Differential effects of opioids on the proliferation of a macrophage cell line, Bac 1.2F5.

Chronic treatment of mice with morphine selectively abrogates the terminal differentiation of committed bone marrow progenitor cells to form macrophage colony forming units. To understand the molecular mechanisms involved in morphine-mediated suppression of myeloid cell differentiation, we investigated the use of a macrophage cell line, Bac 1.2 F5. In vitro proliferation of this cell line is dependent on the exogenous supply of macrophage colony stimulating factor. Treatment of Bac 1.2F5 cells in vitro with morphine showed a dose-dependent inhibition of proliferation which was associated with morphological changes. Characterization of the binding site revealed that the binding site for morphine on these cells is different from the classical opioid receptors described in the brain. In addition to the putative novel class of morphine receptors, Bac 1.2F5 cells also expressed the delta opioid receptors as determined by RT-PCR analyses. These studies show that Bac 1.2F5 cells are suitable for the molecular characterization of opioid effects on the proliferation and differentiation of myeloid progenitor cells.

Expression cloning of a full-length cDNA encoding delta opioid receptor from mouse thymocytes.

A delta opioid receptor complementary DNA (cDNA) was cloned by expression of cDNA library from activated thymocytes in Cos 7 cells. The deduced amino acid sequence of this receptor was similar to that described in the brain. As analyzed by southern blot hybridization, the delta opioid receptor transcripts are constitutively expressed in unactivated thymocytes. In addition, neither kappa nor mu opioid receptor transcripts were detected in thymocytes, suggesting tissue-specific opioid receptor gene expression in the immune system. The studies represent the first report of a full-length opioid receptor in the immune system.

Activation of rat thymocytes selectively upregulates the expression of somatostatin receptor subtype-1.

Somatostatin and other neuropeptides are known to modulate the proliferative capacity of immune cells. In the present study, we investigated the expression of Somatostatin receptor (SSTR) subtypes on rat thymocytes. RT-PCR analysis of fresh thymocytes showed significant levels of transcripts for the SSTR2 whereas transcripts for the SSTR1 and SSTR3 were not detectable. Interestingly, when the thymocytes were activated with low concentration of Phytohemagglutinin and interleukin 1, the transcript for SSTR1 was markedly increased. Lymphokine induced activation of thymocytes selectively upregulated the SSTR1 since, transcripts for SSTR2 remained the same after activation and SSTR3 was not detectable. PCR amplified fragment of SSTR1 from the activated thymocytes showed identical sequence to the rat brain receptor. The physiological significance of the increase of SSTR1 mRNA in thymocytes after activation remains to be elucidated but it may be possible that these two different subsets of receptors (SSTR1 and SSTR2) are involved in the modulation of thymocyte proliferation and differentiation.

Immunostimulating lipopeptide, LtriP (RP 56142): comparison of the effect on hepatic cytochrome P 450 modulation and radioprotection in male and female of three mouse strains.

The sex-dependent effect of lauroyl-L-Ala-D-gamma-Glu-L,L-A2pmNH2 (LtriP, RP 56142) on hepatic microsomal cytochromes P 450 (cyt P 450) was studied in three mouse strains NMRI, C3H/OuJ and C3H/HeJ. In NMRI and C3H/OuJ, strains which are responsive to bacterial lipopolysaccharides (LPS-responsive), regardless of the sex of the mouse, significant decrease in the amount of cyt P 450 was observed after LtriP treatment, with a concomitant reduction in ethoxyresorufin-O-deethylase (cyt P 450 1A-dependent) and 7-ethoxycoumarin-O-deethylase activities. This was not seen in C3H/HeJ (LPS-hyporesponsive) mice. These effects may be related to LtriP-dependent cytokine induction, since neither LtriP nor LPS stimulated interleukin-1 (IL-1) secretion by C3H/HeJ macrophages. 11- and 12-hydroxylations (11- and 12-OH) of lauric acid were compared in C3H/OuJ and C3H/HeJ mice. LtriP depressed the total enzymatic conversion of lauric acid in the two strains without modification of the 11/12-OH ratio for C3H/OuJ or male C3H/HeJ mice. However, in females C3H/HeJ mice this decrease was particularly significant and concerned especially the 12-OH activity (a marker of cyt P450 4A family). Although males of the three strains were more sensitive to irradiation than females, LtriP exerted a sex-independent radioprotection on NMRI and C3H/OuJ mice. Its radioprotective effect was illustrated by the preservation of all the enzymatic activities studied in treated NMRI mice, contrary to irradiated control animals. In contrast, for the C3H/HeJ strain, males were not protected by LtriP treatment and, furthermore, females showed a marked sensitization to irradiation. The effects in CH3/HeJ strain implicate LtriP in the control of cyt P 450 induction and of sensitivity to irradiation independently of IL-1 induction.

Complementary DNA cloning of a mu-opioid receptor from rat peritoneal macrophages.

Treatment with opioid agonists in vitro and in vivo has been shown to affect the function of the immune system. Several investigators have suggested that immune cells may express opioid receptors, but it had been very difficult to demonstrate their presence on these cells by direct binding assays. Our earlier studies have shown that macrophage progenitor cells are highly sensitive to morphine treatment in vitro and in vivo. In the current investigation, we determined, unequivocally, the expression of mu-opioid receptor related transcripts in rat peritoneal macrophages by reverse transcriptase-polymerase chain reaction (RT-PCR) studies. In order to further characterize the transcript, the RT-PCR product was cloned and sequenced. The sequence analyses indicate that the transcripts from rat peritoneal macrophages are identical to those for the mu-opioid receptor described in the rat brain. To further confirm the presence of mu-opioid receptors, immunoreactivity to an antiserum raised against the carboxyl terminal fifteen amino acid residues of the mu-opioid receptor was determined. These studies show for the first time that rat peritoneal macrophages express a mu-opioid receptor.

Radioprotective effects of the immunostimulating lauroylpeptide LtriP (RP 56142).

The lipopeptide lauroyl-L-Ala-gamma-D-Glu-L,L-A2pm (LtriP) increased the resistance of mice to the lethal effect of gamma-ray irradiation. The radioprotective effect was dependent on the doses of LtriP and of radiation. Maximum survival was observed when the lipopeptide was injected on two successive days before irradiation. This activity seems to be related to immunostimulating functions, since the non-immunostimulating analog lauroyl-L-Ala-gamma-D-Glu-D,D-A2pm-Gly, containing D,D-diaminopimelic acid, was not radioprotective. The protective activity might result from an induction of cytokines, such as IL-1, TNF and M-CSF, since LtriP induced the mRNA expression and the secretion of these immunomodulators.