Chronic exposure of human macrophages in vitro to morphine and methadone induces a putative tolerant/dependent state.

We have shown previously that whereas acute exposure of cultured murine peritoneal macrophages inhibits phagocytosis, chronic exposure results in a putative tolerant/dependent state. We now report similar observations using human cultured monocyte-derived macrophages (hMDM) from a control population and from methadone patients. With hMDM, acute exposure to morphine and methadone inhibited phagocytosis in a dose-dependent manner. In contrast, chronic exposure resulted in eventual normalization of phagocytosis, indicating that a putative tolerant state to the opiates had developed. When opiates were withdrawn from chronically-exposed, tolerized hMDM, phagocytosis was once again depressed. The duration of withdrawal-induced depression lasted several hours, which is much longer than evidenced previously with murine macrophages. These data identify well with various in vivo studies on immune effects of opiate withdrawal; and, in so-doing, supplement ongoing speculation that opiate withdrawal is likely to have serious impact on host defenses of street heroin addicts.

Altered subcellular signaling in murine peritoneal macrophages upon chronic morphine exposure.

Alterations in opioid signaling that take place in murine peritoneal macrophages in vitro are variably dependent on opiate exposure conditions. Acute exposure to morphine inhibits Fc-mediated phagocytosis by a pertussis toxin (PT)-sensitive mechanism, but has no effect on cAMP levels. In contrast, chronic exposure to morphine results in a "tolerant" state, wherein test and control values for both phagocytosis and cAMP are equivalent. However, drug withdrawal after chronic exposure to morphine results in inhibition of phagocytosis and a concomitant 4-fold increase in cAMP by a PT-insensitive mechanism. This increase is causally related to inhibition of phagocytosis since an artificial increase in cAMP inhibits phagocytosis in non-withdrawn cells exposed chronically to morphine. We suggest that macrophage opioid receptors signaling switches from a Gi/o-mediated mechanism that does not involve adenylate cyclase in acute exposure to a non-Gi/o-mediated adenylate cyclase superactivation during chronic exposure.

Opioid activity of beta-endorphin-like proteins from Tetrahymena.

Morphine and other opioids have been reported to modulate phagocytosis in the ciliate Tetrahymena. However, the endogenous signaling molecule responsible for these effects remains uncharacterized. In this work we present evidence for the presence of beta-endorphin-like protein(s) in Tetrahymena thermophila. Subcellular extracts and cell-free culture supernatants were fractionated by hydrophobic chromatography on Sep Pack C18 columns and by affinity chromatography on polyclonal anti-beta-endorphin columns. Both preparations exhibited opioid-like effects in two different systems: 1) they inhibited phagocytosis in murine peritoneal macrophages, and 2) they blocked the response to mechanical stimuli in the ciliate Stentor. Both of these effects were reversed by naloxone, consistent with an opioid receptor-mediated mechanism. Chromatographic (HPLC) fractionation of the subcellular extracts resolved a component with beta-endorphin-like immunoreactivity, whose retention time was similar to that of the human beta-endorphin standard. Fractions were also analyzed by immunoblots using a monoclonal antibody that recognizes the N-terminus of human beta-endorphin. This antibody detected two antigenic components (corresponding to Mr 9,000 and Mr 12,000 polypeptides) in subcellular extracts, but only a single antigen (corresponding to a Mr 7,000 polypeptide) in culture supernatants. These results indicate that Tetrahymena produces one or more proteins that share some properties with beta-endorphin and that these may form part of an opioid mechanism that originated early in evolution.

Mu and delta receptors mediate morphine effects on phagocytosis by murine peritoneal macrophages.

Studies with selective opioid agonists show that mu- and delta(2)-opioid receptors, but not kappa, are involved in opioid inhibition of phagocytosis in elicited murine macrophages. All mu and delta(2) agonists tested had similar maximal effects on phagocytosis, and all dose-response curves suggest positive cooperativity. In addition, mu and delta antagonists antagonized the effect of both mu and delta agonists. Furthermore, in mu-opioid receptor knockout mice (MORKO), we observed a decrease in potency and maximal effect for a delta agonist. These data suggest that mu and delta receptors are not only involved in the modulation of phagocytosis in macrophages, but they also affect each other's activity by an unknown cooperative mechanism.