Psychiatry Research 226 (2015) 230–234
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Inflammatory response in heroin addicts undergoing methadone maintenance treatment Yuan-Yu Chan a,b, Szu-Nian Yang a, Jyh-Chyang Lin a, Junn-Liang Chang b, Jaung-Geng Lin c, Wan-Yu Lo d,e,n a
Department of Psychiatry, Taoyuan Armed Forces General Hospital, Taoyuan County, Taiwan Department of Pathology and Laboratory Medicine, Taoyuan Armed Forces General Hospital, Taoyuan County, Taiwan c Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan d Graduate Institute of Integrated Medicine, China Medical University, Taichung County, Taiwan e Department of Life Science, National Chung Hsing University, Taichung County, Taiwan b
art ic l e i nf o
a b s t r a c t
Article history: Received 12 May 2014 Received in revised form 15 November 2014 Accepted 30 December 2014 Available online 13 January 2015
Opioid addiction influences many physiological functions including reactions of the immune system. The objective of this study was to investigate the immune system function in heroin addicted patients undergoing methadone maintenance treatment (MMT) compared to healthy controls. We tested the cytokine production of IL-1β, IL-6, IL-8, IL-10 and tumor necrosis factor (TNF)-α from a group of heroin addicts (n ¼34) and healthy controls (n ¼ 20). The results show that production of IL-1β, IL-6 and IL-8 was significantly higher in the group of methadone-maintained patients than in the healthy control group. Plasma TNF-α and IL-6 levels were significantly correlated with the dairy methadone dosage administered, and the IL-1β level was significantly correlated with the duration of methadone maintenance treatment. These findings suggest that methadone maintenance treatment influences the immune system functions of opioid-dependent patients and may also induce long-term systemic inflammation. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Inflammatory Methadone Cytokine
1. Introduction Opiates addiction, both physiological and psychological, is a chronic medical illness and a major public health concern worldwide. Heroin is the most common opiate worldwide with several million addicts worldwide. The social, medical, and economic problems resulting from heroin dependence are severe and current efforts to wean addicts off heroin have often yielded limited results because of a high relapse rate and troublesome subjective symptoms. For managing heroin dependence, a methadone maintenance treatment (MMT) was introduced in the mid1960s (McLachlan et al., 1993), although methadone has been effective in heroin maintenance treatment as a long-acting opioid receptor agonist that can increase adherence to treatment, and reduce illicit drug use and mortality. The opioid relapse rate after methadone discontinuation still remains high. Some studies have revealed that opioid drugs, such as morphine and heroin, exert
n Correspondence to: Graduate Institute of Integrated Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan. E-mail addresses: [email protected] (Y.-Y. Chan), [email protected] (S.-N. Yang), [email protected] (J.-C. Lin), [email protected] (J.-L. Chang), [email protected] (J.-G. Lin), [email protected] (W.-Y. Lo).
http://dx.doi.org/10.1016/j.psychres.2014.12.053 0165-1781/& 2015 Elsevier Ireland Ltd. All rights reserved.
negative effects on the immune system (Sacerdote, 2006; Vallejo et al., 2004). Heroin can affect various physiological functions of the body, including immune system reactions, by directly acting on the opioid receptors present on lymphocytes and macrophages (Nelson et al., 2000; Stefano et al., 1996) or by indirectly acting on the central nervous system (CNS) (Fecho et al., 1996; McCarthy et al., 2001; Peterson et al., 1993, 1998). Cytokines are small molecules that function as immune system messengers. Several in vitro studies have reported that acute morphine treatment alters the secretion of various cytokines, including tumor necrosis factor (TNF)-α (Kapasi et al., 2000; Zubelewicz et al., 2000). In a neuropathic rat model, transected lumbar spinal cords that were chronically treated with morphine revealed increased proinflammatory cytokine secretion and glial cell overactivation (Raghavendra et al., 2002). Another rat study revealed that a combination of heroin and cocaine caused marked neurotoxicity (Cunha-Oliveira et al., 2010). Chronic opioid treatment markedly increased the plasma and drug addiction related cytokine expression in the brain (Chen et al., 2012b), a phenomenon suggesting that chronic opioid use can cause inflammation, neuronal degeneration, and neuronal damage. Various studies on heroin-dependent patients have repeatedly demonstrated that opioids consistently cause immunosuppression (Donahoe and Vlahov, 1998; Friedman et al., 2003; Neri et al., 2005).
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In addition, a study reported that chronic intrathecal morphine administration upregulated IL-6 expression in the cerebrospinal fluid (Zin et al., 2010). Moreover, inducible nitric oxide synthase (iNOS) and cytokine expression were detected in noradrenergic locus coeruleus cells in the brains of heroin-dependent patients (Dyuizen and Lamash, 2009). Chronic morphine use substantially decreases the number of dopaminergic neurons in the ventral tegmental area (Chu et al., 2008; Sklair-Tavron et al., 1996) and elevates serum IL-6 levels by activating the hypothalamic–pituitary–adrenal and autonomic nervous systems (Bertolucci et al., 1996; Houghtling and Bayer, 2002). In the peripheral nervous system, opioids indirectly affect peripheral cytokine expression that can cross the blood–brain barrier (Simard and Rivest, 2005), and high peripheral cytokine levels induce sustained neuronal inflammation, damage, and degeneration in the central nervous system (Qin et al., 2007). Therefore, the present study aimed to investigate the primary inflammatory cytokine expressions in heroin-dependent patients receiving MMT, and compared with the corresponding expressions in healthy controls. We monitored peripheral plasma cytokine (IL-1β, IL-6, IL-8, IL-10 and TNF-α) levels because monitoring these levels in the human brain is unethical and challenging. The hypothesis was that long-term MMT and higher methadone dosage treatment might affect immune system functions.
2. Methods 2.1. Subjects The study was conducted at the outpatient clinic of the Department of Psychiatry at Taoyuan Armed Forces General Hospital, a regional teaching hospital in Taiwan. Subjects were recruited from January 2014 to November 2014 through referrals from psychiatrists and advertisements at the clinic. Only male participants older than 20 years who satisfied the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria for opiate dependence and who had been receiving MMT for more than 1 month were included in the study. Participants with a history of antidepressant or neuroleptic medication use or who were HIV positive or had any severe physical illness, bleeding disorders, or were taking anticoagulant drugs were excluded. According to the aforementioned criteria, 34 heroin addicts and 20 healthy blood donors (controls) were enrolled in this study for assessment. The study design, consent forms and procedures were approved by the Institutional Review Board of Tri-Service General Hospital National Defense Medical Center in Taiwan (IRB no. TY102-03).
2.2. Data collection The patient inclusion form requested the following information: patient identification, heroin and amphetamine abuse history (years), daily consumption of methadone, duration of methadone maintained (months), legal problem, marital status, Beck Anxiety Inventory (BAI) score, Beck Depression Inventory II (BDI-II) score, Pittsburgh Sleep Quality Index (PSQI) score and opioid urine screen. The anxiety symptom severity was measured using the BAI (Beck et al., 1988), which lists 21 symptoms of anxiety, such as feeling hot, scared, or nervous. Each item was rated on a four-point Likert scale, ranging from 0 (not bothered) to 3 (severely bothered), yielding a maximal total score of 63 points. According to the BAI manual, total scores ranging from 0 to 7 indicated minimal anxiety, those ranging from 8 to 15 indicated mild anxiety, those ranging from 16 to 25 indicated moderate anxiety, and those ranging from 26 to 63 indicated severe anxiety. The symptoms of depression were measured using the BDI-II, which comprised 13 items, rated from 0 to 3, to evaluate depression according to the degree to which it reflected the patient's state during the previous week. The BDI-II has a high reliability and concurrent validity (Beck and Steer, 1984). According to the BDI-II manual, total scores indicated four levels of depression: minimal depression (0–13), mild depression (14–19), moderate depression (20–28), and severe depression (29–63). Sleep quality was assessed using the Taiwanese version of the PSQI (Buysse et al., 1989), which has demonstrated reliability and validity (Tsai et al., 2005); this system was used to evaluate sleep disturbances by using seven subscales. Each subscale was rated on a four-point scoring scale (0–3 points; 3 indicates a greater effect), and all the subscale scores were summed together to yield a global score (0–21). Higher scores indicated severe sleep disturbance, whereas a global score exceeding 5 indicated poor sleep (Buysse et al., 1989).
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2.3. Determination of inflammatory cytokine We collected the blood samples (6 ml) from all patients between 13:00 and 17:00 p.m., after their daily methadone intake. Blood samples were collected and centrifuged at 500g for 5 min at 4 1C to extract the serum. The serum samples from both the patients and healthy donors were stored at 80 1C until analysis. A panel of cytokines was quantified using the Human Inflammatory Cytokines Kit (BD Biosciences Pharmingen, San Diego, CA, USA) by flow cytometry (BD FACSCanto™ System; Becton Dickinson Corp., San Jose, CA, USA). In the analysis, six bead populations with distinct fluorescence intensities were coated with six type specific antibodies to capture and simultaneously determine the various cytokines. The cytokine-captured beads were incubated with the secondary antibodies to form the sandwich complexes. After incubation, washing, and acquisition, the results were determined by BD array software (FCAP Array V 3.0). 2.4. Statistical analysis Statistical analyses were performed using SPSS 18.0 statistical software for Windows. All data are presented as the mean7 standard error of the mean (SEM). Student's t test was used for the statistical evaluations between the methadonemaintained patients (patient group) and the healthy controls (control group). The correlation among the different parameters in the patient group was evaluated using the mean of the Pearson correlation test; p o 0.05 was considered statistically significant for all tests.
3. Result Table 1 summarizes the sociodemographic characteristics of all participants. No significant differences were observed regarding age, employment status, and marital status between the control and the patient groups. In the patient group, 2.9% of the patients reported legal problems. The mean methadone dose was 59.1 7 5.6 mg. The mean duration of MMT was 23.56 7 5.6 months. On average, the reported history of heroin and amphetamine abuse was 8.6 71.1 and 4.2 71.0 years, respectively. In the 30 days before baseline assessment, 50.0% and 17.65% of the patients reported occasional heroin and amphetamine use, respectively. In addition, the average BAI score was 9.65 71.25, which indicated mild anxiety. The average BDI-II score was 15.56 72.36, indicating mild depression, and the average PSQI score was 10.79 71.24, indicating poor sleep. The leukocytes obtained from the control group participants were cultivated alone, and the levels of the cytokines IL-1β, IL-6, IL-8, IL-10 and TNF-α were determined using ELISA. The mean IL1β, IL-6 and IL-8 were significantly higher in the patient group than those in the control group; in addition, similar finding were observed regarding the corresponding median levels in both groups. The median number of IL-1β, IL-6 and IL-8 levels was higher in the patient group (IL-1β:1131.56 fg/ml, IL-6:472.27 fg/ml, and IL-8:15,998.41 fg/ml) than that in the control group
Table 1 Characteristics of the study participants.
Age (years) Employed (%) Married (%) IL-1β (fg/m) IL-6 (fg/m) IL-8 (fg/m) IL-10 (fg/m) TNF-α (fg/m)
Methadone-maintained patients (n¼ 34)
Health controls (n¼20)
40.127 1.20 73.3 41.1 2822.83 7 580.58nn 1834.647 603.55n 57,849.56 7 14,377.92nn 631.75 7260.97 912.82 7465.72
37.85 72.07 70 65 51.08 7 12.33 499.97 207.41 2315.487 292.44 320.357 120.68 38.51 714.24
Values are mean 7 SEM. n
p o 0.05. po 0.01.
nn
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Table 2 The correlation between the different parameters in the methadone-maintained patient group.
IL-1β IL-6 IL-8 IL-10 TNF-α n
Methadone dosage
Duration of methadone maintained (months)
Heroin abuse history Amphetamine abuse BAI (years) history (years)
BDI
PSQI
p Pearson Value correlation
p Value
Pearson correlation
p Pearson Value correlation
p Pearson Value correlation
p Pearson Value correlation
0.18 0.03 0.33 0.17 0.02
o 0.01 0.13 0.29 0.26 0.24
0.45nn 0.26 0.19 0.20 0.21
0.78 0.31 0.67 0.33 0.49
0.24 0.38n 0.17 0.24 0.40n
0.05 0.18 0.08 0.17 0.12
p Pearson Value correlation
p Pearson Value correlation
0.38 0.15 0.93 0.54 0.86
0.43 0.94 0.56 0.21 0.64
0.16 0.25 0.02 0.11 0.03
0.14 0.14 0.10 0.22 0.08
0.46 0.57 0.75 0.94 0.44
0.13 0.10 0.06 0.01 0.14
0.68 0.47 0.68 0.07 0.35
0.07 0.13 0.07 0.31 0.17
po 0.05. p o0.01.
nn
(IL-1β:37.3 fg/ml, IL-6:61.7 fg/ml, and IL-8:2572.5 fg/ml). However, no significant intergroup differences were observed in the IL-10 and TNF-α levels. In the patient group, no significant correlation was observed between the immune system parameters and the heroin or amphetamine abuse history, anxiety symptoms, depression symptoms, sleep quality and age. Moreover, a significant positive correlation was observed between the duration of MMT and the IL-1β levels (correlation coefficient: 0.453, po0.01), between the methadone dosage and the IL-6 levels (correlation coefficient: 0.375, p¼0.03), and between the methadone dosage and the TNF-α levels (correlation coefficient 0.402, p¼ 0.02; Table 2).
4. Discussion In the present study, the IL-1β, IL-6 and IL-8 levels were significantly higher in the patient group than those in the control group. In addition, a significant correlation was observed between the plasma TNF-α and IL-6 levels and the methadone dosage. Moreover, a significant correlation was observed between the IL1β levels and the duration of MMT, indicating that long-term MMT and increased methadone dosage may induce systemic inflammation. Studies have reported that chronic exposure to opioids can cause atrophy of the dendrites, abnormal neurogenesis, and neurodegeneration in rats (Eisch et al., 2000; Robinson and Kolb, 1999). Another study (Wang et al., 2011) reported that methadone exerts neurotoxic effects because it is a long-acting synthetic opioid that impairs brain white-matter integrity of the brain in heroin users receiving long-term MMT; moreover, a significant correlation was observed between the methadone dose and the degree of white matter injury (Wang et al., 2011). Furthermore, methadone has been demonstrated to impair cognitive function and sustained attention (Mintzer and Stitzer, 2002; Prosser et al., 2009) and damage striatal dopamine transporters in humans (Shi et al., 2008). Thus, increasing the methadone dose and duration of MMT might increase the cytokine production and further worsen the functional and structural impairment in the brain among patients receiving long-term MMT. The cytokines that are frequently observed in patients with major psychiatric disorders are IL-1, IL-6 and TNF-α, all of which exert both positive and negative impacts on central nervous system (CNS). All types of brain cells, including astrocytes, microglia, neurons, oligodendrocytes and endothelial cells, produce IL-1; however, microglia are the primary source (Pinteaux et al., 2009). IL-1 is primarily produced by cells from the mononuclear phagocytic linage (Commins et al., 2010) and can induce neurotoxicity by prompting neurotoxins secretion from astrocytes or by direct toxic effects. Thus, IL-1 is a crucial mediator of neurodegeneration caused by cerebral injuries such as ischemia and traumatic brain
damage (Allan and Rothwell, 2001; Arvin et al., 1996; Boutin et al., 2001; Lucas et al., 2006). In addition, exposure to a stressor affects the IL-1 expression in the brain and the periphery. IL-1β expression is frequently elevated in the CNS of animals exposed to an acute psychological stressor and in the plasma of humans exposed to a speech task stressor (Ackerman et al., 1998; Nguyen et al., 1998). The effects of chronic stress on IL-1β expression remain unclear. Some studies have reported that chronic stress increases the IL-1β expression, whereas other studies have demonstrated IL1β suppression (McCarthy et al., 1992; Nguyen et al., 1998). Reportedly, IL-6 was considered to exert both inflammatory and antiinflammatory actions (Gadient and Otten, 1997). IL-6 secretion can be induced by psychological stress in animals and may be neurotoxic at chronically elevated levels (Allan and Rothwell, 2001). This IL-6 elevation is likely induced by other molecules, including cytokines such as IL-1β and TNF-α (Lucas et al., 2006). IL-6 levels were elevated in patients with various CNS disorders, such as epilepsy, depression, Alzheimer's disease, Parkinson's disease and schizophrenia (Lucas et al., 2006) and studies have proposed that IL-6 is a neurodegenerative factor that is involved in the pathomechanisms of Alzheimer's disease (BlumDegen et al., 1995; Hull et al., 1996a, 1996b). Moreover, the neurotoxic effects of IL-6 are not as obvious as TNF-α or IL-1 because of its inability to directly induce iNOS expression in the CNS (Munoz-Fernandez et al., 1994). TNF-α is a central mediator of tissue inflammation and has been implicated in the pathogenesis of several neurological conditions, such as Alzheimer's disease, Parkinson's disease and ischemia, because its expression increases in the CNS after tissue damage or degenerative processes (Arvin et al., 1996; Lucas et al., 2006; Munhoz et al., 2008). In humans, psychological stressors resulted in increased circulating TNF-α levels, and social stress results in TNFR-2 receptor upregulation (Slavich et al., 2010). In addition, studies have confirmed immune activation by IL-1, IL-6 and TNF-α in patients with bipolar disorder and depression (Berk et al., 2011; Drexhage et al., 2010; Maes, 1994; Myint et al., 2005; Sluzewska et al., 1996). Moreover, increased IL-8 levels were reported in patients with fibromyalgia (Rodriguez-Pinto et al., 2014). Previous studies using experimental models have demonstrated that the production of certain cytokines increases within a few minutes after morphine administration (Pacifici et al., 2000) and that this increase is more pronounced among the proinflammatory cytokines (Peng et al., 2000). Furthermore, augmented IL-6 production after morphine treatment has been observed in patients receiving morphine for postoperative pain management (Beilin et al., 2003) and in various experimental models (Zubelewicz et al., 1999). Chen et al. (2012a) discovered that plasma cytokine levels of TNF-α and IL-8 were significantly higher in the heroin-dependent patients receiving MMT than the corresponding levels in healthy controls. Another study reported that the production of IL-2, IFNγ
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and IL-10 was greater in heroin addicts than it was in healthy controls; this increased production was partially or completely normalized in patients receiving MMT (Zajicova et al., 2004). In addition, Zajicova et al. reported that peripheral blood leukocytes from heroin addicts exhibited a significantly enhanced proliferative response after Con A stimulation than did the peripheral blood leukocytes from control donors. However, in the present study, the patients receiving MMT did not exhibit significantly higher production of IL-10, and TNF-α, all of which may have been partially or completely normalized after the MMT. This study has several limitations. First, all the participants were all male and were recruited through a methadone replacement therapy outpatient department in Taiwan; thus, the results must be cautiously generalized to patient populations of both sexes and belonging to other nations. Second, the sample size may have been too small, and thus, larger-scale studies are required to confirm these results. Third, certain participants reported occasionally using other illicit drugs, such as amphetamine. Because amphetamine affects immune responses (Peerzada et al., 2013), a possible interaction of this substance with opioid-induced immune alterations should be considered. Moreover, the relatively small number of patients did not facilitate adequately evaluating the immune effects of polydrug abuse compared to those of single drug abuse. Therefore, additional studies are warranted to address this concern. Finally, some studies have attempted to determine whether the amelioration of immune responses observed with MMT depends on the drug profile or on the life style changes (Alonzo and Bayer, 2002; McLachlan et al., 1993). Several aspects of lifestyle associated with injecting heroin use including numerous infectious disease, malnutrition, stress, sleep deprivation and alcohol consumption are known to impair function (Blank et al., 1991; Glaser and Kiecolt-Glaser, 1988). The present study did not analyze such changes in lifestyle and quality of life, addiction severity, financial problems, duration of imprisonment, or any recent stressful event that could have influenced cytokine production.
5. Conclusion In conclusion, our results revealed that opioid dependence influences the functions of the immune system in patients receiving MMT and may induce long-term systemic inflammation. Although MMT is an effective method for treating heroindependent patients, long-term MMT and increases methadone dosage might exacerbate the consequences of systemic inflammation and cause subsequent neuronal inflammation and damage. Future well-controlled longitudinal studies are required to evaluate the immune response in such patients before and during MMT and assess the benefits of methadone in relationship to immune responses.
Conflicts of interests The author(s) declare that they have no conflict of interests.
Acknowledgments This work was supported by Grant NSC 102-2320-B-039-024 from the National Science Council. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Inflammatory response in heroin addicts undergoing methadone maintenance treatment Yuan-Yu Chan a,b, Szu-Nian Yang a, Jyh-Chyang Lin a, Junn-Liang Chang b, Jaung-Geng Lin c, Wan-Yu Lo d,e,n a
Department of Psychiatry, Taoyuan Armed Forces General Hospital, Taoyuan County, Taiwan Department of Pathology and Laboratory Medicine, Taoyuan Armed Forces General Hospital, Taoyuan County, Taiwan c Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan d Graduate Institute of Integrated Medicine, China Medical University, Taichung County, Taiwan e Department of Life Science, National Chung Hsing University, Taichung County, Taiwan b
art ic l e i nf o
a b s t r a c t
Article history: Received 12 May 2014 Received in revised form 15 November 2014 Accepted 30 December 2014 Available online 13 January 2015
Opioid addiction influences many physiological functions including reactions of the immune system. The objective of this study was to investigate the immune system function in heroin addicted patients undergoing methadone maintenance treatment (MMT) compared to healthy controls. We tested the cytokine production of IL-1β, IL-6, IL-8, IL-10 and tumor necrosis factor (TNF)-α from a group of heroin addicts (n ¼34) and healthy controls (n ¼ 20). The results show that production of IL-1β, IL-6 and IL-8 was significantly higher in the group of methadone-maintained patients than in the healthy control group. Plasma TNF-α and IL-6 levels were significantly correlated with the dairy methadone dosage administered, and the IL-1β level was significantly correlated with the duration of methadone maintenance treatment. These findings suggest that methadone maintenance treatment influences the immune system functions of opioid-dependent patients and may also induce long-term systemic inflammation. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Inflammatory Methadone Cytokine
1. Introduction Opiates addiction, both physiological and psychological, is a chronic medical illness and a major public health concern worldwide. Heroin is the most common opiate worldwide with several million addicts worldwide. The social, medical, and economic problems resulting from heroin dependence are severe and current efforts to wean addicts off heroin have often yielded limited results because of a high relapse rate and troublesome subjective symptoms. For managing heroin dependence, a methadone maintenance treatment (MMT) was introduced in the mid1960s (McLachlan et al., 1993), although methadone has been effective in heroin maintenance treatment as a long-acting opioid receptor agonist that can increase adherence to treatment, and reduce illicit drug use and mortality. The opioid relapse rate after methadone discontinuation still remains high. Some studies have revealed that opioid drugs, such as morphine and heroin, exert
n Correspondence to: Graduate Institute of Integrated Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan. E-mail addresses: [email protected] (Y.-Y. Chan), [email protected] (S.-N. Yang), [email protected] (J.-C. Lin), [email protected] (J.-L. Chang), [email protected] (J.-G. Lin), [email protected] (W.-Y. Lo).
http://dx.doi.org/10.1016/j.psychres.2014.12.053 0165-1781/& 2015 Elsevier Ireland Ltd. All rights reserved.
negative effects on the immune system (Sacerdote, 2006; Vallejo et al., 2004). Heroin can affect various physiological functions of the body, including immune system reactions, by directly acting on the opioid receptors present on lymphocytes and macrophages (Nelson et al., 2000; Stefano et al., 1996) or by indirectly acting on the central nervous system (CNS) (Fecho et al., 1996; McCarthy et al., 2001; Peterson et al., 1993, 1998). Cytokines are small molecules that function as immune system messengers. Several in vitro studies have reported that acute morphine treatment alters the secretion of various cytokines, including tumor necrosis factor (TNF)-α (Kapasi et al., 2000; Zubelewicz et al., 2000). In a neuropathic rat model, transected lumbar spinal cords that were chronically treated with morphine revealed increased proinflammatory cytokine secretion and glial cell overactivation (Raghavendra et al., 2002). Another rat study revealed that a combination of heroin and cocaine caused marked neurotoxicity (Cunha-Oliveira et al., 2010). Chronic opioid treatment markedly increased the plasma and drug addiction related cytokine expression in the brain (Chen et al., 2012b), a phenomenon suggesting that chronic opioid use can cause inflammation, neuronal degeneration, and neuronal damage. Various studies on heroin-dependent patients have repeatedly demonstrated that opioids consistently cause immunosuppression (Donahoe and Vlahov, 1998; Friedman et al., 2003; Neri et al., 2005).
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In addition, a study reported that chronic intrathecal morphine administration upregulated IL-6 expression in the cerebrospinal fluid (Zin et al., 2010). Moreover, inducible nitric oxide synthase (iNOS) and cytokine expression were detected in noradrenergic locus coeruleus cells in the brains of heroin-dependent patients (Dyuizen and Lamash, 2009). Chronic morphine use substantially decreases the number of dopaminergic neurons in the ventral tegmental area (Chu et al., 2008; Sklair-Tavron et al., 1996) and elevates serum IL-6 levels by activating the hypothalamic–pituitary–adrenal and autonomic nervous systems (Bertolucci et al., 1996; Houghtling and Bayer, 2002). In the peripheral nervous system, opioids indirectly affect peripheral cytokine expression that can cross the blood–brain barrier (Simard and Rivest, 2005), and high peripheral cytokine levels induce sustained neuronal inflammation, damage, and degeneration in the central nervous system (Qin et al., 2007). Therefore, the present study aimed to investigate the primary inflammatory cytokine expressions in heroin-dependent patients receiving MMT, and compared with the corresponding expressions in healthy controls. We monitored peripheral plasma cytokine (IL-1β, IL-6, IL-8, IL-10 and TNF-α) levels because monitoring these levels in the human brain is unethical and challenging. The hypothesis was that long-term MMT and higher methadone dosage treatment might affect immune system functions.
2. Methods 2.1. Subjects The study was conducted at the outpatient clinic of the Department of Psychiatry at Taoyuan Armed Forces General Hospital, a regional teaching hospital in Taiwan. Subjects were recruited from January 2014 to November 2014 through referrals from psychiatrists and advertisements at the clinic. Only male participants older than 20 years who satisfied the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria for opiate dependence and who had been receiving MMT for more than 1 month were included in the study. Participants with a history of antidepressant or neuroleptic medication use or who were HIV positive or had any severe physical illness, bleeding disorders, or were taking anticoagulant drugs were excluded. According to the aforementioned criteria, 34 heroin addicts and 20 healthy blood donors (controls) were enrolled in this study for assessment. The study design, consent forms and procedures were approved by the Institutional Review Board of Tri-Service General Hospital National Defense Medical Center in Taiwan (IRB no. TY102-03).
2.2. Data collection The patient inclusion form requested the following information: patient identification, heroin and amphetamine abuse history (years), daily consumption of methadone, duration of methadone maintained (months), legal problem, marital status, Beck Anxiety Inventory (BAI) score, Beck Depression Inventory II (BDI-II) score, Pittsburgh Sleep Quality Index (PSQI) score and opioid urine screen. The anxiety symptom severity was measured using the BAI (Beck et al., 1988), which lists 21 symptoms of anxiety, such as feeling hot, scared, or nervous. Each item was rated on a four-point Likert scale, ranging from 0 (not bothered) to 3 (severely bothered), yielding a maximal total score of 63 points. According to the BAI manual, total scores ranging from 0 to 7 indicated minimal anxiety, those ranging from 8 to 15 indicated mild anxiety, those ranging from 16 to 25 indicated moderate anxiety, and those ranging from 26 to 63 indicated severe anxiety. The symptoms of depression were measured using the BDI-II, which comprised 13 items, rated from 0 to 3, to evaluate depression according to the degree to which it reflected the patient's state during the previous week. The BDI-II has a high reliability and concurrent validity (Beck and Steer, 1984). According to the BDI-II manual, total scores indicated four levels of depression: minimal depression (0–13), mild depression (14–19), moderate depression (20–28), and severe depression (29–63). Sleep quality was assessed using the Taiwanese version of the PSQI (Buysse et al., 1989), which has demonstrated reliability and validity (Tsai et al., 2005); this system was used to evaluate sleep disturbances by using seven subscales. Each subscale was rated on a four-point scoring scale (0–3 points; 3 indicates a greater effect), and all the subscale scores were summed together to yield a global score (0–21). Higher scores indicated severe sleep disturbance, whereas a global score exceeding 5 indicated poor sleep (Buysse et al., 1989).
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2.3. Determination of inflammatory cytokine We collected the blood samples (6 ml) from all patients between 13:00 and 17:00 p.m., after their daily methadone intake. Blood samples were collected and centrifuged at 500g for 5 min at 4 1C to extract the serum. The serum samples from both the patients and healthy donors were stored at 80 1C until analysis. A panel of cytokines was quantified using the Human Inflammatory Cytokines Kit (BD Biosciences Pharmingen, San Diego, CA, USA) by flow cytometry (BD FACSCanto™ System; Becton Dickinson Corp., San Jose, CA, USA). In the analysis, six bead populations with distinct fluorescence intensities were coated with six type specific antibodies to capture and simultaneously determine the various cytokines. The cytokine-captured beads were incubated with the secondary antibodies to form the sandwich complexes. After incubation, washing, and acquisition, the results were determined by BD array software (FCAP Array V 3.0). 2.4. Statistical analysis Statistical analyses were performed using SPSS 18.0 statistical software for Windows. All data are presented as the mean7 standard error of the mean (SEM). Student's t test was used for the statistical evaluations between the methadonemaintained patients (patient group) and the healthy controls (control group). The correlation among the different parameters in the patient group was evaluated using the mean of the Pearson correlation test; p o 0.05 was considered statistically significant for all tests.
3. Result Table 1 summarizes the sociodemographic characteristics of all participants. No significant differences were observed regarding age, employment status, and marital status between the control and the patient groups. In the patient group, 2.9% of the patients reported legal problems. The mean methadone dose was 59.1 7 5.6 mg. The mean duration of MMT was 23.56 7 5.6 months. On average, the reported history of heroin and amphetamine abuse was 8.6 71.1 and 4.2 71.0 years, respectively. In the 30 days before baseline assessment, 50.0% and 17.65% of the patients reported occasional heroin and amphetamine use, respectively. In addition, the average BAI score was 9.65 71.25, which indicated mild anxiety. The average BDI-II score was 15.56 72.36, indicating mild depression, and the average PSQI score was 10.79 71.24, indicating poor sleep. The leukocytes obtained from the control group participants were cultivated alone, and the levels of the cytokines IL-1β, IL-6, IL-8, IL-10 and TNF-α were determined using ELISA. The mean IL1β, IL-6 and IL-8 were significantly higher in the patient group than those in the control group; in addition, similar finding were observed regarding the corresponding median levels in both groups. The median number of IL-1β, IL-6 and IL-8 levels was higher in the patient group (IL-1β:1131.56 fg/ml, IL-6:472.27 fg/ml, and IL-8:15,998.41 fg/ml) than that in the control group
Table 1 Characteristics of the study participants.
Age (years) Employed (%) Married (%) IL-1β (fg/m) IL-6 (fg/m) IL-8 (fg/m) IL-10 (fg/m) TNF-α (fg/m)
Methadone-maintained patients (n¼ 34)
Health controls (n¼20)
40.127 1.20 73.3 41.1 2822.83 7 580.58nn 1834.647 603.55n 57,849.56 7 14,377.92nn 631.75 7260.97 912.82 7465.72
37.85 72.07 70 65 51.08 7 12.33 499.97 207.41 2315.487 292.44 320.357 120.68 38.51 714.24
Values are mean 7 SEM. n
p o 0.05. po 0.01.
nn
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Table 2 The correlation between the different parameters in the methadone-maintained patient group.
IL-1β IL-6 IL-8 IL-10 TNF-α n
Methadone dosage
Duration of methadone maintained (months)
Heroin abuse history Amphetamine abuse BAI (years) history (years)
BDI
PSQI
p Pearson Value correlation
p Value
Pearson correlation
p Pearson Value correlation
p Pearson Value correlation
p Pearson Value correlation
0.18 0.03 0.33 0.17 0.02
o 0.01 0.13 0.29 0.26 0.24
0.45nn 0.26 0.19 0.20 0.21
0.78 0.31 0.67 0.33 0.49
0.24 0.38n 0.17 0.24 0.40n
0.05 0.18 0.08 0.17 0.12
p Pearson Value correlation
p Pearson Value correlation
0.38 0.15 0.93 0.54 0.86
0.43 0.94 0.56 0.21 0.64
0.16 0.25 0.02 0.11 0.03
0.14 0.14 0.10 0.22 0.08
0.46 0.57 0.75 0.94 0.44
0.13 0.10 0.06 0.01 0.14
0.68 0.47 0.68 0.07 0.35
0.07 0.13 0.07 0.31 0.17
po 0.05. p o0.01.
nn
(IL-1β:37.3 fg/ml, IL-6:61.7 fg/ml, and IL-8:2572.5 fg/ml). However, no significant intergroup differences were observed in the IL-10 and TNF-α levels. In the patient group, no significant correlation was observed between the immune system parameters and the heroin or amphetamine abuse history, anxiety symptoms, depression symptoms, sleep quality and age. Moreover, a significant positive correlation was observed between the duration of MMT and the IL-1β levels (correlation coefficient: 0.453, po0.01), between the methadone dosage and the IL-6 levels (correlation coefficient: 0.375, p¼0.03), and between the methadone dosage and the TNF-α levels (correlation coefficient 0.402, p¼ 0.02; Table 2).
4. Discussion In the present study, the IL-1β, IL-6 and IL-8 levels were significantly higher in the patient group than those in the control group. In addition, a significant correlation was observed between the plasma TNF-α and IL-6 levels and the methadone dosage. Moreover, a significant correlation was observed between the IL1β levels and the duration of MMT, indicating that long-term MMT and increased methadone dosage may induce systemic inflammation. Studies have reported that chronic exposure to opioids can cause atrophy of the dendrites, abnormal neurogenesis, and neurodegeneration in rats (Eisch et al., 2000; Robinson and Kolb, 1999). Another study (Wang et al., 2011) reported that methadone exerts neurotoxic effects because it is a long-acting synthetic opioid that impairs brain white-matter integrity of the brain in heroin users receiving long-term MMT; moreover, a significant correlation was observed between the methadone dose and the degree of white matter injury (Wang et al., 2011). Furthermore, methadone has been demonstrated to impair cognitive function and sustained attention (Mintzer and Stitzer, 2002; Prosser et al., 2009) and damage striatal dopamine transporters in humans (Shi et al., 2008). Thus, increasing the methadone dose and duration of MMT might increase the cytokine production and further worsen the functional and structural impairment in the brain among patients receiving long-term MMT. The cytokines that are frequently observed in patients with major psychiatric disorders are IL-1, IL-6 and TNF-α, all of which exert both positive and negative impacts on central nervous system (CNS). All types of brain cells, including astrocytes, microglia, neurons, oligodendrocytes and endothelial cells, produce IL-1; however, microglia are the primary source (Pinteaux et al., 2009). IL-1 is primarily produced by cells from the mononuclear phagocytic linage (Commins et al., 2010) and can induce neurotoxicity by prompting neurotoxins secretion from astrocytes or by direct toxic effects. Thus, IL-1 is a crucial mediator of neurodegeneration caused by cerebral injuries such as ischemia and traumatic brain
damage (Allan and Rothwell, 2001; Arvin et al., 1996; Boutin et al., 2001; Lucas et al., 2006). In addition, exposure to a stressor affects the IL-1 expression in the brain and the periphery. IL-1β expression is frequently elevated in the CNS of animals exposed to an acute psychological stressor and in the plasma of humans exposed to a speech task stressor (Ackerman et al., 1998; Nguyen et al., 1998). The effects of chronic stress on IL-1β expression remain unclear. Some studies have reported that chronic stress increases the IL-1β expression, whereas other studies have demonstrated IL1β suppression (McCarthy et al., 1992; Nguyen et al., 1998). Reportedly, IL-6 was considered to exert both inflammatory and antiinflammatory actions (Gadient and Otten, 1997). IL-6 secretion can be induced by psychological stress in animals and may be neurotoxic at chronically elevated levels (Allan and Rothwell, 2001). This IL-6 elevation is likely induced by other molecules, including cytokines such as IL-1β and TNF-α (Lucas et al., 2006). IL-6 levels were elevated in patients with various CNS disorders, such as epilepsy, depression, Alzheimer's disease, Parkinson's disease and schizophrenia (Lucas et al., 2006) and studies have proposed that IL-6 is a neurodegenerative factor that is involved in the pathomechanisms of Alzheimer's disease (BlumDegen et al., 1995; Hull et al., 1996a, 1996b). Moreover, the neurotoxic effects of IL-6 are not as obvious as TNF-α or IL-1 because of its inability to directly induce iNOS expression in the CNS (Munoz-Fernandez et al., 1994). TNF-α is a central mediator of tissue inflammation and has been implicated in the pathogenesis of several neurological conditions, such as Alzheimer's disease, Parkinson's disease and ischemia, because its expression increases in the CNS after tissue damage or degenerative processes (Arvin et al., 1996; Lucas et al., 2006; Munhoz et al., 2008). In humans, psychological stressors resulted in increased circulating TNF-α levels, and social stress results in TNFR-2 receptor upregulation (Slavich et al., 2010). In addition, studies have confirmed immune activation by IL-1, IL-6 and TNF-α in patients with bipolar disorder and depression (Berk et al., 2011; Drexhage et al., 2010; Maes, 1994; Myint et al., 2005; Sluzewska et al., 1996). Moreover, increased IL-8 levels were reported in patients with fibromyalgia (Rodriguez-Pinto et al., 2014). Previous studies using experimental models have demonstrated that the production of certain cytokines increases within a few minutes after morphine administration (Pacifici et al., 2000) and that this increase is more pronounced among the proinflammatory cytokines (Peng et al., 2000). Furthermore, augmented IL-6 production after morphine treatment has been observed in patients receiving morphine for postoperative pain management (Beilin et al., 2003) and in various experimental models (Zubelewicz et al., 1999). Chen et al. (2012a) discovered that plasma cytokine levels of TNF-α and IL-8 were significantly higher in the heroin-dependent patients receiving MMT than the corresponding levels in healthy controls. Another study reported that the production of IL-2, IFNγ
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and IL-10 was greater in heroin addicts than it was in healthy controls; this increased production was partially or completely normalized in patients receiving MMT (Zajicova et al., 2004). In addition, Zajicova et al. reported that peripheral blood leukocytes from heroin addicts exhibited a significantly enhanced proliferative response after Con A stimulation than did the peripheral blood leukocytes from control donors. However, in the present study, the patients receiving MMT did not exhibit significantly higher production of IL-10, and TNF-α, all of which may have been partially or completely normalized after the MMT. This study has several limitations. First, all the participants were all male and were recruited through a methadone replacement therapy outpatient department in Taiwan; thus, the results must be cautiously generalized to patient populations of both sexes and belonging to other nations. Second, the sample size may have been too small, and thus, larger-scale studies are required to confirm these results. Third, certain participants reported occasionally using other illicit drugs, such as amphetamine. Because amphetamine affects immune responses (Peerzada et al., 2013), a possible interaction of this substance with opioid-induced immune alterations should be considered. Moreover, the relatively small number of patients did not facilitate adequately evaluating the immune effects of polydrug abuse compared to those of single drug abuse. Therefore, additional studies are warranted to address this concern. Finally, some studies have attempted to determine whether the amelioration of immune responses observed with MMT depends on the drug profile or on the life style changes (Alonzo and Bayer, 2002; McLachlan et al., 1993). Several aspects of lifestyle associated with injecting heroin use including numerous infectious disease, malnutrition, stress, sleep deprivation and alcohol consumption are known to impair function (Blank et al., 1991; Glaser and Kiecolt-Glaser, 1988). The present study did not analyze such changes in lifestyle and quality of life, addiction severity, financial problems, duration of imprisonment, or any recent stressful event that could have influenced cytokine production.
5. Conclusion In conclusion, our results revealed that opioid dependence influences the functions of the immune system in patients receiving MMT and may induce long-term systemic inflammation. Although MMT is an effective method for treating heroindependent patients, long-term MMT and increases methadone dosage might exacerbate the consequences of systemic inflammation and cause subsequent neuronal inflammation and damage. Future well-controlled longitudinal studies are required to evaluate the immune response in such patients before and during MMT and assess the benefits of methadone in relationship to immune responses.
Conflicts of interests The author(s) declare that they have no conflict of interests.
Acknowledgments This work was supported by Grant NSC 102-2320-B-039-024 from the National Science Council. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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