Inhibition by opioids of phagocytosis in peritoneal macrophages.

We have tested the effect of prototypic opioid agonists on phagocytosis of sheep erythrocytes by mouse peritoneal macrophages. It was found that morphine and all the opioid peptides tested inhibited phagocytosis by a biphasic, naloxone-reversible mechanism. Delta agonists were the most effective inhibitors, suggesting that the response is mediated by a delta receptor. Chronic exposure to morphine apparently results in the development of tolerance since under these conditions the inhibitory effect of the opiate is abolished. These results are similar to previously reported effects of opioids on endocytosis in other systems, which suggests that this inhibition is part of a basic regulatory mechanism that has been conserved in evolution.     

Myelin phagocytosis by peritoneal macrophages in organ cultures of mouse peripheral nerve. A new model for studying myelin phagocytosis in vitro

Organ cultures of degenerating nerve fascicles were exposed to cultured macrophages obtained by peritoneal lavage. Invasion of the nerve fascicle by phagocytes was shown by prelabeling with carbon and with electron microscopy. There was massive active phagocytosis of degenerating myelin sheaths. The invading phagocytic cell population was identified as Fc receptor-positive, Mac-1-positive macrophages by immunocytochemistry. The Schwann cell population persisted without significant myelin phagocytosis. The vitality of the Schwann cell population was shown by subsequent reimplantation of the organ cultures into host animals. The reimplants had retained their ability to remyelinate regenerating axon sprouts. These observations were made in cultures exposed to cytostatic agents. If cytostatic agents were omitted, there was proliferation of endogenous phagocytes in the nerve fascicles without added peritoneal cells. These endogenous phagocytes were identified as proliferating resident monocytes and were positive for the Fc receptor and Mac-1 markers. This model allows studies on how monocytes recognize and digest degenerating myelin apart from surviving Schwann cells.     

巴曲酶促进小鼠腹腔巨噬细胞吞噬过氧化低密度脂蛋白

采用小鼠腹腔巨噬细胞在含有过氧化低密度脂蛋白地培养液中培养，并用巴曲酶进行干预，运用透射电子显微镜进行观察，旨在了解巴曲酶对巨噬细胞天噬ｐｏｘ－ＬＤＬ的影响。研究结果表明：小鼠腹腔巨噬细胞与纯培养剂ＤＭＥＭ正常低密度脂蛋白，以及ｎ－ＬＤＬ加巴曲酶培养４小时后，细胞的超微结构与培养前相似，而与Ｐｏｘ－ＬＤＬ一起培养４小时后细胞浆内则含大量的脂滴，加入巴曲酶及小鼠腹腔巨噬细胞内脂滴的生成明显加快。     

Anti-macrophage CR3 antibody blocks myelin phagocytosis by macrophages in vitro

Summary Myelin phagocytosis in Wallerian degeneration of peripheral nerves depends on invasion of nerves by non-resident macrophages. The present study was done to clarify the role of the macrophage complement receptor type 3 (CR3) in myelin removal. Myelin phagocytic capacity of invading macrophages was abolished by treatment of cultured nerves and macrophages with anti-CR3 antibody or by serum complement depletion with cobra venom factor. This indicates that myelin phagocytosis is mediated by the macrophage CR3.Supported by grant 609/2-1 from the Deutsche Forschungsgemeinschaft     

Antinociceptive effect produced by intracerebroventricularly administered dynorphin A is potentiated by p-hydroxymercuribenzoate or phosphoramidon in the mouse formalin test

The antinociceptive effects of intracerebroventricularly (i.c.v.) administered dynorphin A, an endogenous agonist for κ-opioid receptors, in combination with various protease inhibitors were examined using the mouse formalin test in order to clarify the nature of the proteases involved in the degradation of dynorphin A in the mouse brain. When administered i.c.v. 15 min before the injection of 2% formalin solution into the dorsal surface of a hindpaw, 1–4 nmol dynorphin A produced a dose-dependent reduction of the nociceptive behavioral response consisting of licking and biting of the injected paw during both the first (0–5 min) and second (10–30 min) phases. When co-administered with p-hydroxymercuribenzoate (PHMB), a cysteine protease inhibitor, dynorphin A at the subthreshold dose of 0.5 nmol significantly produced an antinociceptive effect during the second phase. This effect was significantly antagonized by nor-binaltorphimine, a selective κ-opioid receptor antagonist, but not by naltrindole, a selective δ-opioid receptor antagonist. At the same dose of 0.5 nmol, dynorphin A in combination with phosphoramidon, an endopeptidase 24.11 inhibitor, produced a significant antinociceptive effect during both phases. The antinociceptive effect was significantly antagonized by naltrindole, but not by nor-binaltorphimine. Phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor, bestatin, a general aminopeptidase inhibitor, and captopril, an angiotensin-converting enzyme inhibitor, were all inactive. The degradation of dynorphin A by mouse brain extracts in vitro was significantly inhibited only by the cysteine protease inhibitors PHMB and N-ethylmaleimide, but not by PMSF, phosphoramidon, bestatin or captopril. The present results indicate that cysteine proteases as well as endopeptidase 24.11 are involved in two steps in the degradation of dynorphin A in the mouse brain, and that phosphoramidon inhibits the degradation of intermediary δ-opioid receptor active fragments enkephalins which are formed from dynorphin A.     

Intrathecal dynorphin A1–13 and dynorphin A3–13 reduce rat spinal cord blood flow by non-opioid mechanisms

Radiolabeled microspheres were used to examine the effects of paralytic intrathecal doses of dynorphin A (Dyn A_1–13) and Dyn A_3–13 on rat brain and spinal cord blood flows and cardiac output. Dyn A_1–13 produced significant dose-related reductions in blood flow to lumbosacral and thoracic spinal cord without altering cardiac output and blood flow to brain and cervical spinal cord. Naloxone failed to block these effects. Dyn A_3–13, which lacks opioid activity, also significantly reduced blood flow in lumbosacral spinal cord. Thus, the paralytic effects of Dyn A in the rat may involve reductions in spinal cord resulting from non-opioid actions of Dyn A.     

Macrophage phagocytosis of myelin in vitro determined by flow cytometry: phagocytosis is mediated by CR3 and induces production of tumor necrosis factor-α and nitric oxide

Demyelination of axons in the central nervous system (CNS) during multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE) is a result of phagocytosis and digestion by macrophages (M?) and the local release of inflammatory mediators like tumor necrosis factor-α (TNF-α) and nitric oxide (NO). We have investigated the process of myelin phagocytosis by M? in vitro using flow cytometric analysis. The binding and uptake of CNS-derived myelin was dose dependent, was abolished in the presence of EDTA and was enhanced after opsonization with complement. The phagocytosis of opsonized myelin could be inhibited by antibodies directed against complement receptor type 3 (CR3). Furthermore, CR3 also contributes to phagocytosis of non-opsonized myelin, e.g. under serum-free conditions. The phagocytosis of CNS-derived myelin induced the production of substantial amounts of TNF-α and NO by the M?. Our results indicate an important role for CR3 in myelin phagocytosis. The induction of TNF-α and NO which accompanies this phagocytosis may further contribute to the overall process of demyelination during MS or EAE.     

Adenosine modulation of dynorphin B release by hippocampal synaptosomes

A rat hippocampal preparation enriched in mossy fiber synaptosomes was employed in an attempt to expose any relationship between endogenous adenosine and the release of dynorphin B-like immunoreactivity (DynB-LI). Presumptive blockade of purinergic receptors increased the spontaneous release of DynB-LI, and reducing synaptic adenosine by exogenous adenosine deaminase increased the K(+)-evoked release. Evoked release of DynB-LI was reduced by inhibitors of adenosine uptake and 5'-nucleotidase. Taken together, these data suggest that adenosine endogenous to hippocampal mossy fiber synaptosomes serves to inhibit the release of one of the peptide neuromodulators of this preparation, and provide support for the concept of autoregulation of release.     

Regulation of the concentration of dynorphin A1–8 in the striatonigral pathway by the dopaminergic system

The purpose of this study was to explore the dopaminergic control of the striatonigral dynorphin system by measuring the levels of dynorphin A1-8-like immunoreactivity (DN-LI) after repeated injections of a dopaminergic receptor agonist or antagonist. Seven daily injections of different doses of apomorphine (0.5, 1.0, 2.5 and 5.0 mg/kg, s.c.) caused a significant dose-related increase of DN-LI in the striatum (26, 34, 63, 85% over control at each corresponding dose). Similar increases were observed in the substantia nigra (22, 52, 50 and 62% over control). In another experiment, rats received 5 mg/kg of apomorphine for 1, 3, and 7 days. There was a significant time-related increase in DN-LI both in the striatum (37, 50 and 85% over control at each corresponding period) and in the substantial nigra (32, 78 and 62%). Repeated administration of haloperidol (1 mg/kg, i.p.) failed to change the striatal level of DN-LI, but, when given at the same time as apomorphine, significantly attenuated the effect of apomorphine. These results suggest that dopamine exerts a modulatory influence on the metabolism of dynorphin in the striatonigral pathway.     

A 35 kDa Fos-related antigen is co-localized with substance P and dynorphin in striatal neurons

The rat striatum after dopamine denervation followed by repeated apomorphine treatment was examined for the co-expression of c-fos and Fos-related antigens with dynorphin, substance P and [Met5]enkephalin using Western blot and immunohistochemical techniques. Administration of apomorphine, a dopamine agonist, elevated the level of 35 kDa Fos-related antigen which co-localized with dynorphin and substance P, but not enkephalin, in striatal neurons.     

In vivo microdialysis study on changes in septal dynorphin and β-endorphin activities in active and hibernating Columbian ground squirrels

State-dependent changes in extracellular concentration of endogenous opioids in the septum of Columbian ground squirrels were examined in the hibernating and euthermic states using in vivo microdialysis. The order of estimated extracellular concentration was found to be: hibernating > interbout euthermia > non-hibernating euthermia for dynorphin A and interbout euthermia > hibernating > non-hibernating euthermia for β-endorphin. The apparent turnover rates of dynorphin A during hibernation was 15 times greater than that during euthermic non-hibernation phase and that of β-endorphin was 8-fold greater. These results demonstrate that subfamilies of endogenous opioids may vary differentially in their activities at different stages of an annual hibernation cycle and may reflect their different roles in the regulation of hibernation.     

A glutamate antagonist blocks perforant path stimulation-induced reduction of dynorphin peptide and prodynorphin mRNA levels in rat hippocampus

Stimulation of the perforant path elicits a behavioral response, wet dog shakes (WDS), and reduction in hippocampal dynorphin A(1-8) immunoreactivity (DYN-IR) and prodynorphin mRNA (DYN mRNA) in rats. This study examined whether glutamate, the proposed endogenous transmitter released by perforant fibers, mediated the above responses. A glutamate antagonist, gamma-D-glutamylglycine (DGG, 25 micrograms/0.5 microliters), or artificial cerebrospinal fluid (ACSF, 0.5 microliters) was injected into the ventral hippocampus 10-20 min prior to acute or daily stimulation of the left perforant path in rats. In acute stimulation experiments, 4 consecutive stimulation trials elicited a total of 73 +/- 4 WDS at an average threshold intensity of 0.46 +/- 0.03 mA in ACSF-treated rats. The hippocampal DYN-IR in these animals decreased by more than 40% in both dorsal and ventral hippocampus relative to sham-stimulated rats. DGG injections significantly elevated the threshold for WDS (0.78 +/- 0.05 mA, P less than 0.01), reduced the number of WDS (45 +/- 6, P less than 0.01), and partially antagonized stimulation-induced reduction of DYN-IR in the ventral, but not dorsal, hippocampus. In daily stimulation experiments, rats received a single trial of stimulation once per day for 6 days. Daily DGG pretreatment almost completely abolished WDS at control threshold intensities, and significantly inhibited stimulation-induced decrease of DYN-IR in both dorsal and ventral hippocampus. In situ hybridization using a 35S-labeled oligodeoxyribonucleotide probe demonstrated a clear depletion of DYN mRNA signal in the dentate granule cell layer of ACSF-treated animals. This depletion was completely prevented in DGG-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased dynorphin A (1-17) in the spinal cord of spastic rats after the compressive injury

Spasticity in rat hindlimbs was induced by compressing cervical spinal cord with a wax ball. Ashworth score and H-reflex were measured 1 week after the surgery. The results showed that: (1) muscle spasm was detected in the hindlimbs a week after the operation and maintained at least 8 weeks, (2) in the spastic animals, dynorphin A (1-17)-ir decreased significantly in thoracic and lumbar segments of the spinal cord and (3) peripheral administration of kappa receptor agonist U50488H and electrical stimulation at 100 Hz effectively relieved the muscle spasm. Our data supported the note that the reduction of endogenous dynorphin A (1-17) might play an important role in the pathogenesis of spinally induced muscle spasticity and the replenishment of its shortage might relieve the spasticity.     

Morphine alters macrophage and lymphocyte populations in the spleen and peritoneal cavity

We have previously shown that subcutaneous implantation of a 75 mg morphine pellet results in suppression of the ability of murine splenocytes to mount an antibody response to sheep red blood cells, due in part to a reduction of macrophage function. The present studies used flow cytometry to examine whether the decrement in macrophage function in the spleens of morphine-treated mice results from a reduction in macrophage numbers. Parallel analysis was carried out on non-elicited peritoneal cells. In the spleen, morphine resulted in a reduction in the relative proportion of macrophages and B-cells, with a concomitant increase in the proportion of T-cells. Alteration in the ratio of CD4⁺ to CD8⁺ T-cells was not observed. In contrast, in the peritoneal cavity, morphine increased the number of macrophages and reduced the number of B-cells. Naltrexone blocked all of the changes in cellular composition. These results support the conclusion that an important mechanism in the immunosuppression seen in the spleens of mice implanted with morphine pellets is a differential reduction in the number of macrophages and B-cells as compared with T-cells. Further, these studies show that subsets of cells of the immune system are differentially affected by morphine in different anatomical compartments.     

Binding of α-adrenergic receptors stimulates the anti-mycobacterial activity of murine peritoneal macrophages

The effects of adrenergic stimulation of the anti-mycobacterial activity of peritoneal macrophages was investigated. We found that epinephrine and norepinephrine stimulated macrophages to suppress the growth of Mycobacterium avium. Stimulation was mediated by binding to the α2 adrenergic receptor. The addition of the α2 agonist clonidine to cultures resulted in an inhibition of mycobacterial growth and the effect of epinephrine was blocked by the α-antagonist phentolamine. Treatment of the macrophages with propranolol, a β-antagonist, potentiated the effect of epinephrine. Epinephrine mediates its effect by stimulating the expression of macrophage activation genes.     

Analgesic actions of dynorphin A(1–13) antiserum in the rat brain stem

This study was performed to evaluate the effects of dynorphin A(1–13) antiserum when microinjected into an active hyperalgesic region within the rat brain stem. When administered within the dorsal posterior mesencephalic tegmentum (DPMT) of intact conscious rats, dynorphin A(1–13) antiserum produced rapid onset and persistent prolongation of a low intensity thermally evoked tail avoidance response (LITETAR). These analgesic actions of the dynorphin A(1–13) antiserum appeared to be dose dependent. These studies support previous hypotheses about the existence of tonically active brain stem opioid hyperalgesic processes. Further, the results provide indirect evidence for a potential role of brain stem dynorphin(s) in facilitating pain.     

Effect of macrophage activators on the phagocytosis of mycobacteria by Schwann cells

Concanavalin A-induced lymphokines decreased the phagocytosis of 14C-acetate-labelled Mycobacterium w by 33B rat Schwannoma cells while increasing the phagocytosis by macrophages. This suggests that Schwann cells are responsive to lymphokines. Phorbol myristate acetate decreased the phagocytosis in Schwannoma cells but increased that in macrophages. Schwann cells, thus, respond to stimulants of macrophage phagocytosis in a manner opposite to that of macrophages.     

Opiate modulation of dynorphin conversion in primary cultures of rat cerebral cortex

Rat brain cortical cells in primary culture were used to investigate long-term effects of opiates on endopeptidases acting on dynorphin peptides. Enzyme activity in the soluble fraction of the cells converted dynorphin B to Leu-enkephalin-Arg⁶ and to a lesser extent to Leu-enkephalin. Five day treatment with 10 μM morphine increased the conversion to Leu-enkephalin-Arg⁶ by 370%. This effect was prevented by the presence of naloxone in the culture medium. The opiate-inducible activity was directed to the Arg-Arg bond in dynorphins with preference for dynorphin B>-neoendorphin>>dynorphin A. The K_m for the generation of Leu-enkephalin-Arg⁶ from dynorphin B was 40 μM. Enzyme activity was inhibited by dynorphin fragments, in the following order of potency: dynorphin A(1–13)>A(2–13)>A(1–17)>A(2–17) and by SH-reagents, suggesting the presence of a cysteine-protease. The opiate-stimulated dynorphin-converting enzyme (DCE)-activity affects the balance between dynorphin peptides (selective for κ-opioid receptors) and enkephalin peptides (selective for δ-opioid receptors). Since both types of opioid peptides can influence the development of opiate tolerance, the change in the extent of this transformation may be functionally important.     

Dynamics of actin filaments in microglia during Fc receptor-mediated phagocytosis

The phagocytic ability of mouse microglia during their differentiation in culture and after stimulation with bacterial wall lipopolysaccharide (LPS) has been investigated using Fc receptor-mediated phagocytosis of immunoglobulin (IgG)-coated sheep erythrocytes (SRBCs). We observed that in 10–14 day-confluent neopallial cell cultures some immature microglia are not phagocytic. LPS-stimulated microglia are able to phagocytose larger numbers of IgG-coated SRBCs and at a faster rate than non-stimulated microglia. Within 5–10 min of phagocytosis the actin filaments of the LPS-stimulated microglia become depolymerized, leaving only bundles of actin filaments around the phagocytosed SRBCs (phagosome cups). At 30 min after the start of phagocytosis the actin filaments of the LPS-stimulated microglia begin to polymerize, and within 2 h the original pre-phagocytosis pattern of the actin filament network is re-established. The non-LPS-stimulated microglia exhibit actin filament depolymerization in only a few lamellipodia and polymerization of actin filaments around engulfed particles, but much later during phagocytosis.Supported by Medical Research Council of Canada Grant MT4235 to S. F. and a Centres of Excellence fellowship to E. M.     

Dynorphin B and spinal analgesia: induction of antinociception by the cannabinoids CP55,940, Δ-THC and anandamide

The endogenous opioid dynorphin B was evaluated for its role in cannabinoid-induced antinociception. Previous work in our laboratory has shown that the synthetic, bicyclic cannabinoid, CP55,940, induces the release of dynorphin B whilst the naturally occurring cannabinoid, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), releases dynorphin A. The dynorphins contribute in part to the antinociceptive effects of both cannabinoids at the level of the spinal cord. The present study compares dynorphin B released from perfused rat spinal cord in response to acute administration of anandamide (AEA), Δ⁹-THC and CP55,940 at two time points, 10 min and 30 min post administration, and attempts to correlate such release with antinociceptive effects of the drugs. Dynorphin B was collected from spinal perfusates of rats pretreated with Δ⁹-THC, CP55,940 or AEA. The supernatant was lyophilized and the concentrations of dynorphin B were measured via radioimmunoassay. At a peak time of antinociception (10 min), CP55,940 and Δ⁹-THC induced significant two-fold increases in the release of dynorphin B. AEA did not significantly release dynorphin B. Upon a 30-min pretreatment with the drugs, no significant dynorphin B release was observed, although antinociceptive effects persisted for CP55,940 and Δ⁹-THC. Previous work indicates that Δ⁹-THC releases dynorphin A while AEA releases no dynorphin A. This study confirms that although all three test drugs produced significant antinociception at 10 min, the endocannabinoid, AEA, does not induce antinociception via dynorphin release. Thus, our data indicate a distinct mechanism which underlies AEA-induced antinociception.