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Effect of morphine on Fc-mediated phagocytosis by murine macrophages in vitro
Journal of Neuroimmunology 74 Ž1997. 111–116
Effect of morphine on Fc-mediated phagocytosis by murine macrophages in vitro Enid Z. Tomei a , Fernando L. Renaud a
b,)
Department of Psychiatry, Emory UniÕersity School of Medicine, G.M.H.I. 1256 Briarcliff Rd., N.E., Atlanta, GA 30306, USA b Biology Department, UniÕersity of Puerto Rico, P.O. Box 23360, San Juan 00931-3360, Puerto Rico Received 19 June 1996; revised 21 October 1996; accepted 22 October 1996
Abstract Acute exposure to morphine has been shown to inhibit phagocytosis in murine macrophages, whereas chronic exposure results in apparent desensitization. We now show that morphine may be either inhibitory or stimulatory depending on concentration and exposure time. Furthermore, under some conditions drug withdrawal from putatively desensitized cells will result in inhibition of phagocytosis, suggesting that a state akin to dependence has developed. Desensitization can also develop with intermittent exposures if the opiate-free period between drug exposures is shorter than 4 h. These effects of morphine on macrophages are important in understanding the role of this drug as an immunomodulatory agent. Keywords: Morphine; Murine macrophage; Desensitization; Phagocytosis
1. Introduction There is much recent evidence to show that the nervous and immune systems interact ŽBlalock, 1994; Gilmore et al., 1990.. It is also known that both endogenous opioids and exogenous opiates may have important modulatory effects on cells of the immune system such as T-cells and macrophages ŽTubaro et al., 1983; Donahoe et al., 1985; Fischer, 1988; Peterson et al., 1990, 1993; Rouveix, 1992.. These findings suggest that opiate abuse may be a factor in susceptibiliy to infections such as HIV-1 observed in drug addicts ŽDonahoe, 1992.. However, it is perplexing that this apparent drug-induced susceptibility seems to be less frequent when opiates are administered in a clinical setting. This may be due to the differences in dosage and exposure intervals between a clinical setting and the situation in a drug addict. Morphine and some endogenous opioids have been shown to affect processes in macrophages such as oxidative burst, chemotaxis and phagocytosis ŽPeterson et al., 1987; Perez-Castrillon ´ ´ et al., 1992; Foris ´ et al., 1986; Rojavin et al., 1993; Tubaro et al., 1987.. Work performed previously in our laboratory showed that opioids inhibited )
Corresponding author. Tel.: q1-787-764-0000, Ext. 2037; fax: q1787-7643875; e-mail: [email protected]
phagocytosis in murine peritoneal macrophages in vitro by a naloxone-reversible mechanism ŽCasellas et al., 1991.. Furthermore, chronic exposure to morphine resulted in the development of a state akin to desensitization in these cells, since after exposure for 16 h the drug no longer had any inhibitory effect on phagocytosis ŽCasellas et al., 1991.. Nevertheless, the cellular basis for this observation still needs to be established. Desensitization in nerve cells has been postulated to involve both receptor ŽWang et al., 1994. and post-receptor mechanisms ŽNestler, 1992., but the involvement of these mechanisms in opioid effects on macrophages has not been studied. In this work we show that the effects of morphine on macrophages depend to an important extent on the particular conditions of exposure and drug concentration. Such parameters therefore are important aspects of studies concerning the effect of morphine and other opioids on the immune system. 2. Materials and methods 2.1. Induction and isolation of murine peritoneal macrophages Four to eight week old female C3HeBrFeJ mice ŽJackson Labs., Bar Harbor, ME. were utilized in this study.
0165-5728r97r$17.00 Copyright q 1997 Published by Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 6 . 0 0 2 1 3 - 5
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Animals were injected i.p. with 2 cc of 3% Brewer’s Thioglycollate medium ŽDifco, Detroit, MI.. The mice were euthanized by cervical dislocation five days following the injection, and the peritoneal-elicited cells were recovered by injecting 10 cc of sterile phosphate buffered saline ŽPBS. into the peritoneal cavity. The peritoneum was then gently massaged 2 min and cell exudate aspirated with a syringe. The exudate was transferred to a sterile 50 cc conical centrifuge tube, sterile PBS was added to reach a final volume of 30 cc, and the cell suspension was washed by centrifuging three times at room temperature at 250 g for 10 min. The cells were then resuspended in sterile RPMI 1640 medium supplemented with 10% heatinactivated fetal bovine serum ŽHyClone, UT. and 1% antibioticrantimycotic solution ŽSigma, St. Louis, MO.. This medium will be referred to as complete RPMI. Macrophages were counted in a hemocytometer and cell viability was determined by trypan blue exclusion Ž0.4% trypan blue.. Only cell suspensions with a viability greater than 90% were used in these studies. The cell concentration was adjusted to 5 = 10 3 macrophages mly1 , and 400 m l of this cell suspension were plated per well in 8-well Lab-Tek w culture plates ŽMiles Laboratories, Naperville, IL.. The cultures were incubated overnight at 378C under 5% CO 2 . 2.2. Opsonization of sheep red blood cells Sheep red blood cells ŽSRBC. were purchased from Colorado Serum, CO. An aliquot of SRBC was washed twice in sterile PBS, and after the last wash the pellet was resuspended in complete RPMI. The SRBC were counted in a hemocytometer, and the total number of cells needed were transferred to a sterile centrifuge tube. The aliquot was spun at 250 g for 10 min and the cell pellet was resuspended in 1 cc of the rabbit IgG anti-SRBC ŽDiamedic Cordis Laboratories, FL. at 1:500 to 1:1000 final dilution in complete RPMI. The SRBC were incubated with the immunoglobulin in a shaking water bath for 15 min at 308C. SRBC were then washed once and kept on ice until needed. 2.3. Phagocytosis assay The cultured adherent macrophages were washed twice with complete RPMI to remove non-adherent cells. Cells were incubated with opsonized SRBC at a ratio of 1 macrophage:100 SRBC. To optimize contact between the two cell populations, the Lab-Tek w chambers were carefully placed on top of cold centrifuge carriers and gently spun at 10 g for 2 min at 48C. Phagocytosis was started by transferring slides immediately to a 378C incubator. The process was stopped after 20 min by placing the slides on ice, and non-phagocytosed SRBC were lysed by hypotonic shock ŽBobak et al., 1987.. The culture slides were washed twice with PBS to remove cell debris, and macrophages
were fixed with 1% glutaraldehyde for 5 min at room temperature. Fixed cells were stained with Dif-Quick w and phagocytosis was determined by light microscopy. Direct visual discrimination of ingested erythrocytes was possible since these stain pale red with the Wright stain, while the macrophage’s cytoplasm stains blue with the Giemsa stain. One hundred macrophages were scored per well and each experimental condition was performed in triplicate replicas, each replica being treated and processed independently. The results were expressed in terms of the phagocytic factor: phagocytic factor % phagocytosis in the presence of agonist s
percent phagocytosis of control cells
= 100
where % phagocytosis is defined as the percentage of cells ingesting at least one SRBC. To compare data from different experiments, the results were standardized by giving a value of 100% to the mean of the % phagocytosis observed in control cells, and the experimental values were adjusted accordingly. This was necessary to be able to perform a statistical comparison among data obtained from different experiments, because it was found that the basal level of phagocytosis Ž% phagocytosis of opsonized SRBC by control cells. varied from experiment to experiment, with individual experiments ranging between 40 and 93% Žmean " standard deviations 67 " 17.. However, the results were remarkably consistent within each experiment, as evidenced by the low standard errors obtained for control cells. Analysis of the samples was done using the Student’s t-test. A difference was considered significant when p - 0.05. 2.4. Effect of acute, chronic and intermittent exposures to morphine on FcR-mediated phagocytosis To assess the effect of acute exposure to morphine sulfate ŽSigma, St. Louis, MO. macrophages were washed and incubated for 30 min at 378C in 400 m l complete RPMI, or in complete RPMI supplemented with morphine concentrations ranging from 1 nM to 1 m M; some experiments were also carried out in the presence of equimolar concentrations of naloxone in addition to the opiate. To study the effect of chronic exposure the cells were incubated overnight Ž17 h. at 378C with 1 nM to 1 m M morphine. To assess the effect of intermittent exposures, incubation periods of 2–5 h with 100 nM morphine were alternated with periods of drug withdrawal of 2–5 h in various combinations. Three washes with RPMI were performed prior to the withdrawal periods to remove the opioid from the cell milieu. Following acute, chronic or intermittent incubation with the opiate agonist, opsonized SRBC were added, still in the presence of the opiate, and phagocytosis analysed as described above.
E.Z. Tomei, F.L. Renaudr Journal of Neuroimmunology 74 (1997) 111–116
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3. Results 3.1. Effect of acute exposure to different morphine concentrations on FcR-mediated phagocytosis of SRBC The effect of incubating macrophages for 30 min with various concentrations of morphine prior to the phagocytosis assay is illustrated in Fig. 1. It is characterized by a biphasic response, with significant Ž p - 0.01. inhibition of phagocytosis by 50–100 nM morphine. As reported previously ŽCasellas et al., 1991. this inhibition was naloxonereversible Ždata not shown.. 3.2. Effect of different incubation times with morphine on Fc-mediated phagocytosis To assess the temporal aspects of the ability of morphine to modulate phagocytosis, macrophages were incubated with 100 nM morphine for various times prior to the phagocytosis assay. The results show that the phagocytic effects of morphine depend strongly on exposure time ŽFig. 2.. Up to 5 h incubation resulted in inhibition of phagocytosis Ž p - 0.001., whereas an incubation of between 6 and 8 h resulted in no effect on phagocytosis when compared to control cells Ž p - 0.3.. However, 9 to 10 h of continuous exposure to the opiate results in a moderate, but significant Ž p - 0.05., stimulation of phagocytosis in the presence of the drug.
Fig. 2. Effect of morphine pre-incubation time on phagocytosis of SRBC by murine macrophages. Cells were pre-incubated with 100 nM morphine for periods ranging from 0.5 to 10 h Žfilled circles.; following this, cells were incubated with opsonized SRBC and allowed to phagocytize for 20 min, while still in the presence of the opiate. Control cellss open circles. ) p- 0.01; ) ) p- 0.03; ) ) ) p- 0.02; ns6 Žtwo independent experiments with each variable in triplicate..
3.3. Effect of chronic exposure to different morphine concentrations on Fc-mediated phagocytosis A state akin to desensitization was induced in macrophages exposed for 16 h to 50 nM morphine, since phagocytosis in these cells is no longer inhibited by the opiate ŽCasellas et al., 1991.. Therefore we performed a dose–response study to determine if this effect was concentration-dependent. The results illustrated in Fig. 1 illustrate a biphasic response to chronic morphine: after a 17 h exposure to the opiate, 50 nM and 100 nM concentrations actually caused a modest but significant stimulation of phagocytosis Ž p - 0.05.; whereas lower and higher concentrations had no significant effect, except for 1 m M, which was inhibitory Ž p - 0.05.. The latter finding is in contrast to data from our acute-exposure study ŽFig. 1. wherein 1 m M morphine had no effect on phagocytosis. 3.4. Effect of morphine withdrawal from putatiÕely desensitized cells on Fc-mediated phagocytosis
Fig. 1. Effect of acute Žopen circles. and chronic Žfilled circles. exposure to different concentrations of morphine on phagocytosis of sheep red blood cells ŽSRBC. by murine peritoneal macrophages. Percent phagocytosis is standardized relative to the control value which is assumed to be 100%. In this and in all following figures data are expressed in terms of the phagocytic factor Žsee text for definition. that standardizes results with respect to control values. Each point or bar represents the mean" standard error; the dashed line represents the control level Žopen squares. of phagocytosis. ) p- 0.01, ns9 Žthree independent experiments with each variable in triplicate.; ) ) p- 0.05, ns6 Žtwo independent experiments with each variable in triplicate..
A study was performed to determine if morphine withdrawal had any additional effects on the phagocytic capacity of macrophages that had already demonstrated a diminished capacity to respond to morphine. Macrophages were exposed to morphine for 8 h, and the opiate was withdrawn from the cell milieu 5 h, 1 h, or immediately prior to the phagocytosis assay. To ascertain that the macrophages had been deprived of the opiate, cells were washed three times with RPMI and incubated in complete RPMI at 378C for the deprivation periods indicated above.
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3.5. Effect of intermittent exposure to morphine on phagocytosis
Fig. 3. Effect of morphine withdrawal on phagocytosis of SRBC by murine macrophages. Cells were exposed to 100 nM morphine for 8 h at 378C, and morphine was withdrawn from the medium for the indicated times prior to assaying phagocytosis of opsonized SRBC. Phagocytosis by morphine-deprived macrophages Žhatched bars. was then compared to phagocytosis by macrophages that were not depleted of the opiate Žconstant morphine controls, open bars., and to phagocytosis by opiate-naive controls Žfilled bars.. ) p- 0.01, ns9 Žthree independent experiments with each variable in triplicate..
The phagocytosis assay was carried out as described previously, by addition of opsonized SRBC and incubation for 20 min at 378C, but in the absence of morphine. The results of this study are illustrated in Fig. 3. As expected from the data in Fig. 2, macrophages exposed for 8 h to 100 nM morphine were no longer inhibited by the opiate. However, when they were deprived of morphine for 5 h ŽFig. 3. these macrophages exhibited a significant decrease in phagocytosis Ž p - 0.05. when compared to control macrophages, or to non-deprived macrophages. However, withdrawal of morphine for 1 h, or immediately prior to the assay, did not significantly affect phagocytosis in these cells ŽFig. 3..
This series of experiments was designed to determine if phagocytosis by macrophages exposed intermittently to 100 nM morphine would still be inhibited by the drug, as in acute exposure, or whether intermittent exposure would result in a lack of effect or in a stimulation of phagocytosis, as in chronic exposure ŽFigs. 1 and 2.. When macrophages were depleted of morphine for 2 h intervals in a 10 h assay Žthree 2 h exposure periods separated by two 2 h depletion periods., phagocytosis was not affected by the drug ŽFig. 4A.; similar results were obtained when depletion periods of 3 h were tested Žtwo 3 h exposure periods separated by a three hour depletion period; data not shown.. However, when intervals of 4 h were examined Žtwo 4 h exposure periods separated by a 4 h withdrawal period; Fig. 4B., a significant inhibitory effect of morphine on phagocytosis was observed Ž p - 0.01., when compared to control cells exposed continually for 12 h to 100 nM morphine. The basal level of phagocytosis was not affected by the washes involved in these experiments, since the % phagocytosis of opiate-naive control cells ranged between 47 and 93%, well within the values expected for a typical experiment. We then decided to test longer exposure periods alternated with 4 h withdrawals. Cells were exposed to 100 nM morphine for a 5 h interval, deprived for 4 h, and then re-exposed to 100 nM morphine for 5 h prior to the phagocytosis assay. Under these conditions morphine still inhibited phagocytosis when compared to control cells exposed continuously to the opiate for 14 h ŽFig. 4C; p - 0.01..
4. Discussion
Fig. 4. Effect of intermittent exposures to 100 nM morphine on phagocytosis of SRBC by murine macrophages. Macrophages were incubated for alternated intervals in the presence Žq. and absence Žy. of 100 nM morphine, for a total incubation time of 10 to 14 h ŽA–C. at 378C. After the last exposure to morphine, and while still in the presence of the opiate, opsonized SRBC were added to macrophages and phagocytosis was allowed to proceed for 20 min at 378C. The phagocytic factor of macrophages exposed to morphine for these various intervals Žopen bars. was compared to the phagocytosis of control cells exposed continuously to the opiate Žfilled bars.. ŽA. 2 h Žq., 2 h Žy., 2 h Žq., 2 h Žy., 2 h Žq.; control exposed for 10 h. ŽB. 4 h Žq., 4 h Žy., 4 h Žq.; control exposed for 12 h. ŽC. 5 h Žq., 4 h Žy., 5 h Žq.; control exposed for 14 h. ) p- 0.01, ns6 Žtwo independent experiments with each variable in triplicate..
The results obtained in this work confirm previous findings that opioids inhibit phagocytosis in murine and rat peritoneal macrophages ŽForis ´ et al., 1986; Casellas et al., 1991; Rojavin et al., 1993.. However, in the mouse, the inhibition observed is partial, fluctuating between 20 and 26%. At present we do not know the reason for the partial nature of this inhibition, but phagocytosis may be promoted by a variety of signals, some of which may not be susceptible to opioid-mediated inhibition. On the other hand, macrophage populations may be heterogeneous in terms of expression of the opioid receptor ŽSedqi et al., 1995.; therefore not all cells may be susceptible to morphine effects. Another puzzling aspect of our results is the biphasic nature of the acute exposure dose–response curve. Biphasic dose–response curves had also been obtained in our laboratory for the modulation by opioids of phagocytosis in the protozoan ciliate Tetrahymena ŽDe Jesus ´ and Re-
E.Z. Tomei, F.L. Renaudr Journal of Neuroimmunology 74 (1997) 111–116
naud, 1989.. A biphasic curve could be indicative of rapid desensitization Žtachyphylaxis. during the pre-incubation period at the higher agonist concentrations. This idea is backed by the fact that a sustained inhibition of phagocytosis is obtained with both murine macrophages and Tetrahymena when the particles to be ingested are added simultaneously with morphine, with no pre-incubation period ŽChiesa et al., 1993; Szabo et al., 1993.. Alternatively, the biphasic curve could be explained on the basis of concentration-dependent differential effects of morphine on signal transduction pathways. For instance, it has been reported that different morphine concentrations can either stimulate or inhibit met-enkephalin secretion in the myenteric plexus. These effects seem to be mediated by differential coupling of the receptor to G-proteins ŽGintzler and Xu, 1991; Wang and Gintzler, 1994.. Thus, in the macrophage at low morphine concentrations the receptor could be coupled to an inhibitory pathway, and at higher concentrations it could be coupled to a stimulatory pathway. It is of great interest that the dose–response curve generated when using chronically exposed cells is approximately a mirror image of the dose–response curve obtained with acute exposures, with a stimulatory effect observed in the 50–100 nM range. A similar mirror image relationship was reported earlier in the Tetrahymena opioid mechanism, when comparing dose–response curves in acute versus chronic exposures ŽSalaman ´ et al., 1990.. This is an important observation because it suggests that if these results can be extrapolated to the in vivo situation, chronic exposure to morphine may under some circumstances actually have a stimulatory effect on phagocytes. Our work clearly shows that the effect of morphine on phagocytosis, whether inhibitory or stimulatory, is dependent on the length of the pre-incubation period ŽFig. 2.. Although the lack of inhibition of phagocytosis by morphine after a chronic exposure had been hypothesized to be due to desensitization ŽCasellas et al., 1991., at the moment the basis for these differential effects is unknown. As sta te d p re v io u sly , in th e n e rv o u s sy ste m desensitizationrtolerance may involve mechanisms at the receptor ŽWang et al., 1994. and post-receptor ŽNestler, 1992. levels. Since we do not know if these mechanisms are taking place in vitro in our experiments, we can only use the term desensitization as an operational one until the cellular basis for these variable effects is established. Other alternatives may be considered besides desensitization; perhaps a chronic or prolonged exposure results the accumulation of either cytokines or morphine metabolites that counteract the inhibitory effect of the drug or that stimulate phagocytosis. Nevertheless, it is of interest that upon chronic exposure a high concentration of morphine, namely 1 m M, appears to be inhibitory, whereas it had no effect in an acute exposure ŽFig. 1.. This suggests the existence of a tolerant state, with a shift to the right of the dose–response curve.
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Other differential effects of morphine dependent on concentration and incubation time have been reported ŽXu et al., 1989; Salaman ´ et al., 1990.. Furthermore, in tolerant Guinea pigs morphine seems to be required for stimulation of met-enkephalin secretion ŽGintzler et al., 1987.. These differential effects of morphine in the Guinea pig ileum may perhaps be explained by differential coupling of the opioid receptor to stimulatory or inhibitory G-proteins ŽWang and Gintzler, 1994, 1995.. If these differential effects of morphine are shown to have a common underlying mechanism, the use of the term desensitization to describe the response to the agonist after chronic exposure would be ambiguous; the systems are indeed sensitive to morphine and contrary to an acute exposure they may require the drug to be stimulated. Perhaps the term transsensitization to describe this novel situation may be more descriptive. Our observation that under some conditions withdrawal of morphine will inhibit phagocytosis suggests that a state akin to dependence ŽCollin and Cesselin, 1991. has been induced in these cells by a chronic exposure. It is of interest that the magnitude of the withdrawal-induced inhibition of phagocytosis is comparable to that caused by an acute exposure to morphine. Similar results had been reported earlier in the Tetrahymena opioid mechanism ŽSalaman ´ et al., 1990., and in the Guinea pig myenteric plexus ŽGintzler et al., 1987.. These data suggest that dependence may be manifested at the cellular level in these various systems. It remains to be seen if these observations can be extrapolated to the in vivo situation. If chronically exposed cells are indeed in a dependent state, as suggested by our data, it is tempting to speculate that the inhibition of phagocytosis observed by Rojavin et al., 1993, may be due to withdrawal of the drug, when cells from morphine-pelleted mice are used for ex vivo analysis in an in vitro opiate-free environment. A limitation of most studies on the effect of drugs of abuse in the laboratory is the fact that the timing of administration of the drug is very different from that of the drug addict: in the laboratory drugs are administered at regular intervals, whereas the drug addict gets it erratically. This is why we decided to perform a study of the development of putative desensitization when morphine was administered intermittently. Our results show that a withdrawal period of 4 h is enough to prevent desensitization since under these conditions 100 nM morphine will inhibit phagocytosis, regardless of the total exposure time to the drug. What events take place in the macrophage signal transduction pathways during that 4 h period is unknown at the moment, but these are probably of paramount importance in understanding how desensitization comes about. The idea of opioid withdrawal contributing to immune dysfunction has been previously presented by other investigators. Morphine withdrawal from tolerant mice caused a decrease in cytotoxicity ŽBhargava et al., 1994. and in
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tolerant monkeys infected with SIV drug withdrawal precipitated development of immune dysfunction ŽDonahoe et al., 1993.. Regardless, it still remains to be established if these results can be extrapolated to human beings. It is our premise and that of others ŽDonahoe et al., 1993. that a drug addict may not suffer immune dysfunction when getting the drug on a regular basis. However, phagocytosis and perhaps other functions of macrophages may be affected adversely by withdrawal, perhaps making the addict more susceptible to infections. It also remains to be established how other macrophage functions, such as oxidative burst and cytokine secretion, are affected by prolonged exposure and withdrawal. Nevertheless, it may be concluded that the effect of morphine on murine macrophages is quite complex and the effects observed are strongly dependent on experimental conditions such as concentration and time of exposure and withdrawal.
Acknowledgements The authors wish to thank Dr. Robert M. Donahoe, from the Psychiatry Department, Emory University School of Medicine, and Dr. Jose´ E. Garcıa-Arraras, ´ ´ from the Biology Department, University of Puerto Rico, Rıo ´ Piedras Campus, for valuable suggestions on the original manuscript. This study was supported by the Department of Education grant P120A40059, NIH grant S06 GM08102 and NSF grant MCB-9105024.
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Effect of morphine on Fc-mediated phagocytosis by murine macrophages in vitro Enid Z. Tomei a , Fernando L. Renaud a
b,)
Department of Psychiatry, Emory UniÕersity School of Medicine, G.M.H.I. 1256 Briarcliff Rd., N.E., Atlanta, GA 30306, USA b Biology Department, UniÕersity of Puerto Rico, P.O. Box 23360, San Juan 00931-3360, Puerto Rico Received 19 June 1996; revised 21 October 1996; accepted 22 October 1996
Abstract Acute exposure to morphine has been shown to inhibit phagocytosis in murine macrophages, whereas chronic exposure results in apparent desensitization. We now show that morphine may be either inhibitory or stimulatory depending on concentration and exposure time. Furthermore, under some conditions drug withdrawal from putatively desensitized cells will result in inhibition of phagocytosis, suggesting that a state akin to dependence has developed. Desensitization can also develop with intermittent exposures if the opiate-free period between drug exposures is shorter than 4 h. These effects of morphine on macrophages are important in understanding the role of this drug as an immunomodulatory agent. Keywords: Morphine; Murine macrophage; Desensitization; Phagocytosis
1. Introduction There is much recent evidence to show that the nervous and immune systems interact ŽBlalock, 1994; Gilmore et al., 1990.. It is also known that both endogenous opioids and exogenous opiates may have important modulatory effects on cells of the immune system such as T-cells and macrophages ŽTubaro et al., 1983; Donahoe et al., 1985; Fischer, 1988; Peterson et al., 1990, 1993; Rouveix, 1992.. These findings suggest that opiate abuse may be a factor in susceptibiliy to infections such as HIV-1 observed in drug addicts ŽDonahoe, 1992.. However, it is perplexing that this apparent drug-induced susceptibility seems to be less frequent when opiates are administered in a clinical setting. This may be due to the differences in dosage and exposure intervals between a clinical setting and the situation in a drug addict. Morphine and some endogenous opioids have been shown to affect processes in macrophages such as oxidative burst, chemotaxis and phagocytosis ŽPeterson et al., 1987; Perez-Castrillon ´ ´ et al., 1992; Foris ´ et al., 1986; Rojavin et al., 1993; Tubaro et al., 1987.. Work performed previously in our laboratory showed that opioids inhibited )
Corresponding author. Tel.: q1-787-764-0000, Ext. 2037; fax: q1787-7643875; e-mail: [email protected]
phagocytosis in murine peritoneal macrophages in vitro by a naloxone-reversible mechanism ŽCasellas et al., 1991.. Furthermore, chronic exposure to morphine resulted in the development of a state akin to desensitization in these cells, since after exposure for 16 h the drug no longer had any inhibitory effect on phagocytosis ŽCasellas et al., 1991.. Nevertheless, the cellular basis for this observation still needs to be established. Desensitization in nerve cells has been postulated to involve both receptor ŽWang et al., 1994. and post-receptor mechanisms ŽNestler, 1992., but the involvement of these mechanisms in opioid effects on macrophages has not been studied. In this work we show that the effects of morphine on macrophages depend to an important extent on the particular conditions of exposure and drug concentration. Such parameters therefore are important aspects of studies concerning the effect of morphine and other opioids on the immune system. 2. Materials and methods 2.1. Induction and isolation of murine peritoneal macrophages Four to eight week old female C3HeBrFeJ mice ŽJackson Labs., Bar Harbor, ME. were utilized in this study.
0165-5728r97r$17.00 Copyright q 1997 Published by Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 6 . 0 0 2 1 3 - 5
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Animals were injected i.p. with 2 cc of 3% Brewer’s Thioglycollate medium ŽDifco, Detroit, MI.. The mice were euthanized by cervical dislocation five days following the injection, and the peritoneal-elicited cells were recovered by injecting 10 cc of sterile phosphate buffered saline ŽPBS. into the peritoneal cavity. The peritoneum was then gently massaged 2 min and cell exudate aspirated with a syringe. The exudate was transferred to a sterile 50 cc conical centrifuge tube, sterile PBS was added to reach a final volume of 30 cc, and the cell suspension was washed by centrifuging three times at room temperature at 250 g for 10 min. The cells were then resuspended in sterile RPMI 1640 medium supplemented with 10% heatinactivated fetal bovine serum ŽHyClone, UT. and 1% antibioticrantimycotic solution ŽSigma, St. Louis, MO.. This medium will be referred to as complete RPMI. Macrophages were counted in a hemocytometer and cell viability was determined by trypan blue exclusion Ž0.4% trypan blue.. Only cell suspensions with a viability greater than 90% were used in these studies. The cell concentration was adjusted to 5 = 10 3 macrophages mly1 , and 400 m l of this cell suspension were plated per well in 8-well Lab-Tek w culture plates ŽMiles Laboratories, Naperville, IL.. The cultures were incubated overnight at 378C under 5% CO 2 . 2.2. Opsonization of sheep red blood cells Sheep red blood cells ŽSRBC. were purchased from Colorado Serum, CO. An aliquot of SRBC was washed twice in sterile PBS, and after the last wash the pellet was resuspended in complete RPMI. The SRBC were counted in a hemocytometer, and the total number of cells needed were transferred to a sterile centrifuge tube. The aliquot was spun at 250 g for 10 min and the cell pellet was resuspended in 1 cc of the rabbit IgG anti-SRBC ŽDiamedic Cordis Laboratories, FL. at 1:500 to 1:1000 final dilution in complete RPMI. The SRBC were incubated with the immunoglobulin in a shaking water bath for 15 min at 308C. SRBC were then washed once and kept on ice until needed. 2.3. Phagocytosis assay The cultured adherent macrophages were washed twice with complete RPMI to remove non-adherent cells. Cells were incubated with opsonized SRBC at a ratio of 1 macrophage:100 SRBC. To optimize contact between the two cell populations, the Lab-Tek w chambers were carefully placed on top of cold centrifuge carriers and gently spun at 10 g for 2 min at 48C. Phagocytosis was started by transferring slides immediately to a 378C incubator. The process was stopped after 20 min by placing the slides on ice, and non-phagocytosed SRBC were lysed by hypotonic shock ŽBobak et al., 1987.. The culture slides were washed twice with PBS to remove cell debris, and macrophages
were fixed with 1% glutaraldehyde for 5 min at room temperature. Fixed cells were stained with Dif-Quick w and phagocytosis was determined by light microscopy. Direct visual discrimination of ingested erythrocytes was possible since these stain pale red with the Wright stain, while the macrophage’s cytoplasm stains blue with the Giemsa stain. One hundred macrophages were scored per well and each experimental condition was performed in triplicate replicas, each replica being treated and processed independently. The results were expressed in terms of the phagocytic factor: phagocytic factor % phagocytosis in the presence of agonist s
percent phagocytosis of control cells
= 100
where % phagocytosis is defined as the percentage of cells ingesting at least one SRBC. To compare data from different experiments, the results were standardized by giving a value of 100% to the mean of the % phagocytosis observed in control cells, and the experimental values were adjusted accordingly. This was necessary to be able to perform a statistical comparison among data obtained from different experiments, because it was found that the basal level of phagocytosis Ž% phagocytosis of opsonized SRBC by control cells. varied from experiment to experiment, with individual experiments ranging between 40 and 93% Žmean " standard deviations 67 " 17.. However, the results were remarkably consistent within each experiment, as evidenced by the low standard errors obtained for control cells. Analysis of the samples was done using the Student’s t-test. A difference was considered significant when p - 0.05. 2.4. Effect of acute, chronic and intermittent exposures to morphine on FcR-mediated phagocytosis To assess the effect of acute exposure to morphine sulfate ŽSigma, St. Louis, MO. macrophages were washed and incubated for 30 min at 378C in 400 m l complete RPMI, or in complete RPMI supplemented with morphine concentrations ranging from 1 nM to 1 m M; some experiments were also carried out in the presence of equimolar concentrations of naloxone in addition to the opiate. To study the effect of chronic exposure the cells were incubated overnight Ž17 h. at 378C with 1 nM to 1 m M morphine. To assess the effect of intermittent exposures, incubation periods of 2–5 h with 100 nM morphine were alternated with periods of drug withdrawal of 2–5 h in various combinations. Three washes with RPMI were performed prior to the withdrawal periods to remove the opioid from the cell milieu. Following acute, chronic or intermittent incubation with the opiate agonist, opsonized SRBC were added, still in the presence of the opiate, and phagocytosis analysed as described above.
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3. Results 3.1. Effect of acute exposure to different morphine concentrations on FcR-mediated phagocytosis of SRBC The effect of incubating macrophages for 30 min with various concentrations of morphine prior to the phagocytosis assay is illustrated in Fig. 1. It is characterized by a biphasic response, with significant Ž p - 0.01. inhibition of phagocytosis by 50–100 nM morphine. As reported previously ŽCasellas et al., 1991. this inhibition was naloxonereversible Ždata not shown.. 3.2. Effect of different incubation times with morphine on Fc-mediated phagocytosis To assess the temporal aspects of the ability of morphine to modulate phagocytosis, macrophages were incubated with 100 nM morphine for various times prior to the phagocytosis assay. The results show that the phagocytic effects of morphine depend strongly on exposure time ŽFig. 2.. Up to 5 h incubation resulted in inhibition of phagocytosis Ž p - 0.001., whereas an incubation of between 6 and 8 h resulted in no effect on phagocytosis when compared to control cells Ž p - 0.3.. However, 9 to 10 h of continuous exposure to the opiate results in a moderate, but significant Ž p - 0.05., stimulation of phagocytosis in the presence of the drug.
Fig. 2. Effect of morphine pre-incubation time on phagocytosis of SRBC by murine macrophages. Cells were pre-incubated with 100 nM morphine for periods ranging from 0.5 to 10 h Žfilled circles.; following this, cells were incubated with opsonized SRBC and allowed to phagocytize for 20 min, while still in the presence of the opiate. Control cellss open circles. ) p- 0.01; ) ) p- 0.03; ) ) ) p- 0.02; ns6 Žtwo independent experiments with each variable in triplicate..
3.3. Effect of chronic exposure to different morphine concentrations on Fc-mediated phagocytosis A state akin to desensitization was induced in macrophages exposed for 16 h to 50 nM morphine, since phagocytosis in these cells is no longer inhibited by the opiate ŽCasellas et al., 1991.. Therefore we performed a dose–response study to determine if this effect was concentration-dependent. The results illustrated in Fig. 1 illustrate a biphasic response to chronic morphine: after a 17 h exposure to the opiate, 50 nM and 100 nM concentrations actually caused a modest but significant stimulation of phagocytosis Ž p - 0.05.; whereas lower and higher concentrations had no significant effect, except for 1 m M, which was inhibitory Ž p - 0.05.. The latter finding is in contrast to data from our acute-exposure study ŽFig. 1. wherein 1 m M morphine had no effect on phagocytosis. 3.4. Effect of morphine withdrawal from putatiÕely desensitized cells on Fc-mediated phagocytosis
Fig. 1. Effect of acute Žopen circles. and chronic Žfilled circles. exposure to different concentrations of morphine on phagocytosis of sheep red blood cells ŽSRBC. by murine peritoneal macrophages. Percent phagocytosis is standardized relative to the control value which is assumed to be 100%. In this and in all following figures data are expressed in terms of the phagocytic factor Žsee text for definition. that standardizes results with respect to control values. Each point or bar represents the mean" standard error; the dashed line represents the control level Žopen squares. of phagocytosis. ) p- 0.01, ns9 Žthree independent experiments with each variable in triplicate.; ) ) p- 0.05, ns6 Žtwo independent experiments with each variable in triplicate..
A study was performed to determine if morphine withdrawal had any additional effects on the phagocytic capacity of macrophages that had already demonstrated a diminished capacity to respond to morphine. Macrophages were exposed to morphine for 8 h, and the opiate was withdrawn from the cell milieu 5 h, 1 h, or immediately prior to the phagocytosis assay. To ascertain that the macrophages had been deprived of the opiate, cells were washed three times with RPMI and incubated in complete RPMI at 378C for the deprivation periods indicated above.
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3.5. Effect of intermittent exposure to morphine on phagocytosis
Fig. 3. Effect of morphine withdrawal on phagocytosis of SRBC by murine macrophages. Cells were exposed to 100 nM morphine for 8 h at 378C, and morphine was withdrawn from the medium for the indicated times prior to assaying phagocytosis of opsonized SRBC. Phagocytosis by morphine-deprived macrophages Žhatched bars. was then compared to phagocytosis by macrophages that were not depleted of the opiate Žconstant morphine controls, open bars., and to phagocytosis by opiate-naive controls Žfilled bars.. ) p- 0.01, ns9 Žthree independent experiments with each variable in triplicate..
The phagocytosis assay was carried out as described previously, by addition of opsonized SRBC and incubation for 20 min at 378C, but in the absence of morphine. The results of this study are illustrated in Fig. 3. As expected from the data in Fig. 2, macrophages exposed for 8 h to 100 nM morphine were no longer inhibited by the opiate. However, when they were deprived of morphine for 5 h ŽFig. 3. these macrophages exhibited a significant decrease in phagocytosis Ž p - 0.05. when compared to control macrophages, or to non-deprived macrophages. However, withdrawal of morphine for 1 h, or immediately prior to the assay, did not significantly affect phagocytosis in these cells ŽFig. 3..
This series of experiments was designed to determine if phagocytosis by macrophages exposed intermittently to 100 nM morphine would still be inhibited by the drug, as in acute exposure, or whether intermittent exposure would result in a lack of effect or in a stimulation of phagocytosis, as in chronic exposure ŽFigs. 1 and 2.. When macrophages were depleted of morphine for 2 h intervals in a 10 h assay Žthree 2 h exposure periods separated by two 2 h depletion periods., phagocytosis was not affected by the drug ŽFig. 4A.; similar results were obtained when depletion periods of 3 h were tested Žtwo 3 h exposure periods separated by a three hour depletion period; data not shown.. However, when intervals of 4 h were examined Žtwo 4 h exposure periods separated by a 4 h withdrawal period; Fig. 4B., a significant inhibitory effect of morphine on phagocytosis was observed Ž p - 0.01., when compared to control cells exposed continually for 12 h to 100 nM morphine. The basal level of phagocytosis was not affected by the washes involved in these experiments, since the % phagocytosis of opiate-naive control cells ranged between 47 and 93%, well within the values expected for a typical experiment. We then decided to test longer exposure periods alternated with 4 h withdrawals. Cells were exposed to 100 nM morphine for a 5 h interval, deprived for 4 h, and then re-exposed to 100 nM morphine for 5 h prior to the phagocytosis assay. Under these conditions morphine still inhibited phagocytosis when compared to control cells exposed continuously to the opiate for 14 h ŽFig. 4C; p - 0.01..
4. Discussion
Fig. 4. Effect of intermittent exposures to 100 nM morphine on phagocytosis of SRBC by murine macrophages. Macrophages were incubated for alternated intervals in the presence Žq. and absence Žy. of 100 nM morphine, for a total incubation time of 10 to 14 h ŽA–C. at 378C. After the last exposure to morphine, and while still in the presence of the opiate, opsonized SRBC were added to macrophages and phagocytosis was allowed to proceed for 20 min at 378C. The phagocytic factor of macrophages exposed to morphine for these various intervals Žopen bars. was compared to the phagocytosis of control cells exposed continuously to the opiate Žfilled bars.. ŽA. 2 h Žq., 2 h Žy., 2 h Žq., 2 h Žy., 2 h Žq.; control exposed for 10 h. ŽB. 4 h Žq., 4 h Žy., 4 h Žq.; control exposed for 12 h. ŽC. 5 h Žq., 4 h Žy., 5 h Žq.; control exposed for 14 h. ) p- 0.01, ns6 Žtwo independent experiments with each variable in triplicate..
The results obtained in this work confirm previous findings that opioids inhibit phagocytosis in murine and rat peritoneal macrophages ŽForis ´ et al., 1986; Casellas et al., 1991; Rojavin et al., 1993.. However, in the mouse, the inhibition observed is partial, fluctuating between 20 and 26%. At present we do not know the reason for the partial nature of this inhibition, but phagocytosis may be promoted by a variety of signals, some of which may not be susceptible to opioid-mediated inhibition. On the other hand, macrophage populations may be heterogeneous in terms of expression of the opioid receptor ŽSedqi et al., 1995.; therefore not all cells may be susceptible to morphine effects. Another puzzling aspect of our results is the biphasic nature of the acute exposure dose–response curve. Biphasic dose–response curves had also been obtained in our laboratory for the modulation by opioids of phagocytosis in the protozoan ciliate Tetrahymena ŽDe Jesus ´ and Re-
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naud, 1989.. A biphasic curve could be indicative of rapid desensitization Žtachyphylaxis. during the pre-incubation period at the higher agonist concentrations. This idea is backed by the fact that a sustained inhibition of phagocytosis is obtained with both murine macrophages and Tetrahymena when the particles to be ingested are added simultaneously with morphine, with no pre-incubation period ŽChiesa et al., 1993; Szabo et al., 1993.. Alternatively, the biphasic curve could be explained on the basis of concentration-dependent differential effects of morphine on signal transduction pathways. For instance, it has been reported that different morphine concentrations can either stimulate or inhibit met-enkephalin secretion in the myenteric plexus. These effects seem to be mediated by differential coupling of the receptor to G-proteins ŽGintzler and Xu, 1991; Wang and Gintzler, 1994.. Thus, in the macrophage at low morphine concentrations the receptor could be coupled to an inhibitory pathway, and at higher concentrations it could be coupled to a stimulatory pathway. It is of great interest that the dose–response curve generated when using chronically exposed cells is approximately a mirror image of the dose–response curve obtained with acute exposures, with a stimulatory effect observed in the 50–100 nM range. A similar mirror image relationship was reported earlier in the Tetrahymena opioid mechanism, when comparing dose–response curves in acute versus chronic exposures ŽSalaman ´ et al., 1990.. This is an important observation because it suggests that if these results can be extrapolated to the in vivo situation, chronic exposure to morphine may under some circumstances actually have a stimulatory effect on phagocytes. Our work clearly shows that the effect of morphine on phagocytosis, whether inhibitory or stimulatory, is dependent on the length of the pre-incubation period ŽFig. 2.. Although the lack of inhibition of phagocytosis by morphine after a chronic exposure had been hypothesized to be due to desensitization ŽCasellas et al., 1991., at the moment the basis for these differential effects is unknown. As sta te d p re v io u sly , in th e n e rv o u s sy ste m desensitizationrtolerance may involve mechanisms at the receptor ŽWang et al., 1994. and post-receptor ŽNestler, 1992. levels. Since we do not know if these mechanisms are taking place in vitro in our experiments, we can only use the term desensitization as an operational one until the cellular basis for these variable effects is established. Other alternatives may be considered besides desensitization; perhaps a chronic or prolonged exposure results the accumulation of either cytokines or morphine metabolites that counteract the inhibitory effect of the drug or that stimulate phagocytosis. Nevertheless, it is of interest that upon chronic exposure a high concentration of morphine, namely 1 m M, appears to be inhibitory, whereas it had no effect in an acute exposure ŽFig. 1.. This suggests the existence of a tolerant state, with a shift to the right of the dose–response curve.
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Other differential effects of morphine dependent on concentration and incubation time have been reported ŽXu et al., 1989; Salaman ´ et al., 1990.. Furthermore, in tolerant Guinea pigs morphine seems to be required for stimulation of met-enkephalin secretion ŽGintzler et al., 1987.. These differential effects of morphine in the Guinea pig ileum may perhaps be explained by differential coupling of the opioid receptor to stimulatory or inhibitory G-proteins ŽWang and Gintzler, 1994, 1995.. If these differential effects of morphine are shown to have a common underlying mechanism, the use of the term desensitization to describe the response to the agonist after chronic exposure would be ambiguous; the systems are indeed sensitive to morphine and contrary to an acute exposure they may require the drug to be stimulated. Perhaps the term transsensitization to describe this novel situation may be more descriptive. Our observation that under some conditions withdrawal of morphine will inhibit phagocytosis suggests that a state akin to dependence ŽCollin and Cesselin, 1991. has been induced in these cells by a chronic exposure. It is of interest that the magnitude of the withdrawal-induced inhibition of phagocytosis is comparable to that caused by an acute exposure to morphine. Similar results had been reported earlier in the Tetrahymena opioid mechanism ŽSalaman ´ et al., 1990., and in the Guinea pig myenteric plexus ŽGintzler et al., 1987.. These data suggest that dependence may be manifested at the cellular level in these various systems. It remains to be seen if these observations can be extrapolated to the in vivo situation. If chronically exposed cells are indeed in a dependent state, as suggested by our data, it is tempting to speculate that the inhibition of phagocytosis observed by Rojavin et al., 1993, may be due to withdrawal of the drug, when cells from morphine-pelleted mice are used for ex vivo analysis in an in vitro opiate-free environment. A limitation of most studies on the effect of drugs of abuse in the laboratory is the fact that the timing of administration of the drug is very different from that of the drug addict: in the laboratory drugs are administered at regular intervals, whereas the drug addict gets it erratically. This is why we decided to perform a study of the development of putative desensitization when morphine was administered intermittently. Our results show that a withdrawal period of 4 h is enough to prevent desensitization since under these conditions 100 nM morphine will inhibit phagocytosis, regardless of the total exposure time to the drug. What events take place in the macrophage signal transduction pathways during that 4 h period is unknown at the moment, but these are probably of paramount importance in understanding how desensitization comes about. The idea of opioid withdrawal contributing to immune dysfunction has been previously presented by other investigators. Morphine withdrawal from tolerant mice caused a decrease in cytotoxicity ŽBhargava et al., 1994. and in
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tolerant monkeys infected with SIV drug withdrawal precipitated development of immune dysfunction ŽDonahoe et al., 1993.. Regardless, it still remains to be established if these results can be extrapolated to human beings. It is our premise and that of others ŽDonahoe et al., 1993. that a drug addict may not suffer immune dysfunction when getting the drug on a regular basis. However, phagocytosis and perhaps other functions of macrophages may be affected adversely by withdrawal, perhaps making the addict more susceptible to infections. It also remains to be established how other macrophage functions, such as oxidative burst and cytokine secretion, are affected by prolonged exposure and withdrawal. Nevertheless, it may be concluded that the effect of morphine on murine macrophages is quite complex and the effects observed are strongly dependent on experimental conditions such as concentration and time of exposure and withdrawal.
Acknowledgements The authors wish to thank Dr. Robert M. Donahoe, from the Psychiatry Department, Emory University School of Medicine, and Dr. Jose´ E. Garcıa-Arraras, ´ ´ from the Biology Department, University of Puerto Rico, Rıo ´ Piedras Campus, for valuable suggestions on the original manuscript. This study was supported by the Department of Education grant P120A40059, NIH grant S06 GM08102 and NSF grant MCB-9105024.
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