Schizophrenia Research 42 (2000) 157–164 www.elsevier.com/locate/schres
Immunosuppressive effects of clozapine and haloperidol: enhanced production of the interleukin-1 receptor antagonist Cai Song a,b, Ai-hua Lin a, Gunter Kenis c,d, Eugene Bosmans c, Michael Maes a,e,f,g,* a Clinical Research Centre for Mental Health, 2060 Antwerp, Belgium b Neuroscience Institute, Carleton University, Ottawa, Canada c Eurogenetics, Transportstraat, Tessenderlo, 3650 Belgium d Department of Anesthesiology, ZOL, Oost-Limburg, 3600 Genk, Belgium e Giovanni di Dio Fatebenefratelli, via Pilcesfroni 4, Brescia, Italy f Department of Psychiatry, Vanderbilt University, Nashville, TN, USA g Department of Psychiatry, University of Maastricht, 6202 Maastricht, The Netherlands Received 12 October 1998; accepted 15 June 1999
Abstract In schizophrenic patients, multiple immune abnormalities have been reported, including increased production of proinflammatory cytokines. There is some evidence that antipsychotic drugs may have immunosuppressive effects. The aim of this study was to examine the in-vitro effects of different concentrations of antipsychotic agents on cytokine production by human whole blood. We examined the effects of clozapine and haloperidol, 10−4, 10−6 and 10−8 M, on the unstimulated and stimulated ( lipopolysaccharide+phytohemagglutinin) production of interleukin-6 (IL-6), IL-10, interferon-c (IFNc), and the IL-1 receptor antagonist (IL-1RA). Clozapine, 10−6 and 10−8 M, and haloperidol, 10−4, 10−6, and 10−8 M, significantly increased the unstimulated and stimulated production of IL-1RA. Clozapine 10−6 M significantly increased the stimulated production of IFNc. Clozapine 10−4 M significantly suppressed the unstimulated production of IL-6 and IL-1RA and the stimulated production of IL-6, IL-10, IFNc and IL-1RA. The results suggest that both clozapine and haloperidol, at concentrations within the therapeutic range, may exert immunosuppressive effects through an enhanced production of IL-1RA. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Cytokines; IL-1RA; Immunosuppression; Neuroleptics; Schizophrenia
1. Introduction There is now evidence that schizophrenia is associated with various dysfunctions in the * Corresponding author. Present address: Department of Psychiatry and Neuropsychology, University Hospital of Maastricht, Postbus 5800, 6202 AZ Maastricht, The Netherlands. E-mail address:
[email protected] (M. Maes)
immune system ( Kirch, 1993; Maes et al., 1994, 1997). These changes include increased numbers of neutrophils and monocytes ( Wilke et al., 1996), elevated immunoglobulin concentrations (DeLisi et al., 1985), decreased mitogen-induced lymphocyte proliferation (Chengappa et al., 1995) and decreased numbers of total T and T-helper cells (Muller et al., 1993). Changes in cytokines, cytokine receptors and cytokine activity modifiers have been reported in the blood and cerebro-spinal fluid
0920-9964/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0 9 2 0 -9 9 6 4 ( 9 9 ) 0 0 11 6 - 4
158
C. Song et al. / Schizophrenia Research 42 (2000) 157–164
(CSF ) of schizophrenic patients. For example, significant increases in interleukin (IL)-1b, IL-6 and tumor necrosis factor ( TNF )-a, decreased in-vitro interferon (IFN )-c and IL-2 production, and increased serum IL-2, IL-6 receptor (IL-6R) and IL-1R antagonist (IL-1RA) concentrations have been found in schizophrenic patients (Maes et al., 1994, 1995, 1996, 1997; Sirota et al., 1995; Naudin et al., 1996; Wilke et al., 1996; Monteleone et al., 1997; Lin et al., 1998). Not all authors, however, were able to find alterations in the IRS in schizophrenia (Baker et al., 1996). It is thought that the efficacy of antipsychotic agents to treat schizophrenia is related to their dopaminergic activity. However, more than 20% of schizophrenic patients are refractory to treatment with these typical antipsychotic agents (Davis et al., 1980; Freed, 1988). Clozapine, an atypical antipsychotic drug, has been found to be more effective in non-responders to conventional neuroleptics (Meltzer, 1992). Recently, it has been shown that typical and atypical antipsychotic drugs have effects on the production of cytokines or cytokine activity modifiers (Maes et al., 1994, 1995). Chronic treatment with clozapine significantly increases serum concentrations of the IL-2R (Maes et al., 1994). Clozapine treatment also increases the serum concentrations of endogenous antiinflammatory agents, such as the IL-1RA (Maes et al., 1997). Leykin et al. (1997) reported that clozapine and haloperidol in vitro significantly suppressed mitogen-induced lymphocyte proliferation and the production of cytokines. Treatment with neuroleptics suppresses the serum concentrations of IL-6, IL-6R, and IL-6R in schizophrenic patients and the stimulated production of IL-2 and IL-3 (Bessler et al., 1995; Maes et al., 1995; Frommberger et al., 1997; Monteleone et al., 1997; Muller et al., 1997). To further examine the immunomodulatory effects of typical and atypical antipsychotic agents in vitro, we examined the effects of haloperidol and clozapine on the unstimulated and stimulated production of proinflammatory cytokines, such as IL-6 and IFNc, and negative immunoregulatory cytokines or cytokine activity modulators, such as IL-10 and IL-1RA.
2. Subjects and methods 2.1. Subjects Blood samples for the assay of cytokine production were collected from nine healthy volunteers (mean age=33.1±7.5 years; male/female ratio: 5/4). All subjects gave written informed consent after the study design had been fully explained. Exclusion criteria for subjects were as follows: 1. subjects with a past or present history of psychiatric disorders (axis-I and axis-II ); 2. subjects who ever had been taking major psychotropic medications, e.g. antidepressants and antipsychotics; 3. subjects with drug (alcohol and any other drug of dependence) abuse; 4. smokers; 5. subjects with any medical, e.g. endocrine, immune, metabolic disorders, such as diabetes, autoimmune disorders, inflammatory bowel disease, acquired immunodeficiency syndrome; and 6. subjects who currently (2 weeks prior to the first blood sample) suffered from an infectious, allergic or inflammatory response. The subjects abstained from caffeine, alcohol and nicotine for at least 8 h before blood samplings. 2.2. Methods After an overnight fast, blood samples for assays of cytokines in culture supernatant were taken at 7.30 a.m. (±30 min). Effects of antipsychotic agents on cytokine production were studied by stimulating whole blood with phytohemagglutinin (PHA) and lipopolysaccharide (LPS) and analyzing IL-1RA, IL-6, IFNc and IL-10 production in culture supernatant (De Groote et al., 1992). Diluted whole blood stimulated with LPS+PHA offers a reproducible condition of culture to measure cytokine production, whereas isolated peripheral blood mononuclear cells (PBMC ) cultivated in media or whole blood stimulated with LPS or PHA yield a lower reproducibility (De Groote et al., 1992; Zangerle et al., 1992). Moreover, in diluted whole blood, the natural cell-
C. Song et al. / Schizophrenia Research 42 (2000) 157–164
to-cell interactions are preserved. The methods used to isolate PBMC may modify the lymphocyte/monocyte ratio and may eliminate immunomodulatory agents (De Groote et al., 1992). RPMI-1640 medium (Life Technologies, Belgium) with -glutamine and Phenol Red and containing 1% of penicillin (Sigma) was employed with (stimulated) or without (unstimulated) 5 mg/ml PHA (Murex, Belgium)+25 mg/ml lipopolysaccharide (LPS; Sigma, Belgium). 1.8 ml of either one of these two media were placed into 24-well sterile plates. 0.2 ml of whole blood, 1/10 diluted, from each of the nine healthy volunteers was added. The antipsychotic drugs were dissolved in sterile water, whereas sterile water alone served as the corresponding control. Twenty microliters of each antipsychotic drug solution were added to the wells and gently mixed with the medium. Whole blood was seeded in the 24-well culture plates with clozapine 10−4, 10−6 and 10−8 M and haloperidol 10−4, 10−6 and 10−8 M. For each subject, 14 different wells were plated, i.e. two without drugs (with and without PHA+LPS)+ two (with or without PHA+LPS, i.e. the positive and negative controls)×six (three different concentrations of two antipsychotic agents). Samples were incubated for 72 h in a humidified atmosphere at 37°C, 5% CO . This time point was chosen 2 because under the culture conditions employed, peak cumulative responses for most cytokines, such as IFNc and IL-6, are obtained at 72 h (De Groote et al., 1992; Zangerle et al., 1992; Bosmans et al., unpublished data). After incubation, the plates were centrifuged at 1500 rpm for 8 min. Supernatants were taken off carefully under sterile conditions, divided into Eppendorf tubes, and frozen immediately at −70°C until they were thawed for assay. Clozapine and haloperidol were kind gifts from Sandoz and Janssen Pharmaceuticals (Belgium), respectively. IL-1RA, IL-6, IFN-c and IL-10 were quantified by means of ELISA methods ( Eurogenetics, Tessenderlo, Belgium) based on appropriate and validated sets of monoclonal antibodies (Maes et al., 1999). All samples from the normal volunteers were assayed at the same time, in a single run with a single lot number of reagents and consumables employed by a single operator. The
159
intra-assay CV values for all analytes were less than 8%. In our laboratory, the detection limits were as follows: IFNc: 1.5 U/ml; IL-6: 5 pg/ml; IL-1RA: 0.1 ng/ml; and IL-10: 10 pg/ml. 2.3. Statistics Repeated measure design analyses of variance ( RM ANOVAs) were used to examine the (1) within-subject variability with effects of antipsychotic drugs and/or effects of PHA+LPS treatment as temporal condition; and (2) betweensubject variability with gender as a factor. In order to assess the actual responsivity to PHA+LPS, independently from the unstimulated values, RM design analyses of covariance (RM ANCOVAs) were used with the stimulated cytokine production as a dependent variable and the unstimulated production as a covariate. The results of RM ANOVAs/ANCOVAs were corrected for sphericity. The ratio of IFNc/IL-10 production in culture supernatant is of critical importance in determining the capacity of supernatants to activate or inhibit monocytic and T lymphocytic functions ( Katsikis et al., 1995). The IFNc/IL-10 ratio was computed as: z transformed IFNc−z transformed IL-10. Relationships between variables were ascertained by means of Pearson’s product moment correlation coefficients.
3. Results RM design ANOVAs with the stimulated condition (PHA+LPS stimulated versus unstimulated ) as a within-subject factor and gender as a between subject factor showed (1) significant effects of PHA+LPS stimulation on IL-6 (F=76, df=1/117, p=0.0002), IL-RA (F=12.1 df=1/117, p=0.001), IL-10 (F=67, df=1/117, p=0.0002) and IFNc (F=71, df=1/117, p=0.0002) production; (2) no significant difference in stimulated and/or unstimulated IL-6, IL-1RA, IL-10 or IFNc production between men and women; and (3) no significant interaction pattern between gender and the unstimulated/stimulated condition. Consequently, we have performed the analyses on the combined study groups of men and women. There
UNST ST UNST ST UNST ST UNST ST UNST ST
IL-6 (ng/ml )
7.79 (3.38) 27.3 (11.4) 2.47 (1.63) 3.64 (1.17) 4.98 (6.52) 574 (208) 56 (73) 574 (257) −0.47 −0.173
Control
3.06 (3.08)* 16.0 (4.7)* 2.14 (1.68)* 2.92 (1.28)* 4.68 (10.05) 44 (28)* 37 (33) 108 (59)* −0.043 0.410
7.15 (3.94) 28.4 (10.4) 3.65 (2.47)** 5.70 (2.02)** 8.44 (13.0) 990 (1164)** 59 (51) 547 (197) −0.106 0.669
7.18 (4.02) 29.1 (10.4) 3.25 (1.75)** 5.98 (2.38)** 10.7 (11.2) 568 (136) 60 (46) 550 (223) 0.079 −0.191
6.85 (2.69) 31.5 (11.0) 3.69 (2.86)** 5.56 (1.97)** 6.9 (8.2) 566 (285) 56 (64) 576 (310) −0.181 −0.293
10−4 M
10−8 M
10−4 M
10−6 M
Haloperidol
Clozapine
a All results are expressed as mean (s.d.). UNST: unstimulated whole blood; ST: stimulated with PHA+LPS. * Significantly different from all other conditions. ** Significantly different from the control conditions
IFN-c/IL-10 ratio
IL-10 (pg/ml )
IFNc ( U/ml )
IL-1RA (ng/ml )
Condition
Variables
7.89 (4.12) 31.7 (12.3) 3.64 (2.42)** 6.08 (2.60)** 14.9 (19.3) 671 (259) 49 (59) 572 (238) 0.613 −0.067
10−6 M
Table 1 Effects of different concentrations of clozapine and haloperidol on cytokine and IL-1RA production by whole blood of human subjectsa
7.40 (3.59) 31.9 (12.5) 3.49 (2.35)** 6.43 (3.23)** 11.5 (14.5) 667 (235) 57 (70) 569 (253) 0.183 −0.065
10−8 M 9.3 15.4 12.4 19.4 1.99 19.3 1.21 29.7 1.14 1.44
F
3/20 2/13 2/20 2/17 4/29 1/11 2/19 3/26 3/21 1/11
df
0.0007 0.0006 0.0005 0.0001 0.12 0.001 0.3
