SCHRES-06209; No of Pages 8 Schizophrenia Research xxx (2015) xxx–xxx
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Altered IL-2, IL-6 and IL-8 serum levels in schizophrenia patients with tardive dyskinesia Hui-Mei An a,1, Yun-Long Tan a,1, Jing Shi a, Zhi-Ren Wang a, Jair C. Soars c, Jing Qin Wu d, Fu-De Yang a, Xu-Feng Huang b, Xiang Yang Zhang a,c,⁎ a
Beijing HuiLongGuan Hospital, Peking University, Beijing, China Centre for Translational Neuroscience, School of Medicine, Illawarra Health and Medical Research Institute, University of Wollongong, NSW, Australia Department of Psychiatry and Behavioral Sciences, Harris County Psychiatric Center, The University of Texas Health Science Center at Houston, Houston, TX, USA d School of Biomedical Sciences and Pharmacy, Faculty of Health, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia b c
a r t i c l e
i n f o
Article history: Received 25 June 2014 Received in revised form 29 November 2014 Accepted 29 December 2014 Available online xxxx Keywords: Schizophrenia Tardive dyskinesia Immune Interleukin
a b s t r a c t Immune deregulation has been postulated to be one of the mechanisms underlying the pathogenesis of tardive dyskinesia (TD). We hypothesized that interleukins would have a link with TD in schizophrenia patients. In this study, the serum IL-2, IL-6 and IL-8 levels were examined by enzyme-linked immunosorbent assay (ELISA) in schizophrenia patients with TD (n = 48) and without TD (n = 45), and healthy controls (n = 44). The psychopathological symptoms of schizophrenia were assessed by the Positive and Negative Syndrome Scale (PANSS). The severity of TD was evaluated using Abnormal Involuntary Movement Scale (AIMS). The results showed that serum IL-2, IL-6 and IL-8 levels were significantly different among schizophrenia patients with TD and without TD and normal controls. Moreover, IL-2 level was significantly correlated with PANSS positive subscale and general subscale in patients with TD and without TD. In addition, IL-2 level was positively correlated with AIMS score in TD patients. The results supported that immune disturbance is related to the schizophrenia patients, especially to the patients with TD and ILs might play an important role in the pathophysiology of schizophrenia patients with TD. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The neurodevelopmental and neurodegenerative theories of schizophrenia have been put forward to explain the etiology and clinical course of this disorder (Kochunov and Hong, 2014). Cytokines and their receptors have been considered to be important contributors to the development and progression in neurodegenerative processes (Frank-Cannon et al., 2009). Numerous studies have demonstrated that immune abnormalities may be involved in the pathophysiology of schizophrenia (Potvin et al., 2008; de Witte et al., 2014). Long-term antipsychotic medication for schizophrenia is associated with the emergence of tardive dyskinesia (TD), a motor syndrome consisting of involuntary and hyperkinetic movements (Remington, 2007; Correll and Schenk, 2008). It occurs in 20–50% of patients treated chronically with antipsychotics, and it can manifest months or years after the initiation of antipsychotic treatment and persist even after drug
⁎ Corresponding author at: Department of Psychiatry and Behavioral Sciences, The University of Texas Health Science Center at Houston, UT Houston Medical School, 1941 East Road, Houston, TX 77054, USA. E-mail address: [email protected] (X.Y. Zhang). 1 Hui-Mei An and Yun-Long Tan contributed equally to the study. They should be regarded as joint first authors.
withdrawal (Casey, 2000; Howland, 2011; Trevizol et al., 2011). While its pathophysiology remains unclear, a neurodegenerative hypothesis has been proposed (Lohr et al., 2003). A few previous studies have explored the immune responses in TD, showing that immune disturbance may be involved in the pathophysiology of TD (Rapaport et al., 1997a; Liu et al., 2012). Several hypotheses have been proposed to explain the development of TD, including dopamine supersensitivity (Klawans and Rubovits, 1972; Tarsy and Baldessarini, 1974; Burt et al., 1977), gamma aminobutyric acid (GABA) deficiency (Gale, 1980), serotonin and glutamate receptor dysfunction (Kulkarni et al., 2009), oxidative stress (Kulkarni and Naidu, 2003; Thaakur and Himabindhu, 2009) and neuroinflammation (Bishnoi et al., 2008a, b), suggesting that TD pathogenesis involves multiple pathways including neurotransmitters imbalance, neuroinflammation and oxidative stress. Previous studies have suggested that there was a dynamic interaction between the immune system and central nervous system, mainly mediated by cytokines, hormones and neurotransmitters (Petrovsky, 2001; Masek et al., 2003). In fact, a number of cytokine receptors are expressed in neurons and glial cells, suggesting that cytokine dysfunction may directly affect neurons and brain function (Merrill, 1992; Sawada et al., 1993; Petitto et al., 2012b). Moreover, certain cytokines and/or overloaded oxidative stress could induce the activation of immune system, and subsequently
http://dx.doi.org/10.1016/j.schres.2014.12.037 0920-9964/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: An, H.-M., et al., Altered IL-2, IL-6 and IL-8 serum levels in schizophrenia patients with tardive dyskinesia, Schizophr. Res. (2015), http://dx.doi.org/10.1016/j.schres.2014.12.037
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H.-M. An et al. / Schizophrenia Research xxx (2015) xxx–xxx
influence neurotransmitter metabolism, like dopaminergic system (De Simoni and Imeri, 1998; Larouche et al., 2008; Rankin et al., 2013). All these suggested that there may be inter-relationships among immune system dysfunction, the neurotransmitter metabolism imbalance, and oxidative stress, all of which are involved in the pathophysiology of TD. Cytokines are important mediators of the cross-talk between the central nervous system and immune system. Effective immune response is mainly mediated by two mutually exclusive sets of cytokines: Th1-type and Th2-type. Th1 cells produce interferon-γ (IFN-γ), IL-2 and TNF, and evoke cell-mediated immunity and phagocyte-dependent inflammation. Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, which evoke strong antibody responses but inhibit phagocytic activity (Romagnani et al., 2000; Bocchio Chiavetto et al., 2002). Numerous studies demonstrate various abnormalities in the cytokines and their receptors of schizophrenic patients (Rapaport et al., 1994; Naudin et al., 1996; Rapaport et al., 1997b; Di Nicola et al., 2013; de Witte et al., 2014; Upthegrove et al., 2014). Moreover, typical or atypical antipsychotic drugs may influence the levels of cytokines or cytokine receptors (Maes et al., 2000; Pollmacher et al., 2000; Krause et al., 2013; Tourjman et al., 2013; Klemettila et al., 2014; O'Connell et al., 2014). Interleukin-2 (IL-2) is mainly synthesized by helper CD4+ T lymphocytes, but also by monocytes, macrophages, and endothelial cells. IL-2 plays a crucial role in the regulation of neurotransmitter metabolism, including dopamine metabolism (Kim et al., 2000; Zalcman, 2002; Meyer et al., 2006; Potvin et al., 2008; Rankin et al., 2013). Since dopamine supersensitivity is one of the most important hypotheses to elucidate the pathogenesis of TD, some investigators have proposed possible relationships between IL-2, clinical symptoms, and the dyskinetic severity in schizophrenia patients with TD. For example, Rapaport and Lohr (1994) reported that individuals with schizophrenia that had developed TD had increased serum-soluble IL-2 receptor (sIL2R) levels compared with patients without TD. We recently reported altered serum IL-2 levels in patients with TD compared to those without TD (Liu et al., 2012). IL-6, a 26-kDa protein, is a multiple cytokine released from peripheral immune cells of the monocyte macrophage lineage and acts as a mediator of peripheral acute-phase responses, and also plays a pivotal role in the immune response (T- and B-cell activation or proliferation) (Zhang et al., 2002). It is also produced by both neurons and microglias, and acts as a neurotrophic factor in the central nervous system (CNS) (Maes et al., 1994; Zhang et al., 2002). In addition, IL-8 is a chemokine cytokine produced by monocytes, macrophages, endothelial cells and also by the activated T-cells, which is the best-characterized member of chemokine subfamilies (Zhang et al., 2002). Chemokines, together with adhesion molecules, cytokines and proteases, are essential for the directional migration of leukocytes during normal and inflammatory processes. Chemokines are involved in diseases such as autoimmune diseases, bacterial or viral infection and cancer (Proost et al., 1996). Cross-sectional analyses of the association between serum IL-8 in a number of comparable small studies (n b 50 cases) have produced inconsistent results with a number finding increased serum IL-8 in schizophrenia (Maes et al., 2001; Zhang et al., 2002; Di Nicola et al., 2013), but many others report of no significant association (O'Brien et al., 2008; Reale et al., 2011; Dennison et al., 2012). Interestingly, our previous study found elevated IL-8 serum levels in patients with a chronic form of schizophrenia. Moreover, a significant positive correlation between IL-8 level and PANSS negative subscale was noted in these patients (Zhang et al., 2002). However, to our best knowledge, no study has reported the relationships between TD and IL-6 or IL-8, especially the relationship between these cytokines and the dyskinetic severity in schizophrenia patients with TD. In this study, we postulated that cytokine abnormalities are one of the mechanisms underlying the pathogenesis of TD. The purposes of the present study were to investigate (1) whether serum IL-2, IL-6 and IL-8 levels were altered in schizophrenia patients with and without TD compared with normal controls, (2) whether there was a correlation
between IL-2, IL-6 and IL-8 levels and clinical symptoms in these patients, and (3) whether there was a correlation between serum IL-2, IL-6 and IL-8 levels and dyskinetic severity in patients with TD. 2. Materials and methods 2.1. Subjects Ninety-three schizophrenia inpatients were recruited from Beijing Hui-Long-Guan hospital, a Beijing-city owned psychiatric hospital. All patients met the following inclusion criteria: 1) age 35–75 years, Han Chinese; 2) confirmed DSM-IV diagnosis of schizophrenia based on the Structured Clinical Interview for DSM-IV (SCID) by consensus of two psychiatrists; 4) with at least 5 years of illness; 5) had been receiving stable doses of oral antipsychotic drugs for at least 12 months before entry into the study; 6) had not received any immunomodulators or anti-oxidants in the past 12 weeks. The mean age of the patients was 47.6 ± 9.5 years. All patients were of the chronic type, with a mean illness course of 23.3 ± 7.6 years. TD was diagnosed using the criteria of Schooler and Kane (1982). 48 patients with TD and 45 without TD were recruited. The medications that patients had been taking were either clozapine (25 TD vs. 23 without TD), risperidone (8 TD vs. 8 without TD), perphenazine (5 TD vs. 6 without TD), haloperidol (4 TD vs. 3 without TD), chlorpromazine (3 TD vs. 2 without TD), and sulpiride (3 TD vs. 3 without TD). Forty-four healthy controls were selectively recruited by advertisements at the local community in Beijing. They were recruited about 1 ~ 2 weeks later than the patient groups to match for gender and age. None of them had received any immunomodulators or anti-oxidants in the past 12 weeks. Current mental status and personal or family history of any mental disorder were assessed by unstructured interviews. None of the healthy control subjects presented a personal or family history of psychiatric disorder. A complete medical history, physical examination and laboratory tests were obtained from patients and healthy controls. Any subjects with major medical illness were excluded. None of the subjects met criteria for drug or alcohol abuse or dependence. Both patients and healthy controls who received any immunomodulators or anti-oxidants in the last 12 weeks were excluded from the study. Before this current study began, the research protocol was approved by the Institutional Review Board (IRB), Beijing Hui-Long-Guan hospital (BHLGH), a committee that is composed by 10 members (7 males and 3 females) including physicians, researchers, statisticians, community advocates and a lawyer who are not affiliated with BHLGH and who are not part of the immediate family of a person who is affiliated with BHLGH. The IRB pays attention to both the scientific and the ethic concerns of the studies. A psychiatrist explained the research protocol and procedures to the potential subject. The description of the study was tailored to maximize the understanding of the subject using language appropriate to the subject's level of comprehension, and emotional readiness. All subjects gave signed, informed consent to participate in the study. 2.2. Clinical measurement TD severity was assessed using the Abnormal Involuntary Movement Scale (AIMS). The patients' psychopathology was determined using the Positive and Negative Syndrome Scale (PANSS) by two psychiatrists, who had simultaneously attended a training session in the use of the PANSS. Repeated assessments for the PANSS total score maintained an interrater correlation coefficient greater than 0.8. 2.3. Measurement of IL-2, IL-6 and IL-8 levels in serum Blood samples from both patients and healthy controls were collected between 7 and 9 a.m. following an overnight fast. The serum was
Please cite this article as: An, H.-M., et al., Altered IL-2, IL-6 and IL-8 serum levels in schizophrenia patients with tardive dyskinesia, Schizophr. Res. (2015), http://dx.doi.org/10.1016/j.schres.2014.12.037
H.-M. An et al. / Schizophrenia Research xxx (2015) xxx–xxx
separated, aliquoted, and stored at −70 °C before use. Serum IL-2, IL-6 and IL-8 levels were measured in duplicate by sandwich enzymelinked immunosorbent assay (ELISA) using a commercially available kit (NeoBioscience Technology, Beijing, China) (Kim et al., 2000; Zhang et al., 2004). The kits were purchased from NeoBioscience Technology directly. The sample diluent buffer provided in the kits was used to dilute the sample in a 1:2 ratio (Add 50 μl sample to 50 μl sample diluent buffer). Duplicate assays were performed for both sample and standard testing. All samples were assayed by the same investigator, who was blind to the clinical situation. Totally, 12 assays have been made. The sensitivities for IL-2, IL-6 and IL-8 were 7 pg/ml, 0.2 ng/ml and 0.4 ng/ml, respectively. The inter-assay coefficients were 7%, 7% and 4%, respectively. The intra-assay variation coefficients were 5%, 9% and 8%, respectively. 2.4. Data analysis Demographic and clinical variables of the patients with and without TD were compared using t-tests or analysis of variance (ANOVA) for the continuous variables and chi-square tests for the categorical variables. Since the majority of the interleukin variables were not normally distributed in patients and healthy controls (Shapiro–Wilk test, IL-2: z = 0.944, p = 0.001; IL-6: z = 0.988, p = 0.603; IL-8: z = 0.918, p = 0.000 for patients; IL-2: z = 0.957, p = 0.104; IL-6: z = 0.798, p = 0.000; IL-8: z = 0.589, p = 0.000 for controls), the principal outcome analysis consisted of nonparametric test for comparison between patients and healthy control (Mann–Whitney test). Only one-way analysis of variance (ANOVA) was used to compare the IL-6 levels between the TD and non-TD patients. Further, analysis of covariance (ANCOVA) was also performed to further compare the difference in IL-6 between the TD and non-TD patients after controlling for gender, age, education, duration of illness, antipsychotic treatment (type, dose and duration of treatment) and the PANSS total and its subscale scores. Relationships between clinical ratings and IL-2, IL-6 and IL-8 levels were examined with Spearman correlation in view of the skewness of IL measures in patients. Furthermore, partial correlation was used for controlling for the effects of additional variables. Bonferroni corrections were applied to each test to adjust for multiple testing. The Bonferroni procedure refers to all applied independent statistical tests. This approach was chosen because it allows for stringent control for multiple testing and a reduction of type I errors. The corrections accounted for the number of PANSS scale and the IL parameters. Since we tested 3 cytokines (IL-2, IL-6 and IL-8) in both the healthy controls and the patients, as well as 4 items of PANSS (1 total score and 3 subscale scores) in only patient groups, the Bonferroni-adjusted significance level was set at p b 0.05/3 = 0.017 for healthy controls, or at p b 0.05/12 = 0.0042 for patients.
3
Further, we used stepwise multiple regression analysis with AIMS total score as the dependent variable to investigate the impact of age, gender, education, duration of illness, antipsychotic treatment (type, dose and duration of treatment), the PANSS total score and its subscales, together with IL-2, IL-6 and IL-8. 3. Results 3.1. Demographic data Table 1 shows the demographic data of the subjects in the present study. There was no significant difference in age, gender among the three groups. The age of onset, duration of treatment, the PANSS total score, general psychopathology subscore and positive symptom subscore were significantly different between TD and non-TD patients. In the TD patients, no gender difference in the AIMS total score was noted. Age of onset, and daily antipsychotic dose (chlorpromazine equivalent) did not significantly correlate with the AIMS total score. However, there was a significant correlation of AIMS total score with duration of illness (r = 0.287, n = 48, p = 0.048), or with age (r = 0.315, n = 48, p = 0.029). 3.2. Serum IL-2, IL-6, IL-8 levels in TD and non-TD patients versus normal control subjects 3.2.1. Serum IL-2 level Nonparametric Mann–Whitney test showed that the serum IL-2 levels was higher in schizophrenia patients [median (QR): 6.39 (3.01)] than normal controls [median (QR): 4.10 (2.72); (z = − 4.81; p b 0.001)] (Fig. 1). This difference remained significant after covarying for gender, age, and education (p b 0.01). Similarly, patients with TD [median (QR): 6.80 (3.33)] had significantly higher levels of IL-2 than the patients without TD [median (QR): 5.89 (3.40); Mann–Whitney test, (z = − 2.812; p b 0.005)]. This difference remained significant after covarying for gender, age, education, duration of illness, antipsychotic treatment (type, dose and duration of treatment) and the PANSS total and its subscale scores (p b 0.01). 3.2.2. Serum IL-6 level Nonparametric Mann–Whitney test showed that the serum IL-6 levels was lower in schizophrenia patients [median (QR): 0.31 (0.14)] than normal controls [median (QR):0.44(0.12); z = −4.69; p b 0.001] (Fig. 1). This difference remained significant after covarying for gender, age, and education (p b 0.01). Similarly, patients with TD (mean ± SD = 0.28 ± 0.09) had significantly lower levels of IL-6 than the patients without TD (mean ± SD = 0.34 ± 0.11; F = 10.37, df = 1,89; p b 0.005). ANCOVA analysis showed that this difference remained
Table 1 Demographics of patients with TD and without TD and normal control subjects.
Male/female Age (years) Education (years) Age at onset No. of hospitalization Duration of treatment Daily AP dose (mg) (CPZ equivalent) PANSS total score P subscore N subscore G subscore
Normal controls (n = 44)
Patients without TD (n = 45)
Patients with TD (n = 48)
T or χ2 or F
p value
32/12 47.7 ± 4.5 9.8 ± 3.1
35/10 47.3 ± 4.2 10.2 ± 2.3 25.7 ± 6.8 3.4 ± 1.9 21.5 ± 7.3 443.7 ± 300.3
40/8 47.9 ± 5.0 9.0 ± 1.9 23.0 ± 5.4 3.7 ± 2.1 24.9 ± 7.1 435.4 ± 426.6
1.514 0.222 −2.595 −2.095 0.733 2.267 −0.102
0.469 0.801 0.011 0.039 0.465 0.026 0.919
68.3 ± 15.9 14.3 ± 6.1 23.0 ± 5.2 31.0 ± 8.1
80.0 ± 16.6 17.0 ± 5.6 25.3 ± 6.6 37.3 ± 9.3
3.334 2.182 1.905 3.446
0.001 0.032 0.06 0.001
TD—tardive dyskinesia; AP—antipsychotic; CPZ—chlorpromazine; PANSS—Positive and Negative Syndrome Scale; P—positive symptom subscore; N—negative symptom subscore; G—general psychopathology subscore
Please cite this article as: An, H.-M., et al., Altered IL-2, IL-6 and IL-8 serum levels in schizophrenia patients with tardive dyskinesia, Schizophr. Res. (2015), http://dx.doi.org/10.1016/j.schres.2014.12.037
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H.-M. An et al. / Schizophrenia Research xxx (2015) xxx–xxx ***
***
***
***
***
0.8
15
***
***
***
2.0
ng/ml
0.4
5
ng/ml
ng/ml 10
0.6 1.5 1.0
0.2 0.5
0
0.0 With TD Without TD Control IL-2
With TD Without TD Control IL-8
With TD Without TD Control IL-6
Fig. 1. Scattergrams of serum IL-2, IL-6 and IL-8 levels for schizophrenia patients with TD, without TD and normal controls. P b 0.05 was considered significant.
significant after covarying for gender, age, education, duration of illness, antipsychotic treatment (type, dose and duration of treatment) and the PANSS total and its subscale scores (p b 0.01). 3.2.3. Serum IL-8 level Nonparametric Mann–Whitney test showed that the serum IL-8 levels was higher in schizophrenia patients [median (QR), 0.90 (0.35)] than normal controls [median (QR), 0.75 (0.12); z = − 4.70; p b 0.001] (Fig. 1). This difference remained significant after covarying for gender, age, and education (p b 0.01). However, there was no significant difference in IL-8 between the patients with TD [median (QR), 0.92 (0.35)] and without TD [median (QR), 0.88 (0.40); Mann–Whitney test, z = −1.20; p = 0.23]. 3.3. The correlation between serum levels of IL-2, IL-6, and IL-8 in TD and non-TD patients and normal controls Fig. 2 showed that in the whole sample of schizophrenia patients, Spearman correlation analysis showed no significant correlations between IL-2, IL-6 and IL-8 (all p N 0.05). Further Spearman correlation analysis showed that IL-6 was significantly correlated with IL-8 (r = 0.47, n = 47, p = 0.002; Bonferroni corrected p b 0.05) in TD patients, but no significant correlation was observed between IL-2, IL-6, and IL8 in non-TD patients (all p N 0.05). In addition, there was a significant correlation between IL-2 and IL-6 (r = 0.48, n = 44, p b 0.01; Bonferroni corrected p b 0.05), or between IL-6 and IL-8 (r = 0.32, n = 44, p = 0.037; Bonferroni corrected p N 0.05) in the healthy controls. These results suggest that there was no correlation between IL-6 and IL-8 in healthy controls, since it did not pass the Bonferroni correction. 3.4. The relationship between serum levels of IL-2, IL-6 and IL-8 and clinical symptoms in TD and non-TD patients Fig. 3 showed that in the whole sample of schizophrenia patients, Spearman correlation analysis showed a significantly negative
correlations between IL-2 and the PANSS positive symptom subscore (r = − 0.32, n = 92, p = 0.002; Bonferroni corrected p b 0.05). Further Spearman correlation analysis showed a significant negative correlation between IL-2 and the PANSS positive subscore (r = − 0.36, n = 47, p = 0.009; Bonferroni corrected p N 0.05) in TD patients, and also showed a significant negative correlation between IL-2 and the PANSS positive subscore (r = − 0.53, n = 45, p b 0.001; Bonferroni corrected p b 0.001), the PANSS general psychopathology subscore (r = − 0.44, n = 45, p = 0.003; Bonferroni corrected p b 0.05), or the PANSS total score (r = − 0.49, n = 45, P = 0.001; Bonferroni corrected p b 0.05) in non-TD patients. However, no correlation between the PANSS total score or subscore and IL-6 or IL-8 was found in all patients (all p N 0.05).
3.5. The relationship between serum levels of IL-2, IL-6 and IL-8 and dyskinetic severity of patients Fig. 4 showed that Spearman correlation analysis also showed a significantly positive correlation between IL-2 with the AIMS total score (r = 0.53, n = 47, P b 0.001; Bonferroni corrected p b 0.05) in TD patients. Since there was a significant negative correlation between IL-2 and the PANSS positive subscale, a further partial correlation analysis still showed a significantly positive correlation between IL-2 with the AIMS total score (r = 0.60, n = 44, p b 0.001). However, no correlation between the AIMS total score and IL-6 or IL-8 was found in patients with TD (all p N 0.05). Further, stepwise multiple regression analysis including AIMS total score (as the dependent variable) and PANSS and its subscales with age, gender, education, duration of illness, antipsychotic treatment (type, dose and duration of treatment), together with IL-2, IL-6 and IL8 as independent covariates identified IL-2 (beta = 0.66, t = 4.65, p b 0.001) and the PANSS positive symptom subscore as significant predictor factors for AIMS total score (adjust R2 = 0.34), whereas the other variables showed no effects (p N 0.05).
The correlation between IL-6 and IL-8 in TD patients 2.0
The correlation between IL-2 and IL-6 in normal controls
r=0.47, p=0.002
8 IL-2
IL-8
1.5 1.0
r=0.48, p
Contents lists available at ScienceDirect
Schizophrenia Research journal homepage: www.elsevier.com/locate/schres
Altered IL-2, IL-6 and IL-8 serum levels in schizophrenia patients with tardive dyskinesia Hui-Mei An a,1, Yun-Long Tan a,1, Jing Shi a, Zhi-Ren Wang a, Jair C. Soars c, Jing Qin Wu d, Fu-De Yang a, Xu-Feng Huang b, Xiang Yang Zhang a,c,⁎ a
Beijing HuiLongGuan Hospital, Peking University, Beijing, China Centre for Translational Neuroscience, School of Medicine, Illawarra Health and Medical Research Institute, University of Wollongong, NSW, Australia Department of Psychiatry and Behavioral Sciences, Harris County Psychiatric Center, The University of Texas Health Science Center at Houston, Houston, TX, USA d School of Biomedical Sciences and Pharmacy, Faculty of Health, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia b c
a r t i c l e
i n f o
Article history: Received 25 June 2014 Received in revised form 29 November 2014 Accepted 29 December 2014 Available online xxxx Keywords: Schizophrenia Tardive dyskinesia Immune Interleukin
a b s t r a c t Immune deregulation has been postulated to be one of the mechanisms underlying the pathogenesis of tardive dyskinesia (TD). We hypothesized that interleukins would have a link with TD in schizophrenia patients. In this study, the serum IL-2, IL-6 and IL-8 levels were examined by enzyme-linked immunosorbent assay (ELISA) in schizophrenia patients with TD (n = 48) and without TD (n = 45), and healthy controls (n = 44). The psychopathological symptoms of schizophrenia were assessed by the Positive and Negative Syndrome Scale (PANSS). The severity of TD was evaluated using Abnormal Involuntary Movement Scale (AIMS). The results showed that serum IL-2, IL-6 and IL-8 levels were significantly different among schizophrenia patients with TD and without TD and normal controls. Moreover, IL-2 level was significantly correlated with PANSS positive subscale and general subscale in patients with TD and without TD. In addition, IL-2 level was positively correlated with AIMS score in TD patients. The results supported that immune disturbance is related to the schizophrenia patients, especially to the patients with TD and ILs might play an important role in the pathophysiology of schizophrenia patients with TD. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The neurodevelopmental and neurodegenerative theories of schizophrenia have been put forward to explain the etiology and clinical course of this disorder (Kochunov and Hong, 2014). Cytokines and their receptors have been considered to be important contributors to the development and progression in neurodegenerative processes (Frank-Cannon et al., 2009). Numerous studies have demonstrated that immune abnormalities may be involved in the pathophysiology of schizophrenia (Potvin et al., 2008; de Witte et al., 2014). Long-term antipsychotic medication for schizophrenia is associated with the emergence of tardive dyskinesia (TD), a motor syndrome consisting of involuntary and hyperkinetic movements (Remington, 2007; Correll and Schenk, 2008). It occurs in 20–50% of patients treated chronically with antipsychotics, and it can manifest months or years after the initiation of antipsychotic treatment and persist even after drug
⁎ Corresponding author at: Department of Psychiatry and Behavioral Sciences, The University of Texas Health Science Center at Houston, UT Houston Medical School, 1941 East Road, Houston, TX 77054, USA. E-mail address: [email protected] (X.Y. Zhang). 1 Hui-Mei An and Yun-Long Tan contributed equally to the study. They should be regarded as joint first authors.
withdrawal (Casey, 2000; Howland, 2011; Trevizol et al., 2011). While its pathophysiology remains unclear, a neurodegenerative hypothesis has been proposed (Lohr et al., 2003). A few previous studies have explored the immune responses in TD, showing that immune disturbance may be involved in the pathophysiology of TD (Rapaport et al., 1997a; Liu et al., 2012). Several hypotheses have been proposed to explain the development of TD, including dopamine supersensitivity (Klawans and Rubovits, 1972; Tarsy and Baldessarini, 1974; Burt et al., 1977), gamma aminobutyric acid (GABA) deficiency (Gale, 1980), serotonin and glutamate receptor dysfunction (Kulkarni et al., 2009), oxidative stress (Kulkarni and Naidu, 2003; Thaakur and Himabindhu, 2009) and neuroinflammation (Bishnoi et al., 2008a, b), suggesting that TD pathogenesis involves multiple pathways including neurotransmitters imbalance, neuroinflammation and oxidative stress. Previous studies have suggested that there was a dynamic interaction between the immune system and central nervous system, mainly mediated by cytokines, hormones and neurotransmitters (Petrovsky, 2001; Masek et al., 2003). In fact, a number of cytokine receptors are expressed in neurons and glial cells, suggesting that cytokine dysfunction may directly affect neurons and brain function (Merrill, 1992; Sawada et al., 1993; Petitto et al., 2012b). Moreover, certain cytokines and/or overloaded oxidative stress could induce the activation of immune system, and subsequently
http://dx.doi.org/10.1016/j.schres.2014.12.037 0920-9964/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: An, H.-M., et al., Altered IL-2, IL-6 and IL-8 serum levels in schizophrenia patients with tardive dyskinesia, Schizophr. Res. (2015), http://dx.doi.org/10.1016/j.schres.2014.12.037
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influence neurotransmitter metabolism, like dopaminergic system (De Simoni and Imeri, 1998; Larouche et al., 2008; Rankin et al., 2013). All these suggested that there may be inter-relationships among immune system dysfunction, the neurotransmitter metabolism imbalance, and oxidative stress, all of which are involved in the pathophysiology of TD. Cytokines are important mediators of the cross-talk between the central nervous system and immune system. Effective immune response is mainly mediated by two mutually exclusive sets of cytokines: Th1-type and Th2-type. Th1 cells produce interferon-γ (IFN-γ), IL-2 and TNF, and evoke cell-mediated immunity and phagocyte-dependent inflammation. Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, which evoke strong antibody responses but inhibit phagocytic activity (Romagnani et al., 2000; Bocchio Chiavetto et al., 2002). Numerous studies demonstrate various abnormalities in the cytokines and their receptors of schizophrenic patients (Rapaport et al., 1994; Naudin et al., 1996; Rapaport et al., 1997b; Di Nicola et al., 2013; de Witte et al., 2014; Upthegrove et al., 2014). Moreover, typical or atypical antipsychotic drugs may influence the levels of cytokines or cytokine receptors (Maes et al., 2000; Pollmacher et al., 2000; Krause et al., 2013; Tourjman et al., 2013; Klemettila et al., 2014; O'Connell et al., 2014). Interleukin-2 (IL-2) is mainly synthesized by helper CD4+ T lymphocytes, but also by monocytes, macrophages, and endothelial cells. IL-2 plays a crucial role in the regulation of neurotransmitter metabolism, including dopamine metabolism (Kim et al., 2000; Zalcman, 2002; Meyer et al., 2006; Potvin et al., 2008; Rankin et al., 2013). Since dopamine supersensitivity is one of the most important hypotheses to elucidate the pathogenesis of TD, some investigators have proposed possible relationships between IL-2, clinical symptoms, and the dyskinetic severity in schizophrenia patients with TD. For example, Rapaport and Lohr (1994) reported that individuals with schizophrenia that had developed TD had increased serum-soluble IL-2 receptor (sIL2R) levels compared with patients without TD. We recently reported altered serum IL-2 levels in patients with TD compared to those without TD (Liu et al., 2012). IL-6, a 26-kDa protein, is a multiple cytokine released from peripheral immune cells of the monocyte macrophage lineage and acts as a mediator of peripheral acute-phase responses, and also plays a pivotal role in the immune response (T- and B-cell activation or proliferation) (Zhang et al., 2002). It is also produced by both neurons and microglias, and acts as a neurotrophic factor in the central nervous system (CNS) (Maes et al., 1994; Zhang et al., 2002). In addition, IL-8 is a chemokine cytokine produced by monocytes, macrophages, endothelial cells and also by the activated T-cells, which is the best-characterized member of chemokine subfamilies (Zhang et al., 2002). Chemokines, together with adhesion molecules, cytokines and proteases, are essential for the directional migration of leukocytes during normal and inflammatory processes. Chemokines are involved in diseases such as autoimmune diseases, bacterial or viral infection and cancer (Proost et al., 1996). Cross-sectional analyses of the association between serum IL-8 in a number of comparable small studies (n b 50 cases) have produced inconsistent results with a number finding increased serum IL-8 in schizophrenia (Maes et al., 2001; Zhang et al., 2002; Di Nicola et al., 2013), but many others report of no significant association (O'Brien et al., 2008; Reale et al., 2011; Dennison et al., 2012). Interestingly, our previous study found elevated IL-8 serum levels in patients with a chronic form of schizophrenia. Moreover, a significant positive correlation between IL-8 level and PANSS negative subscale was noted in these patients (Zhang et al., 2002). However, to our best knowledge, no study has reported the relationships between TD and IL-6 or IL-8, especially the relationship between these cytokines and the dyskinetic severity in schizophrenia patients with TD. In this study, we postulated that cytokine abnormalities are one of the mechanisms underlying the pathogenesis of TD. The purposes of the present study were to investigate (1) whether serum IL-2, IL-6 and IL-8 levels were altered in schizophrenia patients with and without TD compared with normal controls, (2) whether there was a correlation
between IL-2, IL-6 and IL-8 levels and clinical symptoms in these patients, and (3) whether there was a correlation between serum IL-2, IL-6 and IL-8 levels and dyskinetic severity in patients with TD. 2. Materials and methods 2.1. Subjects Ninety-three schizophrenia inpatients were recruited from Beijing Hui-Long-Guan hospital, a Beijing-city owned psychiatric hospital. All patients met the following inclusion criteria: 1) age 35–75 years, Han Chinese; 2) confirmed DSM-IV diagnosis of schizophrenia based on the Structured Clinical Interview for DSM-IV (SCID) by consensus of two psychiatrists; 4) with at least 5 years of illness; 5) had been receiving stable doses of oral antipsychotic drugs for at least 12 months before entry into the study; 6) had not received any immunomodulators or anti-oxidants in the past 12 weeks. The mean age of the patients was 47.6 ± 9.5 years. All patients were of the chronic type, with a mean illness course of 23.3 ± 7.6 years. TD was diagnosed using the criteria of Schooler and Kane (1982). 48 patients with TD and 45 without TD were recruited. The medications that patients had been taking were either clozapine (25 TD vs. 23 without TD), risperidone (8 TD vs. 8 without TD), perphenazine (5 TD vs. 6 without TD), haloperidol (4 TD vs. 3 without TD), chlorpromazine (3 TD vs. 2 without TD), and sulpiride (3 TD vs. 3 without TD). Forty-four healthy controls were selectively recruited by advertisements at the local community in Beijing. They were recruited about 1 ~ 2 weeks later than the patient groups to match for gender and age. None of them had received any immunomodulators or anti-oxidants in the past 12 weeks. Current mental status and personal or family history of any mental disorder were assessed by unstructured interviews. None of the healthy control subjects presented a personal or family history of psychiatric disorder. A complete medical history, physical examination and laboratory tests were obtained from patients and healthy controls. Any subjects with major medical illness were excluded. None of the subjects met criteria for drug or alcohol abuse or dependence. Both patients and healthy controls who received any immunomodulators or anti-oxidants in the last 12 weeks were excluded from the study. Before this current study began, the research protocol was approved by the Institutional Review Board (IRB), Beijing Hui-Long-Guan hospital (BHLGH), a committee that is composed by 10 members (7 males and 3 females) including physicians, researchers, statisticians, community advocates and a lawyer who are not affiliated with BHLGH and who are not part of the immediate family of a person who is affiliated with BHLGH. The IRB pays attention to both the scientific and the ethic concerns of the studies. A psychiatrist explained the research protocol and procedures to the potential subject. The description of the study was tailored to maximize the understanding of the subject using language appropriate to the subject's level of comprehension, and emotional readiness. All subjects gave signed, informed consent to participate in the study. 2.2. Clinical measurement TD severity was assessed using the Abnormal Involuntary Movement Scale (AIMS). The patients' psychopathology was determined using the Positive and Negative Syndrome Scale (PANSS) by two psychiatrists, who had simultaneously attended a training session in the use of the PANSS. Repeated assessments for the PANSS total score maintained an interrater correlation coefficient greater than 0.8. 2.3. Measurement of IL-2, IL-6 and IL-8 levels in serum Blood samples from both patients and healthy controls were collected between 7 and 9 a.m. following an overnight fast. The serum was
Please cite this article as: An, H.-M., et al., Altered IL-2, IL-6 and IL-8 serum levels in schizophrenia patients with tardive dyskinesia, Schizophr. Res. (2015), http://dx.doi.org/10.1016/j.schres.2014.12.037
H.-M. An et al. / Schizophrenia Research xxx (2015) xxx–xxx
separated, aliquoted, and stored at −70 °C before use. Serum IL-2, IL-6 and IL-8 levels were measured in duplicate by sandwich enzymelinked immunosorbent assay (ELISA) using a commercially available kit (NeoBioscience Technology, Beijing, China) (Kim et al., 2000; Zhang et al., 2004). The kits were purchased from NeoBioscience Technology directly. The sample diluent buffer provided in the kits was used to dilute the sample in a 1:2 ratio (Add 50 μl sample to 50 μl sample diluent buffer). Duplicate assays were performed for both sample and standard testing. All samples were assayed by the same investigator, who was blind to the clinical situation. Totally, 12 assays have been made. The sensitivities for IL-2, IL-6 and IL-8 were 7 pg/ml, 0.2 ng/ml and 0.4 ng/ml, respectively. The inter-assay coefficients were 7%, 7% and 4%, respectively. The intra-assay variation coefficients were 5%, 9% and 8%, respectively. 2.4. Data analysis Demographic and clinical variables of the patients with and without TD were compared using t-tests or analysis of variance (ANOVA) for the continuous variables and chi-square tests for the categorical variables. Since the majority of the interleukin variables were not normally distributed in patients and healthy controls (Shapiro–Wilk test, IL-2: z = 0.944, p = 0.001; IL-6: z = 0.988, p = 0.603; IL-8: z = 0.918, p = 0.000 for patients; IL-2: z = 0.957, p = 0.104; IL-6: z = 0.798, p = 0.000; IL-8: z = 0.589, p = 0.000 for controls), the principal outcome analysis consisted of nonparametric test for comparison between patients and healthy control (Mann–Whitney test). Only one-way analysis of variance (ANOVA) was used to compare the IL-6 levels between the TD and non-TD patients. Further, analysis of covariance (ANCOVA) was also performed to further compare the difference in IL-6 between the TD and non-TD patients after controlling for gender, age, education, duration of illness, antipsychotic treatment (type, dose and duration of treatment) and the PANSS total and its subscale scores. Relationships between clinical ratings and IL-2, IL-6 and IL-8 levels were examined with Spearman correlation in view of the skewness of IL measures in patients. Furthermore, partial correlation was used for controlling for the effects of additional variables. Bonferroni corrections were applied to each test to adjust for multiple testing. The Bonferroni procedure refers to all applied independent statistical tests. This approach was chosen because it allows for stringent control for multiple testing and a reduction of type I errors. The corrections accounted for the number of PANSS scale and the IL parameters. Since we tested 3 cytokines (IL-2, IL-6 and IL-8) in both the healthy controls and the patients, as well as 4 items of PANSS (1 total score and 3 subscale scores) in only patient groups, the Bonferroni-adjusted significance level was set at p b 0.05/3 = 0.017 for healthy controls, or at p b 0.05/12 = 0.0042 for patients.
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Further, we used stepwise multiple regression analysis with AIMS total score as the dependent variable to investigate the impact of age, gender, education, duration of illness, antipsychotic treatment (type, dose and duration of treatment), the PANSS total score and its subscales, together with IL-2, IL-6 and IL-8. 3. Results 3.1. Demographic data Table 1 shows the demographic data of the subjects in the present study. There was no significant difference in age, gender among the three groups. The age of onset, duration of treatment, the PANSS total score, general psychopathology subscore and positive symptom subscore were significantly different between TD and non-TD patients. In the TD patients, no gender difference in the AIMS total score was noted. Age of onset, and daily antipsychotic dose (chlorpromazine equivalent) did not significantly correlate with the AIMS total score. However, there was a significant correlation of AIMS total score with duration of illness (r = 0.287, n = 48, p = 0.048), or with age (r = 0.315, n = 48, p = 0.029). 3.2. Serum IL-2, IL-6, IL-8 levels in TD and non-TD patients versus normal control subjects 3.2.1. Serum IL-2 level Nonparametric Mann–Whitney test showed that the serum IL-2 levels was higher in schizophrenia patients [median (QR): 6.39 (3.01)] than normal controls [median (QR): 4.10 (2.72); (z = − 4.81; p b 0.001)] (Fig. 1). This difference remained significant after covarying for gender, age, and education (p b 0.01). Similarly, patients with TD [median (QR): 6.80 (3.33)] had significantly higher levels of IL-2 than the patients without TD [median (QR): 5.89 (3.40); Mann–Whitney test, (z = − 2.812; p b 0.005)]. This difference remained significant after covarying for gender, age, education, duration of illness, antipsychotic treatment (type, dose and duration of treatment) and the PANSS total and its subscale scores (p b 0.01). 3.2.2. Serum IL-6 level Nonparametric Mann–Whitney test showed that the serum IL-6 levels was lower in schizophrenia patients [median (QR): 0.31 (0.14)] than normal controls [median (QR):0.44(0.12); z = −4.69; p b 0.001] (Fig. 1). This difference remained significant after covarying for gender, age, and education (p b 0.01). Similarly, patients with TD (mean ± SD = 0.28 ± 0.09) had significantly lower levels of IL-6 than the patients without TD (mean ± SD = 0.34 ± 0.11; F = 10.37, df = 1,89; p b 0.005). ANCOVA analysis showed that this difference remained
Table 1 Demographics of patients with TD and without TD and normal control subjects.
Male/female Age (years) Education (years) Age at onset No. of hospitalization Duration of treatment Daily AP dose (mg) (CPZ equivalent) PANSS total score P subscore N subscore G subscore
Normal controls (n = 44)
Patients without TD (n = 45)
Patients with TD (n = 48)
T or χ2 or F
p value
32/12 47.7 ± 4.5 9.8 ± 3.1
35/10 47.3 ± 4.2 10.2 ± 2.3 25.7 ± 6.8 3.4 ± 1.9 21.5 ± 7.3 443.7 ± 300.3
40/8 47.9 ± 5.0 9.0 ± 1.9 23.0 ± 5.4 3.7 ± 2.1 24.9 ± 7.1 435.4 ± 426.6
1.514 0.222 −2.595 −2.095 0.733 2.267 −0.102
0.469 0.801 0.011 0.039 0.465 0.026 0.919
68.3 ± 15.9 14.3 ± 6.1 23.0 ± 5.2 31.0 ± 8.1
80.0 ± 16.6 17.0 ± 5.6 25.3 ± 6.6 37.3 ± 9.3
3.334 2.182 1.905 3.446
0.001 0.032 0.06 0.001
TD—tardive dyskinesia; AP—antipsychotic; CPZ—chlorpromazine; PANSS—Positive and Negative Syndrome Scale; P—positive symptom subscore; N—negative symptom subscore; G—general psychopathology subscore
Please cite this article as: An, H.-M., et al., Altered IL-2, IL-6 and IL-8 serum levels in schizophrenia patients with tardive dyskinesia, Schizophr. Res. (2015), http://dx.doi.org/10.1016/j.schres.2014.12.037
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H.-M. An et al. / Schizophrenia Research xxx (2015) xxx–xxx ***
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0.8
15
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2.0
ng/ml
0.4
5
ng/ml
ng/ml 10
0.6 1.5 1.0
0.2 0.5
0
0.0 With TD Without TD Control IL-2
With TD Without TD Control IL-8
With TD Without TD Control IL-6
Fig. 1. Scattergrams of serum IL-2, IL-6 and IL-8 levels for schizophrenia patients with TD, without TD and normal controls. P b 0.05 was considered significant.
significant after covarying for gender, age, education, duration of illness, antipsychotic treatment (type, dose and duration of treatment) and the PANSS total and its subscale scores (p b 0.01). 3.2.3. Serum IL-8 level Nonparametric Mann–Whitney test showed that the serum IL-8 levels was higher in schizophrenia patients [median (QR), 0.90 (0.35)] than normal controls [median (QR), 0.75 (0.12); z = − 4.70; p b 0.001] (Fig. 1). This difference remained significant after covarying for gender, age, and education (p b 0.01). However, there was no significant difference in IL-8 between the patients with TD [median (QR), 0.92 (0.35)] and without TD [median (QR), 0.88 (0.40); Mann–Whitney test, z = −1.20; p = 0.23]. 3.3. The correlation between serum levels of IL-2, IL-6, and IL-8 in TD and non-TD patients and normal controls Fig. 2 showed that in the whole sample of schizophrenia patients, Spearman correlation analysis showed no significant correlations between IL-2, IL-6 and IL-8 (all p N 0.05). Further Spearman correlation analysis showed that IL-6 was significantly correlated with IL-8 (r = 0.47, n = 47, p = 0.002; Bonferroni corrected p b 0.05) in TD patients, but no significant correlation was observed between IL-2, IL-6, and IL8 in non-TD patients (all p N 0.05). In addition, there was a significant correlation between IL-2 and IL-6 (r = 0.48, n = 44, p b 0.01; Bonferroni corrected p b 0.05), or between IL-6 and IL-8 (r = 0.32, n = 44, p = 0.037; Bonferroni corrected p N 0.05) in the healthy controls. These results suggest that there was no correlation between IL-6 and IL-8 in healthy controls, since it did not pass the Bonferroni correction. 3.4. The relationship between serum levels of IL-2, IL-6 and IL-8 and clinical symptoms in TD and non-TD patients Fig. 3 showed that in the whole sample of schizophrenia patients, Spearman correlation analysis showed a significantly negative
correlations between IL-2 and the PANSS positive symptom subscore (r = − 0.32, n = 92, p = 0.002; Bonferroni corrected p b 0.05). Further Spearman correlation analysis showed a significant negative correlation between IL-2 and the PANSS positive subscore (r = − 0.36, n = 47, p = 0.009; Bonferroni corrected p N 0.05) in TD patients, and also showed a significant negative correlation between IL-2 and the PANSS positive subscore (r = − 0.53, n = 45, p b 0.001; Bonferroni corrected p b 0.001), the PANSS general psychopathology subscore (r = − 0.44, n = 45, p = 0.003; Bonferroni corrected p b 0.05), or the PANSS total score (r = − 0.49, n = 45, P = 0.001; Bonferroni corrected p b 0.05) in non-TD patients. However, no correlation between the PANSS total score or subscore and IL-6 or IL-8 was found in all patients (all p N 0.05).
3.5. The relationship between serum levels of IL-2, IL-6 and IL-8 and dyskinetic severity of patients Fig. 4 showed that Spearman correlation analysis also showed a significantly positive correlation between IL-2 with the AIMS total score (r = 0.53, n = 47, P b 0.001; Bonferroni corrected p b 0.05) in TD patients. Since there was a significant negative correlation between IL-2 and the PANSS positive subscale, a further partial correlation analysis still showed a significantly positive correlation between IL-2 with the AIMS total score (r = 0.60, n = 44, p b 0.001). However, no correlation between the AIMS total score and IL-6 or IL-8 was found in patients with TD (all p N 0.05). Further, stepwise multiple regression analysis including AIMS total score (as the dependent variable) and PANSS and its subscales with age, gender, education, duration of illness, antipsychotic treatment (type, dose and duration of treatment), together with IL-2, IL-6 and IL8 as independent covariates identified IL-2 (beta = 0.66, t = 4.65, p b 0.001) and the PANSS positive symptom subscore as significant predictor factors for AIMS total score (adjust R2 = 0.34), whereas the other variables showed no effects (p N 0.05).
The correlation between IL-6 and IL-8 in TD patients 2.0
The correlation between IL-2 and IL-6 in normal controls
r=0.47, p=0.002
8 IL-2
IL-8
1.5 1.0
r=0.48, p
