid peptides elicit their biological actions through three highly homologous G protein-coupled receptors, -, -, and (for review, see Kieffer, 1997), which are differently implicated in opioid function. Highly interesting was the observation that the prototypic -opioid antagonist naltrindole (Portoghese et al., 1988) reduces graft rejection in vivo and inhibits allogeneic mixed lymphocyte reaction (MLR) in vitro (Arakawa et al., 1993). In the latter assay, naltrindole showed a potency comparable to that of cyclosporin A, considered to be the most efficient immunosuppressant in the graft rejection process and used clinically for more than two decades (Bush, 1999). This striking finding brought naltrindole as a potential immunosuppressive agent in organ transplantation and prompted further characterization of this activity. Because the existence of two -receptor subtypes ( 1 and 2) had been suggested by the pharmacology (Zaki et al., 1996), another study used naltrindole ( 1 and 2), 7-benzylidene-7-dehydronaltrexone HCl (BNTX, 1; Portoghese et al., 1992), and naltriben methanesulfonate (naltriben, 2; Sofuoglu et al., 1991). The results indicated that naltrindole and BNTX, but not naltriben, suppress several immune responses in vitro (House et al., 1995). In similar experiments, peptidic -antagonists proved little active (House et al., 1997). Another BNTX-related 1opioid receptor antagonist, 7-benzylspiroindanylnaltrexone, also prolonged renal allograft survival in the rat (Linner et al., 1998). Together, the data suggest that selective blockade of -receptors, mainly of the 1 subtype, by nonpeptidic opioid compounds could inhibit the graft rejection process. The purpose of this study was to clarify the molecular basis for naltrindole immunosuppressive activity in the MLR reaction, considered to be a well accepted in vitro model of T-lymphocyte response to allogeneic transplantation. In this assay, we have used novel genetic and pharmacological tools. We first have determined whether mice lacking the -opioid receptor (DOR) gene (Filliol et al., 2000), as well as mice lacking all three opioid receptor genes that we have generated (Kieffer, 1999; Simonin et al., 2001), respond to naltrindole, BNTX, and naltriben in the allogeneic MLR reaction. This work was funded by the Center National de la Recherché Scientifique, Association pour la Recherché sur le Cancer, The Foundation UPSA pour la Douleur. ABBREVIATIONS: MLR, mixed lymphocyte reaction; BNTX, 7-benzylidene-7-dehydronaltrexone; DOR, -opioid receptor; MOR, -opioid receptor; KOR, -opioid receptor; IL-2, interleukin-2. 0022-3565/01/2983-1193–1198$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 298, No. 3 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 3923/926400 JPET 298:1193–1198, 2001 Printed in U.S.A. 1193 at A PE T Jornals on N ovem er 4, 2017 jpet.asjournals.org D ow nladed from We also have examined the activity of a novel -selective antagonist, N,N-dimethyl-Dmt-Tic-OH, which displays a -selectivity far greater than naltrindole (Bryant et al., 1998; Lazarus et al., 1998). Finally, we have investigated the effect of naltrexone, a general opioid antagonist. Together, our results demonstrate that the immunosuppressive activity of naltrindole in the MLR reaction is not mediated by opioid