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Department of Medicine, Long Island Jewish Medical Center, New Hyde Park, and the Long Island Campus for Albert Einstein College of Medicine, Bronx, New York
Received 30 May 2002; revised 25 September 2002; electronically published 13 December 2002.
We examined the role of heme oxygenase (HO)1 in morphine-induced decrease in macrophage migration. Morphine promoted expression of HO-1 in murine macrophages. Morphine-receiving mice (MRCs) showed decreased (P < .001) macrophage migration and increased (P < .001) occurrence of macrophage apoptosis. In in vitro studies, peritoneal macrophages harvested from MRCs also showed decreased (P < .001) migration, compared with those from control mice. Bone marrow cells isolated from MRCs showed not only decreased (P < .001) migration but also increased apoptosis. Pretreatment of MRCs with hemin not only decreased migration of macrophages further but also enhanced the apoptosis of peritoneal macrophages. On the other hand, pretreatment of MRCs with zinc protoporphyrin attenuated the effect of morphine on both macrophage migration and the occurrence of apoptosis. In in vitro studies, pretreatment of macrophages with hemin exacerbated morphine-induced apoptosis, whereas pretreatment with zinc protoporphyrin attenuated morphine-induced macrophage apoptosis.
Financial support: National Institutes on Drug Abuse, Bethesda, Maryland (grant DA 12111).
Presented in part: Meeting of the Society of Neuro Immune Pharmacology, Clearwater, Florida, October 2002 (poster 37).
Reprints or correspondence: Dr. Pravin C. Singhal, Div. of Kidney Diseases and Hypertension, Long Island Jewish Medical Center, New Hyde Park, NY 11040.
Opiate addicts are prone to infections, and at times these infections may be fatal [1, 2]. The increased incidence of infection was once attributed to the use of nonsterile and contaminated needles by drug addicts. Other investigators have emphasized the role of impurities in street heroin [3, 4]. Because mononuclear phagocytes have been demonstrated to possess opiate receptors, the direct effect of opiates on these cells is increasingly recognized [5, 8]. Moreover, morphine, an active metabolite of heroin, has been reported to modulate immune function in multiple ways. In in vitro and in vivo studies, morphine has been demonstrated to suppress the phagocytic capabilities of macrophages [9, 10] and chemotaxis of human monocytes [11]. Because morphine has been demonstrated to induce many of its biological effects through oxidative stress, it may be important to develop strategies to modulate morphine-induced oxidative stress.
Microsomal heme oxygenase (HO) is the rate-limiting enzyme for heme degradation in mammals [12]. HO cleaves heme into biliverdin and releases free iron and carbon monoxide. Two isoforms have been identified that show differences in regulation and localization [13, 15]. HO-1 is an inducible form previously known as "heat-shock protein 32." It is an integral part of the antioxidant response element of cells. It is up-regulated by a variety of factors, including hypoxia, hyperoxia, heat shock, cytokines, heavy metals, hydrogen peroxide, UV irradiation, and its substrate, heme [16, 22]. Because heme synthesis and degradation are essential for cytochromes and other heme-containing enzymes, such as catalase and nitric oxide synthase, HO-1 is expressed in many cells. Moreover, heme is pro-oxidant and induces cytotoxicity [23]. Thus, expression of HO-1 after oxidative stress may be a protective response. Furthermore, the products of heme cleavage (bilirubin and biliverdin) may act as antioxidants [24, 25]. Because 1112 mol of NADPH are consumed in an HO reaction to reduce heme iron to activate molecular oxygen, it appears that HO-1 may play a protective role under conditions of oxidative stress [26].
It is increasingly recognized that HO-1 down-regulates the inflammatory response [27, 29]. In a rat model of pleurisy, up-regulation of HO-1 significantly attenuated acute cellular inflammation [30]. Similarly, up-regulation of HO-1 led to the amelioration of chronic vascular inflammation associated with vascular rejection in antibody-induced transplant arteriosclerosis [31]. On the other hand, in a model of cardiac xenotransplant rejection, the deficiency of HO-1 exacerbated the inflammatory response [32]. Similarly, in a rat model of tubulointerstitial inflammation, deficiency of HO-1 enhanced the chronic inflammation [33]. Because monocyte migration plays an important role in the development of the inflammatory response, it appears that HO-1 may be modulating the migration of monocytes.
Some investigators have reported that morphine inhibits the migration of monocytes/macrophages [11]. This effect of morphine has been reported to be mediated by the inhibition of chemotaxis [11]. We suggest that morphine-induced macrophage apoptosis might also be contributing to morphine-induced inhibition of migration. In the present study, we have evaluated the effect of HO-1 expression on the morphine-induced modulation of macrophage apoptosis and migration.
MATERIAL AND METHODS
Mice. Male FVB/N mice were housed in groups of 4 in a laminar-flow facility (Small Animal Facility, Long Island Jewish Medical Center, New Hyde Park, NY). Rodent Diet 5001 (PMI Nutrition) and fresh water were available ad libitum. The pathogen-free status of each mouse was determined by culture of peritoneal fluid and blood samples for any bacterial growth before the start of the experimental protocols.
Experimental protocols. Eight FVB/N mice (weighing 25 g each) were used in each set of experiments. Three sets of experiments were carried out. Each set involved 2 groups of mice: 4 mice received normal saline, and the other 4 mice received normal saline containing 20 mg of morphine (donated by the National Institute on Drug Abuse) daily for 10 days; doses were administered subcutaneously under sterile conditions. To stimulate macrophage migration into the peritoneal cavity, on the eighth day, 3 mL of thioglycollate was administered intraperitoneally. Forty-eight hours later, macrophages were harvested from the peritoneal cavity, as described elsewhere [34, 36], and counted in a hemocytometer. Morphological (staining with Hoechst 33342 [H-33342; Molecular Probes] and propidium iodide [PI; Sigma Chemical]) and DNA fragmentation assays [34, 37] were carried out to evaluate apoptosis. In addition, bone marrow cells were harvested as described elsewhere [35]. Morphological assay of bone marrow cells was carried out by staining with H-33342 and PI.
To determine the role of HO-1 in the migration of macrophages into the peritoneal cavity, 24 mice in groups of 4 received either normal saline, normal saline containing morphine (20 mg/kg), normal saline containing hemin (50 M/kg; Sigma), normal saline containing zinc protoporphyrin (ZnP, 50 M/kg; Sigma), normal saline containing morphine and hemin, or normal saline containing morphine and ZnP daily for 10 days. However, mice that received a combined therapy (hemin and morphine or ZnP and morphine) received either hemin or ZnP 1 day prior to the administration of morphine. On the eighth day, 3 mL of thioglycollate was administered intraperitoneally. Forty-eight hours later, peritoneal macrophages were harvested, counted, and assayed for the occurrence of apoptosis, and the results were recorded.
Apoptosis studies. Morphological evaluation of macrophage apoptosis was performed by staining cells with H-33342 and PI, as described elsewhere [34, 36]. Cells were prepared under control and experimental conditions. At the end of the incubation period, cells were stained with H-33342 and PI, and the percentage of live, apoptotic, and necrosed cells was recorded in 8 random fields by 2 observers unaware of the experimental conditions.
To determine the effect of HO-1 modulation on morphine-induced macrophage apoptosis, equal numbers of murine macrophages (J774; American Type Culture Collection) were incubated in medium containing either buffer (control), morphine (10-8 M), hemin (5 M), ZnP (50 M), morphine and hemin, or ZnP and morphine (when morphine was used in combination, cells were primed for 16 h with either hemin or ZnP prior to morphine treatment) for 16 h, followed by the apoptosis assay. Four series of experiments were performed.
DNA fragmentation assay: gel electrophoresis. Gel electrophoresis is a simple method that is specific for isolation and confirmation of DNA fragments from apoptotic cells [37]. Because this method only picks up DNA fragments, loading of samples that do not contain any DNA fragments is not seen. DNA was isolated from equal numbers of macrophages harvested under control and experimental conditions. DNA was dissolved in gel loading buffer and separated by electrophoresis in 1.6% agarose gels.
Protein extraction and Western blotting for expression of HO-1. To determine the effect of morphine on macrophage HO-1 expression, equal numbers of macrophages (J774) were incubated in medium containing either vehicle or variable concentrations of morphine (10-1410-6 M) for 16 h. At the end of the incubation period, cells were washed, and protein extraction and blotting were performed as described elsewhere [34, 36]. The blots were treated with 0.5% bovine serum albumin and 0.1% Tween 20 in 1× PBS for 60 min at room temperature and then incubated with the primary monoclonal antibody to HO-1 (Stressgen) for 2 h at 25°C. A horseradish peroxidase