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Originally published online as doi:10.1189/jlb.1203639 on March 23, 2004

Published online before print March 23, 2004
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(Journal of Leukocyte Biology. 2004;75:1131-1138.)
© 2004 by Society for Leukocyte Biology

Morphine-induced macrophage apoptosis: oxidative stress and strategies for modulation

Rajani S. Bhat, Madhu Bhaskaran, Anil Mongia, Naoko Hitosugi and Pravin C. Singhal1

Department of Medicine, Long Island Jewish Medical Center and North Shore University Hospital, New Hyde Park, New York

1 Correspondence: Division of Kidney Diseases and Hypertension, Long Island Jewish Medical Center, 410 Lakeville Road, Suite #207, New Hyde Park, NY 11040. E-mail: singhal{at}lij.edu


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ABSTRACT
 
Occurrence of macrophage apoptosis has been implicated for the altered immune function found in an opiate milieu. In the present study, we evaluated the role of oxidative stress in morphine-induced macrophage apoptosis. Morphine promoted the apoptosis of macrophages. This effect of morphine was associated with the production of superoxide and nitric oxide (NO). Antioxidants provided protection against morphine-induced macrophage injury. In addition, diphenyleneiodonium chloride, an inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, attenuated the proapoptotic effect of morphine. Antitransforming growth factor-ß (anti-TGF-ß) antibody and propranolol (an inhibitor of the phospholipase D pathway) inhibited morphine-induced superoxide generation as well as apoptosis. N'-Tetraacetic acid tetra (acetoxymethyl) ester, a calcium-chelating agent, inhibited morphine-induced apoptosis, whereas thapsigargin (a calcium agonist) stimulated macrophage apoptosis under basal as well as morphine-stimulated states. These studies suggest that morphine-induced macrophage apoptosis is mediated through downstream signaling involving TGF-ß and NO production. Moreover, there is NADPH oxidation activation involving phospholipase D and Ca2+, leading to the generation of superoxide. In in vivo studies, administration of N-acetyl cysteine and preinduction of heme oxygenase activity and epoetin {alpha} prevented morphine-induced peritoneal macrophage apoptosis, thus further confirming the role of oxidative stress in morphine-induced macrophage apoptosis.

Key Words: opiates • signal transduction • nitric oxide • reactive oxygen species


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INTRODUCTION
 
Opiate addicts are prone to infections [1 2 3 4 ]. The mononuclear phagocyte system plays an important role in the host defense. As mononuclear phagocytes have been demonstrated to possess opiate receptors, the direct effect of opiates on these cells has been increasingly recognized [5 6 7 8 ]. For example, morphine, an active metabolite of heroin, has been reported to modulate immune function by suppressing macrophage phagocytic and migration capabilities [9 10 11 ]. We previously reported that morphine, at higher concentrations, promoted macrophage apoptosis [12 , 13 ]. In experimental animal models, Hilburger et al. [14 ] and Roy et al. [15 , 16 ] demonstrated that morphine induced sepsis in mice. This effect of morphine has been suggested to be linked to morphine-induced macrophage apoptosis [17 ].

The role of opiates in the progression of human immunodeficiency virus (HIV) infection remains a controversial issue [18 19 20 21 ]. In in vitro studies, morphine has been shown to enhance the replication of HIV in peripheral blood mononuclear cells (PBMC) [19 , 20 ]. However, epidemiological studies do not support this notion [21 ]. Conversely, synthetic {kappa}-opioid receptor ligand, U50, 488, or U69, 593, inhibited HIV expression by brain macrophages and monocyte-derived macrophages [22 , 23 ].

Oxidative stress has also been reported to play a role in the pathogenesis of AIDS [24 , 25 ]. In in vitro studies, addition of hydrogen peroxide to HIV-infected cells increased apoptosis [26 ]. Conversely, antioxidants inhibited apoptosis [26 ]. In in vivo studies, administration of an antioxidant to patients with HIV infection decreased apoptosis of peripheral blood lymphocytes [25 ]. Thus, it appears that oxidative stress may be contributing to the occurrence of apoptosis in patients with HIV infection. As a significant number of patients with HIV infections have also been reported to have opiate addiction, it is likely that opiates might have contributed to ongoing oxidative stress. However, the contribution of the opiate milieu was not investigated in these studies.

Morphine has been demonstrated to exert oxidative stress in various cells [27 , 28 ]. Moreover, morphine has been shown to up-regulate the expression of heme oxygenase (HO), a biological marker of oxidative stress [29 ]. We recently proposed that morphine may be inducing macrophage injury in mice and humans by different pathways [30 ].

There is no definitive data in the literature about morphine-induced human monocyte apoptosis in vivo. However, it does not mean that this phenomenon does not exist but may well be a reflection of the difficulty in studying ongoing monocyte/macrophage apoptosis in vivo in humans. In murine macrophages, various investigators have demonstrated the role of reactive nitrogen intermediates (RNI) in the induction of macrophage apoptosis [31 ]; whereas, human monocyte/macrophages have inefficient inducible nitric oxide synthase (iNOS). Neverheless, we observed that morphine-induced monocyte apoptosis was blocked by superoxide dismutase (SOD) and catalase but not by NG-nitro-L-arginine methyl ester (L-NAME) [30 ]. These studies proposed that in human monocytes, morphine-induced injury may not be mediated by NO but by reactive oxygen species (ROS) such as superoxide and hydrogen peroxide [30 ].

In the present study, we evaluated the effects of morphine on superoxide and NO production by murine macrophages. In addition, we studied the effect of antioxidants on morphine-induced macrophage injury. To evaluate the involved downstream signaling, we studied the effect of various blockers of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathway.


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MATERIALS AND METHODS
 
Macrophage culture
We used a murine macrophage cell line (J 774.16 cells, American Type Culture Collection, Manassas, VA). Confluent macrophages were subcultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY) containing 10% fetal calf serum (FCS; Atlanta Biologicals, Norcross, GA), 50 U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies).

Experimental agents
Morphine was obtained from the National Institute on Drug Abuse (Rockville, MD). Ascorbic acid, diphenyleneiodonium chloride (DPI), N-acetyl L-cysteine (NAC), and hemin were obtained from Sigma Chemical Co. (St. Louis, MO). Thapsigargin (THPS) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA) were obtained from Calbiochem (San Diego, CA). Epoetin {alpha} was obtained from Ortho Biotech (New Brunswick, NJ). L-NAME was used in concentration 1 mM, and NG-monomethyl-L-arginine monoacetate (L-NMMA) was used in concentration 10–6M (Sigma Chemical Co.).

Apoptosis studies
To determine the occurrence of apoptosis in macrophages, we used Hoechst (H)-33342 (Molecular Probes, Eugene, OR) and propidium iodide (PI; Sigma Chemical Co.) stains. H-33342 stains the nuclei of live cells and identifies apoptotic cells by increased fluorescence, whereas PI stains only the necrosed cells [12 , 13 ]. Equal numbers of macrophages (105 cells/well) were plated in Petri dishes with media (RPMI 1640, Gibco, Grand Island, NY) containing 10% FCS. Once attached, aliquots of methanol containing H-33342 (final concentration, 1 µg/ml) were added and incubated for 10 min at 37°C. Subsequently, cells (without a wash) were placed on ice, and PI (final concentration, 1 µg/ml) was added to each well. Cells were incubated with the dyes for 10 min on ice, protected from light, and then examined under UV light using a Hoechst filter (Nikon, Melville, NY). Percentage of live, apoptotic, and necrosed cells was recorded in eight random fields by two observers unaware of the experimental conditions.

DNA fragmentation assay: gel electrophoresis
This is a simple method that is specific for isolation and confirmation of DNA fragments from apoptotic cells [32 ]. Equal numbers (108 cells/Petri dish) of murine macrophages under control and experimental conditions were trypsinized and centrifuged at 1600 g for 10 min at room temperature, and the pellets were resuspended in DNA lysis buffer (1% Nonidet P-40 in 20 mM EDTA, 50 mM Tris-HCL, pH 7.5, 10 µl per 106 cells). After centrifugation, the supernatant was collected, and the extraction was repeated. Sodium dodecyl sulfate in a final concentration of 1% was added to the supernatants before the samples were treated with RNase A (final concentration, 5 µg/µl) at 56°C. This was followed by digestion with proteinase K (Promega, Madison, WI) for 2 h at 37°C. After addition of one-half volume 10 M ammonium acetate, DNA was precipitated with 2.5 volume ethanol, dissolved in gel loading buffer, and separated by electrophoresis in 1.6% agarose gels.

Superoxide assay
Equal numbers of macrophages were plated in 100 mm Petri dishes and grown to subconfluence. The cells were washed twice with normal saline and incubated in serum-free media under control and experimental conditions. Supernatants were collected at 0, 30, 45, 60, and 120 min into precooled microcentrifuge test tubes. Superoxide assay was performed subsequently. In brief, 50 µl each supernatant was pipeted into a 96-well plate, kept on ice, and mixed with 100 µl cytochrome C (160 µM final concentration, ICN Biomedical, Costa Mesa, CA) diluted with Hanks’ balanced salt solution (Gibco). Incubation was performed at 37°C for 45, 90, and 150 min, and optical density read at 550 nm. Results are expressed in arbitrary units, and experiments are repeated four times each in triplicate.

NO assay
Equal numbers of macrophages were plated in 24-well Petri dishes and grown to subconfluence. The cells were washed twice with normal saline and incubated in serum-free media under control and experimental conditions. Supernatants were collected at 0, 30, 45, 60, and 120 min into precooled microcentrifuge test tubes. Sievers 280 i NO analyzer (Ionics, Watertown, MA) performed the NO assay.

In vivo studies
Mice in groups of three were administered normal saline or morphine [20 mg/kg body weight (B.W.)] subcutaneously (s.c.; every 8 h) for 72 h. In another set of mice, mice were pretreated (intraperitoneally) with hemin (5 µg), NAC (150 mg/kg B.W.), or epoetin {alpha} (1000 U) 16 h before normal saline or morphine administration. At the end of scheduled protocols, peritoneal macrophages were harvested and evaluated for apoptosis as described previously [12 ].

Statistical analysis
Comparison of macrophage apoptosis between control and experimental conditions was performed by an unpaired Students’ t-test. When more than two groups were involved, intergroup comparisons were performed by ANOVA. A Newman-Keuls multiple-range test was used to calculate a P value. Results are represented as means ± SEM.


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RESULTS
 
To evaluate the effect of morphine on murine macrophage apoptosis, equal numbers of macrophages were incubated in media containing buffer or morphine (10–8 and 10–6 M) for 24 h followed by DNA fragmentation assay. As shown in Figure 1A , morphine-treated macrophages showed DNA fragmentation in a ladder pattern.



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  		Figure 1. (A) Effect of morphine on macrophage injury. Equal numbers of macrophages were incubated in media containing buffer (control) or morphine (10–8 and 10–6 M) for 24 h followed by DNA fragmentation assay. Control macrophages are shown in lane 1. Morphine, 10–8- and 10–6 M-treated, macrophages are shown in lanes 2 and 3, respectively. A molecular marker is shown in lane 4. (B) Effect of antioxidants on morphine (Mor)-induced macrophage apoptosis. Equal numbers of macrophages were incubated in media containing buffer [control (C)], DPI (10 µ M), ascorbic acid (AA; 100 µM), or NAC (50 µM) with or without morphine (10–6M) for 24 h. Subsequently, cells were assayed for apoptosis. Results (means±SEM) are from four sets of experiments. *, P < 0.001, compared with all other variables; **, P < 0.01, compared with respective antioxidants. (C) Effect of cytosolic Ca2+ modulation on morphine-induced macrophage apoptosis. Equal numbers of macrophages were incubated in media containing buffer [control (C)], morphine (Mor; 10–6 M), THPS (10 nM), BAPTA (50 µM), morphine + THPS, or morphine + BAPTA for 24 h. Subsequently, cells were assayed for apoptosis. Results (means±SEM) are from four series of experiments. *, P < 0.001, compared with control, BAPTA alone, Mor + BAPTA; **, P < 0.001, compared with THPS alone.

To determine the role of oxidative stress, we studied the effect of antioxidants including DPI (inhibitor of NADPH oxidase), ascorbic acid, and NAC on morphine-induced macrophage apoptosis. Equal numbers of macrophages were incubated in media containing buffer (control), DPI (10 µM), ascorbic acid (100 µM), or NAC (50 µM) with or without morphine (10–6 M) for 24 h. At the end of the incubation period, cells were assayed for apoptosis and necrosis. Morphine promoted (P<0.001) macrophage apoptosis (Fig. 1B) . Ascorbic acid, DPI, and NAC partially inhibited (P<0.001) the effect of morphine, which also promoted macrophage necrosis (control, 3.9±0.7%; morphine, 10–8 M, 6.7±0.5%; morphine, 10–6 M, 9.7±0.5% necrosed cells/field; *, P<0.001, compared with control; **, P<0.001, compared with morphine, 10–8 M). This effect of morphine was not attenuated by antioxidants (data not shown).

Alterations in cytosolic Ca2+ have been shown to play a role in the induction of apoptosis. To determine the role of cytosolic Ca2+ in morphine-induced macrophage apoptosis, equal numbers of macrophages were incubated in media containing buffer (control), morphine (10–6 M), THPS (10 nM; Ca2+ agonist), BAPTA (50 µM; Ca2+ chelator), morphine + THPS, or morphine + BAPTA for 24 h. Subsequently, cells were assayed for apoptosis. As shown in Figure 1C , morphine promoted (P<0.001) macrophage apoptosis. THPS also promoted (P<0.001) macrophage apoptosis. However, BAPTA inhibited the effect of morphine. Conversely, THPS, in combination with morphine, induced greater macrophage apoptosis when compared with morphine or THPS alone (Fig. 1C) . These results suggest that alterations in cytosolic Ca2+ modulate morphine-induced macrophage apoptosis.

We previously reported that morphine promotes macrophage iNOS expression [12 ]. To determine whether morphine stimulates macrophage NO production, equal numbers of macrophages were incubated in media containing buffer or morphine (10–6 M) for 120 min. Aliquots of supernatants were collected at 0, 30, 60, 90, and 120 min followed by NO assay. Morphine stimulated (P<0.001) macrophage NO production (Fig. 2A ). This effect of morphine peaked at 60 min.



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  		Figure 2. (A) Time-course effect of morphine on macrophage NO production. Equal numbers of macrophages were incubated in media containing buffer or morphine (10–6 M) for 120 min. Aliquots of supernatants were collected at 0, 30, 60, 90, and 120 min. NO contents were measured. Results (means±SEM) are from four sets of experiments. *, P < 0.001, compared with control at respective time-points. (B) Dose-response effect of morphine (Mor) on macrophage NO production. Equal numbers of macrophages were incubated in media containing buffer (Control) or variable concentrations of morphine (10–10–10–6 M) for 60 min. Subsequently, supernatants were collected and measured for NO content. Results (means±SEM) are from four series of experiments. *, P < 0.05, compared with control; ** P < 0.001, compared with control and morphine, 10–10 M; ***, P < 0.05, compared with morphine, 10–8 M; a, P < 0.001, compared with control and morphine, 10–10 M. (C) Role of NOS in morphine-induced NO production. Equal numbers of macrophages were incubated in media containing buffer [control (C)], L-NMMA (10–6 M), or L-NAME (1 mM) with or without morphine (Mor; 10–6 M) for 60 min. Subsequently, supernatants were collected, and NO content was measured. Results (means±SEM) are from three sets of experiments. *, P < 0.001, compared with all other variables. (D) Role of transforming growth factor-ß (TGF-ß) in morphine-induced macrophage NO production. Equal numbers of macrophages were incubated in media containing buffer [control (C)] or anti-TGF-ß antibody (A-TGFab; 1 µg/ml) with or without morphine (Mor; 10–8 M) for 60 min. Subsequently, supernatants were collected and assayed for NO. Results (means±SEM) are from three sets of experiments. *, P < 0.001, compared with control and A-TGFab + Mor; **, P < 0.001, compared with morphine alone.

To determine the dose-response effect of morphine, equal numbers of macrophages were incubated in media containing buffer (control) or variable concentrations of morphine (10–10–10–6 M) for 60 min. Subsequently, supernatants were collected and measured for NO content. As shown in Figure 2B , morphine stimulated macrophage NO production in a dose-dependent manner.

To determine the role of NOS in morphine-induced NO production, equal numbers of macrophages were incubated in media containing buffer, L-NMMA (10–6 M, NOS inhibitor), or L-NAME (1 mM, NOS inhibitor) with or without morphine (10–6 M) for 60 min. At the end of the incubation period, supernatants were collected, and NO content was measured. As shown in Figure 2C , morphine promoted (P<0.001) NO production by macrophages. However, this effect of morphine was inhibited by L-NAME as well as by L-NMMA.

TGF-ß has been shown to promote apoptosis in various types of cells [13 ]. We reported previously that morphine-induced macrophage apoptosis is mediated through TGF-ß [13 ]. To determine whether TGF-ß plays a role in morphine-induced NO production, equal numbers of macrophages were incubated in media containing buffer (control) or anti-TGF-ß antibody (1 µg/ml) with or without morphine (10–8 M) for 60 min. Subsequently, supernatants were collected and assayed for NO. As shown in Figure 2C , morphine stimulated (P<0.001) macrophage NO production. However, this effect of morphine was partially inhibited by anti-TGF-ß antibody. These results suggest that TGF-ß plays a role in morphine-stimulated macrophage NO production.

To study the effect of morphine on macrophage superoxide production, equal numbers of macrophages were incubated in media containing buffer (control) or morphine (10–8 and 10–6 M) for 120 min. Aliquots of supernatants were collected at 0, 30, 60, 90, and 120 min. Morphine stimulated the production of superoxide by macrophages from 30 min onward. This effect of morphine peaked at 60 min. The effect of morphine on macrophage superoxide production at 60 min is shown in Figure 3A .



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  		Figure 3. (A) Effect of morphine on macrophage superoxide production. Equal numbers of macrophages were incubated in media containing buffer or morphine (Mor; 10–8 and 10–6 M) for 120 min. Aliquots of supernatants were collected at 0, 30, 60, 90, and 120 min and assayed for superoxide. Results (means±SEM at 60 min) are from three sets of experiments. *, P < 0.01, compared with control; **, P < 0.001, compared with control and Mor, 10–8 M. (B) Role of NADPH oxidase activation and phospholipase D pathway in morphine-induced superoxide production. Equal numbers of macrophages were incubated in media containing buffer [control (C)], DPI (10 µM), or propranolol (Prop; 50 µM) with or without morphine (Mor; 10–6 M) for 60 min. Subsequently, supernatants were collected and assayed for superoxide. Results (means±SEM at 60 min) are from three series of experiments. *, P < 0.001, compared with other variables. (C) Role of cytosolic Ca2+ in morphine-induced macrophage superoxide production. Equal numbers of macrophages were incubated in media containing buffer [control (C)], THPS (10 nM), or BAPTA (50 µM) with or without morphine (Mor; 10–6 M) for 60 min. Subsequently, supernatants were collected and assayed for superoxide. Results (means±SEM at 60 min) are from three sets of experiments. *, P < 0.001, compared with control, BAPTA alone, and BAPTA + Mor; **, P < 0.001, compared with morphine alone and THPS alone. (D) Role of TGF-ß in morphine-induced macrophage superoxide production. Equal numbers of macrophages were incubated in media containing buffer [control (C)] or anti-TGF-ß antibody (A-TGFab; 1 µg/ml) with or without morphine (Mor; 10–8 M) for 60 min. Subsequently, supernatants were collected and assayed for superoxide. Results (means±SEM at 60 min) are from three sets of experiments. *, P < 0.01, compared with control and A-TGFab + Mor; **, P < 0.01, compared with Mor alone.

To determine the role of NADPH oxidase activation and the phospholipase D pathway in morphine-induced superoxideproduction, equal numbers of macrophages were incubated in media-containing buffer, DPI (10 µM, an inhibitor of NADPH oxidase), or propranolol (50 µM, an inhibitor of phospholipase D pathway) with or without morphine (10–6 M) for 60 min. At the end of the incubation period, supernatants were collected and assayed for superoxide. As shown in Figure 3B , morphine promoted (P<0.001) macrophage superoxide production. DPI inhibited this effect of morphine. Similarly, propranolol attenuated the morphine-induced superoxide production. These findings suggest that the activation of NADPH oxidase as well as the phospholipase D pathway plays a role in morphine-mediated superoxide production.

Diacyl glycerol (a product of phospholipase D activation) and Ca2+ have been shown to play a role in NADPH oxidase activation [33 ]. To determine the role of cytosolic Ca2+ in morphine-induced macrophage NADPH oxidase activation, equal numbers of macrophages were incubated in media containing buffer (control), THPS (10 nM), or BAPTA (50 µM) with or without morphine (10–6 M) for 60 min. Subsequently, supernatants were collected and assayed for superoxide. As shown in Figure 3C , morphine promoted (P<0.001) macrophage superoxide production. THPS also stimulated (P<0.001) macrophage superoxide production. BAPTA inhibited (P<0.001), but THPS accentuated (P<0.001) this effect of morphine. These results suggest that morphine-induced macrophage superoxide is modulated with alterations in cytosolic Ca2+.

To explore whether TGF-ß also modulates morphine-induced macrophage superoxide production, equal numbers of macrophages were incubated in media containing buffer (control) or anti-TGF-ß antibody (1 µg/ml) with or without morphine (10–8 M) for 60 min. Subsequently, supernatants were collected and assayed for superoxide. As shown in Figure 3D , morphine stimulated (P<0.01) macrophage superoxide production. However, this effect of morphine was partially inhibited by anti-TGF-ß antibody. These results suggest that TGF-ß also participates in morphine-induced macrophage superoxide production.

To study the role of Fas and FasL interaction in morphine-induced macrophage superoxide production, equal numbers of macrophages were incubated in media containing buffer or anti-FasL antibody (1 µg/ml) with or without morphine (10–6 M) for 120 min. Aliquots of supernatants were collected at 30, 60, 90, and 120 min. The effect of morphine peaked at 60 min. Morphine stimulated (P<0.001) the production of superoxide by macrophages (Fig. 4A ). However, this effect of morphine was partially blocked by anti-FasL antibody.



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  		Figure 4. (A) Effect of anti-FasL antibody on morphine-induced macrophage superoxide production. Equal numbers of macrophages were incubated in media containing buffer (Control) or anti-FasL antibody (Anti-FasL; 1 µg/ml), with or without morphine (10–6 M) for 60 min, followed by collection of incubation media for superoxide assay. Results (means±SEM) are from three sets of experiments. *, P < 0.001, compared with all other variables. **, P < 0.05, compared with anti-FasL. (B) Effect of NAC on morphine-induced peritoneal macrophage injury. Mice in groups of three were administered (s.c.) normal saline or morphine (20 mg/kg, B.W.) every 8 h for 72 h with or without prior treatment with normal saline or NAC. At the end of the experimental protocols, peritoneal macrophages were harvested and assayed for apoptosis. *, P < 0.001, compared with control and NAC alone; **, P < 0.01, compared with morphine alone. (C) Effect of hemin on morphine-induced peritoneal macrophage injury. Mice in groups of three were administered (s.c.) normal saline or morphine (20 mg/kg, B.W.) every 8 h for 72 h with or without prior treatment with normal saline or hemin (5 µM). At the end of the experimental protocols, peritoneal macrophages were harvested and assayed for apoptosis. *, P < 0.001, compared with control and hemin alone; **, P < 0.001, compared with morphine alone.

In vivo studies
In various models of oxidative stress-induced injury, antioxidants such as NAC have been used to provide protection against oxidative damage. To determine the role of morphine-induced oxidative injury in vivo and a possible protective role of antioxidants, mice in groups were administered with normal saline or morphine with or without prior treatment with NAC. At the end of the experimental protocols (described in Materials and Methods), peritoneal macrophages were harvested and assayed for apoptosis. Peritoneal macrophages harvested from morphine-receiving mice showed greater (P<0.001) apoptosis when compared with control mice (Fig. 4A) . This effect of morphine was partially attenuated (P<0.01) by pretreatment of morphine-receiving mice with NAC (Fig. 4B) . These findings suggest that antioxidants also protect against morphine-induced macrophage injury in vivo.

Preinduction of HO-1 has also been used to provide protection against oxidative injury in various animal models of inflammatory diseases [34 35 36 ]. To determine the role of HO-1 preinduction in morphine-induced oxidative stress, mice in groups were administered normal saline or morphine with or without prior treatment with hemin (an inducer of HO-1) or normal saline. Subsequently, peritoneal macrophages were isolated and assayed for apoptosis. Peritoneal macrophages isolated from morphine-receiving mice, pretreated with hemin, showed lower (P<0.001) apoptosis when compared with morphine-receiving mice pretreated with normal saline (Fig. 4C) . These results indicate that preinduction of HO activity provides protection against the proapoptotic effect of morphine in vivo, thus further substantiating that morphine-induced macrophage injury is mediated through oxidative stress in vivo.

Epoetin {alpha} has also been shown to act as an antioxidant in premature rabbits [37 ]. To determine the effect of epoetin {alpha} in providing cytoprotection against morphine-induced macrophage injury in vivo, mice were administered normal saline or morphine with or without prior treatment with epoetin {alpha} or normal saline. Subsequently, peritoneal macrophages were harvested and assayed for apoptosis. Peritoneal macrophages isolated from morphine-receiving mice showed greater (P<0.001) apoptosis when compared with morphine-receiving mice pretreated with epoetin {alpha} (Fig. 5 ). These findings suggest that epoetin {alpha} exerts a cytoprotective effect on morphine-induced macrophage injury in vivo.



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  		Figure 5. Effect of epoetin {alpha} on morphine-induced peritoneal macrophage injury. Mice in groups of three were administered (s.c.) normal saline or morphine (20 mg/kg, B.W.) every 8 h for 72 h with or without prior treatment with normal saline or epoetin {alpha} (Epo; 1000 U). At the end of the experimental protocols, peritoneal macrophages were harvested and assayed for apoptosis. *, P < 0.001, compared with control and Epo alone; **, P < 0.001, compared with morphine alone.


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DISCUSSION
 
In the present study, morphine promoted macrophage apoptosis, whereas antioxidants inhibited this effect of morphine, which stimulated the production of superoxide and NO by macrophages. As DPI (an inhibitor of NADPH oxidase activation) attenuated morphine-induced macrophage apoptosis, it appears that the proapoptotic effect of morphine may be mediated through the activation of NADPH oxidase. It is interesting that THPS, a Ca2+ agonist, not only enhanced morphine-induced superoxide production but also exacerbated the proapoptotic effect of morphine. Conversely, BAPTA, a Ca2+ chelator, diminished macrophage superoxide production as well as provided protection against morphine-induced macrophage injury. Propranolol, an inhibitor of the phospholipase D pathway, also inhibited morphine-induced macrophage injury, thus suggesting a role for the phospholipase pathway in NADPH oxidase activation. In addition, anti-FasL prevented morphine-induced macrophage superoxide production, thus suggesting the role of Fas in NADPH oxidase activation. As L-NAME, a NOS inhibitor, not only attenuated the morphine-induced production of NO but also inhibited macrophage apoptosis, it seems that NO is also mediating morphine-induced macrophage apoptosis. In in vivo studies, NAC, preinduction of HO activity, and pretreatment with epoetin {alpha} provided protection against morphine-induced peritoneal macrophage apoptosis.

The generation or addition of ROS/RNI triggers cell death by two ways, apoptosis and necrosis [38 ]. Necrosis occurs in response to severe injury to the cell and is accompanied by cytoplasmic and mitochondrial swelling, plasma membrane rupturing, and release of organelles into the extracellular space, triggering an inflammatory response. Conversely, apoptosis is induced by relatively milder injury and is a highly regulated process and accompanied by morphologic and biochemical processes. These include mitochondrial depolarization and alteration in phospholipid asymmetry, chromatin condensation and nuclear fragmentation, membrane blebbing, cell shrinkage, and formation of apoptotic bodies.

The role of oxidative stress in the initiation and propagation of apoptosis is indicated by studies where a rather low dose of ROS triggered apoptosis directly, and antioxidants such NAC attenuated the proapoptotic effect of ROS [39 , 40 ]. Activation of membrane-bound NADH and NADPH oxidase has been shown to be an important source of superoxide generation in a variety of cells including macrophages. The NADH–NADPH oxidase system may induce an accumulation of superoxide by multiple ways, including increased activation of xanthine oxidase, auto-oxidation of NADH, and inactivation of SOD [41 ]. In macrophage apoptosis, the role of membrane NADPH oxidase has been increasingly recognized. Assembly and activation of NADPH oxidase have also been demonstrated in response to various stimuli during the immune process [42 ]. In the present study, DPI, an inhibitor of NADPH oxidase activation, not only inhibited morphine-induced macrophage superoxide production but also provided protection from the proapoptotic effect of morphine. This suggests a role of NADPH oxidase activation in morphine-induced macrophage superoxide production and apoptosis.

Diacylglycerol and cytosolic Ca2+ have been demonstrated to play a role in the activation of NADPH oxidase [33 ]. Diacylglycerol, an arachidonic acid metabolite, is formed through activation of the phospholipase D pathway. In the present study, propranolol, an inhibitor of the phospholipase D pathway, not only inhibited morphine-induced superoxide production but also prevented morphine-induced apoptosis. Similarly, BAPTA, a cytosolic Ca2+ chelator, inhibited morphine-induced macrophage superoxide generation as well as macrophage apoptosis. Conversely, THPS, a Ca2+ agonist, enhanced macrophage superoxide production under basal- and morphine-stimulated states. These findings suggest that phospholipase D pathway activation and Ca2+ may act upstream to NADPH oxidase activation.

We previously demonstrated that morphine enhances macrophage Fas expression [43 ]. Morphine has also been shown to promote the production of FasL by macrophages. Interaction between Fas and FasL was incriminated for the induction of macrophage apoptosis [43 ]. Other investigators have demonstrated the role of NADPH oxidase activation and associated generation of ROS in Fas-mediated apoptosis [44 ]. In the present study, anti-FasL antibody prevented morphine-induced macrophage superoxide production. These findings suggest that the Fas-mediated pathway may also contribute to morphine-induced superoxide generation.

Chao et al. [45 ] reported that morphine enhanced the release of TGF-ß from PBMC. We previously reported that morphine-induced macrophage apoptosis was mediated through macrophage TGF-ß generation [12 , 13 ]. In the present study, anti-TGF-ß antibody blocked the production of NO as well as superoxide by macrophages. These findings highlight TGF-ß-mediated, downstream signaling involved in the induction of macrophage apoptosis.

Welters et al. [46 ] demonstrated the role of NO in morphine-modulated nuclear factor (NF)-{kappa}B nuclear binding in human monocytes. In these studies, morphine inhibited lipopolysaccharide-induced NF-{kappa}B nuclear binding in monocytes in a time-, concentration-, and naloxone-sensitive, dependent manner. As human monocytes have inefficient iNOS, it may be worth studying this phenomenon in murine macrophages in future studies.

In in vivo studies, administration of antioxidants such as NAC prevented the proapoptotic effect of morphine on peritoneal macrophage apoptosis. These findings further suggest that oxidative stress plays a role in macrophage apoptosis in vivo. As preinduction of HO-1 has been shown to galvanize antioxidant machinery in various animal models of oxidative stress, we used this strategy to prevent morphine-induced peritoneal macrophage injury. As expected, preinduction of HO-1 prevented morphine-induced macrophage injury. Similarly, epoetin {alpha} also prevented morphine-induced peritoneal macrophage injury.

Morphine has been demonstrated to promote lymphocyte injury in in vivo and in vitro studies [47 , 48 ]. In in vivo studies, morphine-stimulated release of glucocorticoids has also been shown to contrtibute to lymphocyte injury [48 ]. However, there are no reported data about the effect of glucocorticoids on macrophage apoptosis. It may be worth investigating in future studies.

In the present study, we propose the role of superoxide and NO in morphine-induced macrophage apoptosis. Morphine enhances the production of NO through the activation of µ receptors, TGF-ß generation, and up-regulation of macrophage iNOS. We propose that NO induces up-regulation of p53, which acts as a transcription factor for Bax, leading to translocation of cytochrome and cleavage of caspase-9 followed by activation of caspase-3 [43 ]. Conversely, morphine-induced superoxide production seems to occur as a result of activation of µ receptors, leading to the activation of the phospholipase D pathway, and an increase in cytosolic Ca2+, leading to the activation of NADPH oxidase. Accumulation of superoxide might have contributed to macrophage injury by multiple ways including the generation of hydrogen peroxide and production of peroxynitrite (a stable and toxic compound) with an interaction with NO. Alternatively, morphine-induced macrophage Fas activation may have also enhanced NADPH oxidase activation and subsequent superoxide production. In addition, Fas and FasL interaction might have led to cleavage of caspase-8, which may have induced activation of caspase-3. Thus, it appears that morphine may be contributing to the activation of caspase-3 by multiple ways. As caspase-3 activation is the final and irreversible phase of apoptosis, no wonder morphine has a great potential to direct macrophages into an apoptotic cascade.

We conclude that morphine induces oxidative stress, which contributes to macrophage injury. Strategies, which neutralize oxidative stress, can be used to provide protection against morphine-induced macrophage injury in vitro as well as in vivo.


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ACKNOWLEDGEMENTS
 
Grant RO1 DA 12111 from the National Institutes of Health (Bethesda, MD) supported this work.

Received December 17, 2003; revised January 28, 2004; accepted February 16, 2004.


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