Molecular Psychiatry (2010) 15, 364–371 & 2010 Nature Publishing Group All rights reserved 1359-4184/10 $32.00 www.nature.com/mp ORIGINAL ARTICLE Differential effects on T-cell function following exposure to serum from schizophrenia smokers M Herberth1, DN Krzyszton1, D Koethe2, MR Craddock1, E Bulger1, E Schwarz1, P Guest1, FM Leweke2 and S Bahn1 1Institute of Biotechnology, University of Cambridge, Cambridge, UK and 2Department of Psychiatry and Psychotherapy, University of Cologne, Cologne, Germany Cigarette smoking is more prevalent in subjects with schizophrenia compared to those with other psychiatric disorders or the general population and could therefore affect molecular pathways that impact the pathophysiology of this disorder. As smoking is also known to suppress immune responses, we investigated the effects of ‘smoking-conditioned’ serum obtained from schizophrenia and control subjects on healthy T cell in vitro. We found that T-cell proliferation was significantly increased following exposure to serum from smoking schizophrenia patients whereas no effect was observed when using serum from smoking control subjects or non-smoking patients and controls. We eliminated the possibility that these effects were due to quantitative differences in cigarette consumption as serum levels of the stable nicotine metabolite cotinine were similar in schizophrenic and control smokers. Molecular characterization showed that serum from patient smokers increased expression of T-cell activation markers CD69high, CD25high, co-stimulatory molecules CD26 þ , CD27 þ and CD28 þ , and decreased T-cell receptor complex components TCRa/b and CD3. Moreover, analysis of supernatants collected after T-cell exposure to serum from smoking patients showed a time-dependent decline in interleukin (IL)-2 levels, suggesting that the proliferation effect is promoted by enhanced IL-2 processing. These results suggest that cigarette smoking has selective effects on serum components that, in turn, lead to altered immune function in schizophrenia patients relative to healthy subjects. Further studies aimed at characterizing these components could result in a better understanding of the onset and aetiology of schizophrenia and potentially lead to novel therapeutic strategies. Molecular Psychiatry (2010) 15, 364–371; doi:10.1038/mp.2008.120; published online 11 November 2008 Keywords: schizophrenia; T-cell signaling; smoking; cytokine; cotinine; activation marker Introduction Schizophrenia patients have a reduced life expec- tancy of approximately 20% compared to the general Compared to the general population and other population with coronary heart disease accounting for psychiatric disorders, cigarette consumption is high- 10 1,2 about two-thirds of the mortality rate. This disease est among schizophrenia patients. Their depen- susceptibility may be exacerbated through the sup- dence on nicotine is approximately three times that of pressive effect of cigarette smoking on the immune the general population with a prevalence reaching 11,12 3 system. almost 90%. Investigations of smoking behaviour Nicotine, the major active chemical in cigarettes, among schizophrenia patients have shown that they has been shown to impair antigen-driven T-cell smoke more, exhibit stronger addictive behaviours response and inhibit proliferation in rodents as well and encounter more difficulties with smoking cessa- 13–16 4,5 as humans. T lymphocytes have integral function tion compared to healthy cigarette consumers. in cell-mediated immunity and are involved in host Patients may use cigarettes as a form of self-medica- 6 defence against infectious agents. Peripheral changes tion, as a consequence of the stimulatory effect of in the immune system of schizophrenia patients, nicotine on the mesolimbic dopamine system that such as decreased cellular immune response17,18 and improves positive and negative symptoms as well as 19 7,8 altered cytokineexpression, have been reported. cognitive functions such as memory and attention. However, these immunological findings are incon- However, cigarette smoking is a well-known risk 9 sistent and controversial due to different methodo- factor of cardiovascular and respiratory disease. logical approaches, the heterogeneity of sample sourcing and acquisition, as well as lack of controls Correspondence: Dr S Bahn, Institute of Biotechnology, Univer- for confounding factors such as smoking.20 sity of Cambridge, Tennis Court Road, Cambridge, CB2 1QT UK. E-mail: [email protected] To investigate whether smoking has a distinct effect Received 29 July 2008; revised 18 September 2008; accepted 9 on the molecular pathways associated with the October 2008; published online 11 November 2008 immune response in schizophrenia, we examined Healthy T-cell response to in vitro stimulation M Herberth et al 365 the functional response of normal T cells in vitro to Syndrome Scale (PANSS) rating scale (PANSS total: serum obtained from first-onset anti-psychotic naive 100.7 (±24.0), positive symptoms: 25.4 (±4.6), patients and healthy controls (HCs). The main objec- negative symptoms: 22.7 (±8.8), general symptoms: tive was to determine whether cell-extrinsic serum 52.6 (±11.6)). Serum samples were produced by factors from schizophrenia smokers evoke distinct allowing blood to coagulate for 2 h at room disease-associated differential T-cell responses. temperature, centrifuged at 4000 g, 5 min and the supernatant was collected. Samples were stored at À80 1C until use. Materials and methods Sample collection T-cell isolation and stimulation Peripheral blood was collected from consenting Subjects for T-cell isolation. CD3 þ pan T cells were participants into 9 ml EDTA S Monovette tubes isolated from peripheral blood from healthy (Sarstedt). PBMCs (peripheral blood mononuclear volunteers recruited at the University of Cambridge cells) were isolated by centrifugation over Ficoll- showing no comorbidities such as diabetes, heart Paque Plus (GE Healthcare) and washed in Dulbecco’s disease, thyroid disease, autoimmune disease or any phosphate-buffered saline (DPBS; Invitrogen, Paisley, recent infections. Volunteers with a psychotic family Renfrewshire, UK). T cells were obtained by negative history or drug abuse were also excluded. All subjects selection using the MACS human pan T-cell isolation gave written consent. kit II in association with LS MACS separation columns (Miltenyi Biotech, UK) according to the Subjects for serum collection. The study was manufacturer’s specifications. T-cell purity (routinely approved by the local ethics committee and above 95%) was assessed by flow cytometry (CyAn; subsequently performed at the University Hospital DakoCytomation, UK) measuring expression of the of Cologne. All patients and healthy volunteers gave T-cell marker CD3-e. CD3 þ T cells from healthy their written informed consent. We consecutively donors were cultured in triplicate in RPMI 1640 recruited 24 subjects, comprising 12 HCs with no medium (Sigma, Poole, Dorset, UK) supplemented family history of schizophrenia or detectable medical, with 1% glutamine, penicillin, streptomycin (referred psychiatric or neurological history and 12 patients to as incomplete medium) in the absence or presence suffering from first-episode paranoid psychosis of 1 mgmlÀ1 anti-CD3 (clone OKT3). Serum (10%) (Table 1). All patients were drug naive for anti- from a panel of schizophrenia patients and matched psychotic medication. HCs were matched for age, controls was added to each well before incubation at gender, smoking and ethnicity. None of the HCs 37 1C, 5% CO2 for 48 h. Cells were pulsed with developed schizophrenic symptoms during the 0.037 MBq 3H-thymidine (Amersham Biosciences, follow-up period of at least 1 year. HCs were Little Chalfont, Bucks, UK) allowing incorporation carefully screened for medical disorders (such as into DNA for a further 24 h incubation and DNA diabetes, heart disease, thyroid disease, autoimmune harvested onto 96-well filter plates (PerkinElmer, Seer disease or any recent infections) and current or Green, Bucks, UK). T-cell proliferation was deter- previous psychiatric illnesses using the severe mined by measuring the incorporation of 3H-thymi- combined immunodeficiency for Diagnostic and dine into progeny DNA using a liquid scintillation Statistical Manual of Mental Disorders, fourth counter (Packard, Isotech, Chesterfield, Derbe, UK). edition; no previous or current diagnosis of illicit The intra- and inter-assay variability was < 15% drug abuse or dependence and no cannabis use coefficient of variation (CV). within the past 6 months before the study were allowed. Psychopathology of the patients was T-cell surface marker analysis assessed on the day of the blood withdrawal and To corroborate the difference in proliferation between measured by using the Positive and Negative the patient and control smokers we treated T cells Table 1 Demographic characteristics of serum and T-cell samples Demographic parameter Study 1 Study 2 T cells Serum samples T cells Serum samples Patient Control Patient Control Number (n)18661166 Age (year, mean±s.d.) 27.9±8.1 28.3±2.9 28.2±3.3 28.4±6.8 33.2±4.8 32.2±5.0 Sex (male/female) 9/9 3/3 3/3 4/7 3/3 3/3 Race (Caucasian/Asian) 17/1 6/0 6/0 10/1 6/0 6/0 Smoking (yes/no) 3/15 4/2 4/2 3/8 4/2 3/3 Molecular Psychiatry Healthy T-cell response to in vitro stimulation M Herberth et al 366 from 12 healthy donors with four representative Cotinine analysis serum samples from these two categories. Owning to Cotinine serum levels of 12 patient