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The inflammatory response system in treatment-resistant schizophrenia: increased serum interleukin-6
Schizophrenia Research 32 (1998) 9–15
The inflammatory response system in treatment-resistant schizophrenia: increased serum interleukin-6 A. Lin a,b,*, G. Kenis c,d, S. Bignotti e, G.-J.-B. Tura e, R. De Jong c, E. Bosmans d, R. Pioli e, C. Altamura e,f, S. Scharpe´ b, M. Maes a,e,g a Clinical Research Center for Mental Health (CRC-MH), University Department of Psychiatry, 267 Lange Beeldekensstraat, 2060 Antwerp, Belgium b Department of Medical Biochemistry, University of Antwerp, Antwerp, Belgium c Department of Anesthesiology, AZ Genk, Belgium d Eurogenetics, Tessenderlo, Belgium e IRCCS, Istituto Fatebenefratelli, Brescia, Italy f Department of Psychiatry, University of Milan, Milan, Italy g Department of Psychiatry, Vanderbilt University, Nashville, USA Received 15 October 1997; accepted 23 March 1998
Abstract There is some evidence that the pathophysiology of schizophrenia is related to activation of the inflammatory response system (IRS ), as indicated by increased serum concentrations of interleukin-6 (IL-6), IL-6 receptor (IL-6R), IL-1R antagonist (IL-1RA) and IL-2R and lower serum concentrations of CC16, an endogenous anti-inflammatory protein with immunosuppressive and anti-inflammatory effects. The aims of the present study were to examine serum CC16 in relation to IL-6, IL-6R and gp130, the IL-6 transducing signal protein, in schizophrenia and in treatmentresistant schizophrenia (TRS ). Serum IL-6 and sIL-6R were significantly higher in medicated schizophrenic patients than in normal controls. Serum IL-6 was significantly higher in TRS than in normal volunteers, whereas schizophrenic patients without TRS showed intermediate values. Serum CC16 was significantly lower in schizophrenic patients with a positive family history for psychoses than in normal volunteers and patients without a positive family history. There was a significant inverse relationship between serum CC16 and serum IL-6 or sIL-6R in schizophrenic patients, but not in normal volunteers. The results suggest that the inflammatory response in schizophrenia, as indicated by increased serum IL-6 and sIL-6R, may be causally related to lower serum CC16 and that the latter might be a trait marker for schizophrenia. © 1998 Elsevier Science B.V. All rights reserved. Keywords: CC16; Cytokines; Inflammation; Interleukin-6; Neuroleptics; Treatment-resistant schizophrenia
* Corresponding author. Fax: +32 3 4483265; e-mail: [email protected] 0920-9964/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 9 20 - 9 96 4 ( 98 ) 00 0 3 4 -6
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A. Lin et al. / Schizophrenia Research 32 (1998) 9–15
1. Introduction There is now some evidence that schizophrenia is related to activation of the inflammatory response system (IRS ) (Smith, 1991, 1992; Smith and Maes, 1995; Maes et al., 1997b). Plasma concentrations of interleukin-6 (IL-6) and the soluble IL-6 receptor (sIL-6R) are higher in schizophrenic patients than in normal volunteers (Maes et al., 1994a, 1995, 1996; Naudin et al., 1996). IL-6 is a pleiotropic cytokine that plays an important role in the immune and acute phase response, hematopoiesis, and the function of the (central ) nervous system [for a review, see Maes et al. (1993)]. IL-6 exerts its biological effects by binding to a cell surface receptor complex consisting of two subunits, i.e. the IL-6R and gp130, a signaltransducing molecule ( Yamasaki et al., 1988). Gp130 is a common signal transducer for IL-6, leukemia inhibitory factor, oncostatin M, IL-11 and ciliary neurotrophic factor (Gearing et al., 1992; Ip et al., 1992; Yin et al., 1993). The sIL-6R in serum has the potential to mediate IL-6 signals (even in IL-6-insensitive cells) by forming a complex with IL-6, which, in turn, associates with gp130 on the responding cells (Saito et al., 1993; Benigni et al., 1996). Serum sgp130 may compete with its membrane-bound counterpart for binding to the sIL-6/IL-6R complex, which may result in an inhibition of IL-6 signalling in some responding cells (Murakami-Mori et al., 1996). Additional evidence for the IRS activation hypothesis of schizophrenia is that the plasma levels of interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1RA) are increased in patients with schizophrenia ( Katila et al., 1994; Sirota et al., 1995; Maes et al., 1996, 1997a). Some studies have demonstrated that the plasma concentrations of the soluble interleukin 2 receptor (sIL-2R) are significantly increased in schizophrenia (Rapaport et al., 1989; Maes et al., 1994a), indicating a moderate T-cell activation in that illness (Caruso et al., 1993). Plasma concentrations of CC16 are significantly lower in unmedicated schizophrenic patients than in normal controls (Maes et al., 1996). CC16 or Clara cell 16KD protein is a product of the Clara cells lining the bronchiolar epithelium (Singh and Katyal, 1984) and cells of
the urogenital tract (Bernard et al., 1992). CC16 has anti-inflammatory and immunosuppressive functions (Dierynck et al., 1995). Since CC16 is an endogenous anti-inflammatory protein, lower serum CC16 concentrations may be causally related to activation of the IRS in schizophrenia. Thus, it could be hypothesized that lower serum CC16 concentrations in schizophrenic patients might be related to increased plasma IL-6 and sIL-6R concentrations. There is now evidence that typical and atypical antipsychotic drugs have complex in-vivo immunomodulatory effects in schizophrenic subjects (Maes et al., 1994a, 1995, 1996; Pollmacher et al., 1996). Repeated administration of typical antipsychotic drugs may suppress plasma IL-6 and sIL-6R, whereas repeated administration of these drugs does not significantly alter plasma sIL-2R concentrations. These findings suggest that typical antipsychotic drugs have immunosuppressive effects in vivo through suppression of IL-6 related mechanisms. The aims of the present study were to examine (1) whether serum IL-6, sIL-6R and gp130 are increased and serum CC16 is decreased in medicated schizophrenic patients, divided into these with and without treatment resistance (TRS ) or with and without a positive family history for schizophrenia; and (2) whether there are any inverse relationships between serum CC16 and IL-6 or sIL-6R in schizophrenic patients and normal controls.
2. Subjects and methods 2.1. Subjects Forty-two subjects participated in this study: 15 healthy volunteers and 27 schizophrenic subjects. Table 1 shows the demographic data. Schizophrenic patients were admitted to the Psychiatric Institute Fatebenefratelli (Brescia, Italy). Patients were diagnosed according to DSMIII-R criteria (American Psychiatric Association, 1987). All schizophrenic patients were of the chronic or subchronic type. The patients were classified as being treatment-resistant on the basis
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A. Lin et al. / Schizophrenia Research 32 (1998) 9–15 Table 1 Demographic data of the 15 normal volunteers and 27 schizophrenic patients included in this study Groups
Age (years)
Men: women
BPRS
Duration of illness (years)
Haloperidol dose (mg/day)
Length of treatment (days)
Normal controls Schizophrenia
45.1 (11.4) 47.1 (11.6)
8:7 18:9
— 71.0 (17.5)
— 24.5 (11.4)
— 26.4 (16.7)
— 162 (201)
All results are shown as mean±s.d.
of Kane’s criteria ( Kane et al., 1988). Fifteen TRS patients and 12 non-TRS patients were included. Patients were divided into those with a positive family history for schizophrenia in first- or seconddegree relatives (n=7) and those with a negative family history of schizophrenia (n=20). Severity of illness was assessed by means of the Brief Psychiatric Rating Scale (BPRS; 1–7 version; Rhoades and Overall, 1988). Exclusionary criteria for patients were other axis I diagnoses, such as organic mental disorder and substance use disorder (6 months before participating in the studies). The healthy volunteers were free of any medication for at least 1 month prior to blood sampling. None of the volunteers was a regular drinker or had ever taken psychotropic drugs. Controls were excluded for a present, past and family history (first-degree relatives) of psychiatric disorders. Exclusionary criteria for patients and controls were: (1) use of non-psychotropic drugs, which are known to interfere with immune or endocrine function, e.g. corticosteroids and cyclosporin A; (2) chronic illnesses known to affect the immune system; and (3) acute infectious or inflammatory reactions for at least 2 weeks prior to the study. All subjects had a normal physical examination, normal values of blood and urine tests, such as SGOT, SGPT, cGT, serum electrolytes, thyroid function tests and renal tests (blood urea and creatinine). 2.2. Methods Following an overnight fast, plasma for assay of the IRS variables was sampled at 8.00 a.m. and stored at −70°C until thawed for assay. All blood samples in normal volunteers and schizophrenic subjects were collected in October 1996 in order to control for seasonal any variations in immune
variables, such as serum IL-6 and sIL-6R (Maes et al., 1994b). Serum concentrations of IL-6, sIL-6R, CC16 and sgp130 were measured by quantitative enzyme-linked immunosorbent assay ( ELISA) techniques. Monoclonal antibodies specific for each component have been pre-coated on to 96-well microtiter plates. Standards and samples were pipetted into the wells and then incubated at 37°C. Each cytokine receptor or soluble protein was bound by the immobilized antibody and incubated at 37°C. After washing away all unbound substances, an enzyme-linked polyclonal antibody specific for each of these components was added to the wells and incubated at 37°C. Following a wash to remove all of the unbound antibody–enzyme reagent, a substrate solution was added to the wells for 10 min for colour to develop in proportion to the amount of receptor or protein bound in the initial step. The colour development was stopped by sulphuric acid and the intensity of the colour measured by a microtiter plate reader (absorbency at 450 nm). Each assay was carried out at the same time and the same run by the same investigator (AL). The intra-assay coefficients were less than 8% for all variables. 2.3. Statistics Group mean differences were checked by means of analysis of variance (ANOVA) or covariance (ANCOVA). Multiple a priori comparisons among treatment means were checked with the Dunn test, taking into account Bonferroni’s p correction (Howell, 1982). The independence of classification systems was ascertained by means of an analysis of contingence (x2 test). Relationships between variables were assessed with Pearson’s product
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moment correlations or with multiple regression analyses. The normality of the distribution was checked using the Smirnov–Kolmogorov test. Box–Cox transformations were used in order to normalize the distribution of the data or to adjust for heterogeneity of variance. The diagnostic performance of the IRS variables for schizophrenia was assessed by means of ROC (receiver operating characteristics) analysis and computation of sensitivity, specificity and predictive value of a positive test result (PV+) with kappa statistics.
3. Results 3.1. Demographic data Table 1 shows the demographic data of the subjects in the present study. There were no significant differences in age (F=0.3, df=1/40, p= 0.6) or men/women ratio (x2=0.7, df=1, p=0.4) between normal volunteers and schizophrenic patients. There were no significant correlations between age and serum IL-6 (r=0.18, p=0.2), sIL-6R (r=0.12, p=0.5) or sgp130 (r=−0.18, p= 0.3) (all results of regression analyses pooled over the study groups of the normal controls and schizophrenic subjects). In normal volunteers (r=−0.51, p=0.049), but not in schizophrenic patients (r= 0.02, p=0.9), there was a significant and inverse relationship between age and serum sgp130. The regression analysis, pooled over both study groups, showed a significantly negative correlation between age and serum CC16 (r=−0.32, p=0.04). By means of ANOVAs, no significant differences between men and women could be found in serum IL-6 (F=0.9, df=1/37, p=0.7), sIL-6R (F=0.4, df=1/37, p=0.5), sgp130 (F=2.2, df=1/37, p= 0.1) or CC16 (F=0.00, df=1/37, p=0.96). In any case, we have adjusted subsequent statistical analyses for possible effects of age and gender by entering these variables as covariates in regression analyses. Table 1 shows the mean BPRS, duration of illness, and mean dose and length of treatment with haloperidol.
3.2. IRS variables and schizophrenia Table 2 shows that serum IL-6 and sIL-6R were significantly higher in schizophrenic patients than in normal controls. The area under the ROC curve for IL-6 was 82.0%; at the optimal cut-off value, i.e. IL-6≥1 pg/ml, the diagnostic performance was: sensitivity, 65.4%; specificity, 86.7%; and PV+89.5% (k=0.47, t=3.49, p=0.002). There was a trend toward a higher serum sgp130 and lower CC16 in schizophrenic patients. Regression analyses showed significant and positive relationships between serum IL-6 and sIL-6R (r=0.35, p=0.02) in the two study groups combined. There were no significant correlations between serum sgp130 and either IL-6 (r=−0.10, p=0.5) or sIL-6R (r=0.11, p=0.5). There were significant and inverse relationships between serum CC16 and either IL-6 (r=−0.50, p=0.001) or sIL-6R (r=−0.42, p=0.007). The inverse relationship between serum CC16 and IL-6 was significant in schizophrenic patients (r=−0.43, p= 0.03), but not in normal volunteers (r=−0.31, p=0.2). The inverse relationship between serum CC16 and sIL-6R was significant in schizophrenic subjects (r=−0.44, p=0.02), but not in normal controls (r=−0.07, p=0.8). No significant correlations were found between dose of haloperidol, length of treatment with haloperidol or duration of illness, on the one hand, and serum IL-6, sIL-6R or sgp130, on the other. There was a significant and inverse relationship between serum CC16 and duration of illness (r= −0.40, p=0.03). There were no significant relationships between serum CC16 and dose or length of treatment with haloperidol. There were no significant correlations between the total BPRS score and serum IL-6, sIL-6R, sgp130 or CC16. By means of ANCOVAs (with age and gender as covariates), no significant differences could be found in serum IL-6, sIL-6R or sgp130 between schizophrenic subjects with (n=7) and without (n=20) a positive family history for psychoses. By means of ANCOVA (with age and gender as covariates), significantly lower serum CC16 values were found in schizophrenic patients with a positive family history for psychoses (mean= 25.3±13.6 ng/ml ) than in patients without a pos-
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A. Lin et al. / Schizophrenia Research 32 (1998) 9–15 Table 2 Measurements of serum IL-6, sIL-6R, gp130 and CC16 in normal controls and schizophrenic patients Groups
IL-6 (pg/ml )
sIL-6R (ng/ml )
gp130 (ng/ml )
CC16 (ng/ml )
Normal controls Schizophrenia Fa df p
0.38 (0.65) 4.85 (6.13) 12.7 1/37 0.001
188 (43) 279 (199) 10.1 1/37 0.003
1030 (206) 1157 (210) 3.3 1/37 0.07
41.5 (13.8) 34.6 (19.4) 2.7 1/37 0.1
All results are shown as mean±s.d. aAll results of ANCOVAS with age and sex as covariates. All variables were processed in the Box–Cox transformation.
itive family history (mean=37.9±20.4 ng/ml ) or normal controls (mean=41.5±13.8 ng/ml ) (ANCOVA: F=3.5, df=2/36, p=0.03). In schizophrenic patients, it was found that 33.1% of the variance in serum CC16 could be explained by the multiple regression on age (F=7.1, p=0.01) and a positive family history for psychoses (F=4.3, p=0.04) (overall regression: F=5.7, df=2/23, p=0.009). By means of ANCOVAs (with age and gender as covariates), no significant differences in serum sIL-6R, sgp130 or CC16 could be found between TRS and non-TRS patients. Serum IL-6 was significantly higher (mean=6.68±7.31 pg/ml ) in TRS patients (t=4.0, p=0.0006) than in normal volunteers (mean=0.39±0.65 pg/ml ), whereas non-TRS patients (mean=2.72±3.62 pg/ml ) took up an intermediate position. The latter had serum IL-6 values that were not significantly different from normal volunteers (t=2.1, p=0.03) or patients with TRS (t=1.6, p=0.1) (ANCOVA: F=7.8, df=2/36, p=0.002).
4. Discussion Our finding that serum IL-6 is significantly higher in medicated schizophrenic patients than in normal controls is in accordance with previous studies (Shintani et al., 1991; Maes et al., 1995). Increased serum IL-6 (≥1 pg/ml ) had a good diagnostic performance for schizophrenia and detected 65.4% of the patients with a specificity of 86.7%. Increased serum IL-6 and sIL-6R have also been observed in unmedicated schizophrenic patients (Maes et al., 1994a, 1995; Naudin et al., 1996). Subchronic treatment with typical antipsy-
chotic agents may, in part, suppress elevated serum concentrations of IL-6 and sIL-6R in schizophrenic patients (Maes et al., 1995). The above results suggest that (sub)chronic treatment with typical antipsychotics only partially suppresses the initial elevations in serum IL-6 and sIL-6R in schizophrenia. A major finding of this study is that patients with TRS had significantly higher serum IL-6 levels than healthy volunteers, whereas patients with non-TRS did not differ in serum IL-6 levels from controls. In this study, we did not find a correlation between the dose of haloperidol, length of treatment with haloperidol or duration of illness and serum IL-6, sIL-6R or gp130, whereas there were no differences in dose of haloperidol or treatment duration between TRS and non-TRS patients. Taken together, these findings corroborate the hypothesis that schizophrenia is accompanied by IRS activation and that the latter is related to treatment resistance to typical antipsychotic agents. Another major finding of this study is the significant inverse relationship between serum CC16 and serum IL-6 or sIL-6R in schizophrenic patients, but not in healthy volunteers. CC16 can inhibit the biological activity of interferon-c (IFNc) (Dierynck et al., 1995). IFN-c is produced by activated T cells and natural killer cells, and is one of the important activators of monocyte/ macrophage functions and IL-6 secretion. Thus, lower concentrations of CC16 in schizophrenia may be causally related to IRS activation, as indicated by increased serum IL-6 and sIL-6R concentrations. In a previous study, the age at the onset of schizophrenia was positively and significantly related to plasma CC16 (Maes et al., 1996).
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In the present study, we found a significant and inverse relationship between serum CC16 and duration of illness. Lowered serum CC16 was a characteristic of schizophrenic patients with a positive family history for schizophrenia, whereas patients without a family history for schizophrenia had values that were not significantly different from those of normal volunteers. Moreover, in the present study, no significant relationships were found between serum CC16 and the dose of haloperidol or length of treatment. Therefore, it is possible that lower plasma CC16 is a trait marker for schizophrenia. Whether activation of the IRS is related to the pathophysiology rather than to the pathoetiology of schizophrenia remains elusive. In any case, the activation of the IRS found in schizophrenia could be (1) involved in the development of autoimmune responses in schizophrenia (DeLisi and Crow, 1986); (2) related to a viral infection or reactivation occurring in schizophrenia ( Kirch, 1993); or (3) an aspecific response to, for example, psychological stress (Maes et al., in press). In summary, the results of the present study suggest that TRS is accompanied by increased serum IL-6 concentrations and that lower CC16 levels in schizophrenia may constitute a trait marker for schizophrenia and may predispose these patients to IRS activation, such as increased production of IL-6 and IL-6R.
Acknowledgment The research reported was supported in part by the Staglin Investigator Award (NARSAD) to Dr M. Maes, the Clinical Research Center for Mental Health, Antwerp, Belgium, and the Clinical Research Center for Mental Health, Brescia, Italy. The secretarial assistance of Mrs M. Maes is greatly appreciated.
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The inflammatory response system in treatment-resistant schizophrenia: increased serum interleukin-6 A. Lin a,b,*, G. Kenis c,d, S. Bignotti e, G.-J.-B. Tura e, R. De Jong c, E. Bosmans d, R. Pioli e, C. Altamura e,f, S. Scharpe´ b, M. Maes a,e,g a Clinical Research Center for Mental Health (CRC-MH), University Department of Psychiatry, 267 Lange Beeldekensstraat, 2060 Antwerp, Belgium b Department of Medical Biochemistry, University of Antwerp, Antwerp, Belgium c Department of Anesthesiology, AZ Genk, Belgium d Eurogenetics, Tessenderlo, Belgium e IRCCS, Istituto Fatebenefratelli, Brescia, Italy f Department of Psychiatry, University of Milan, Milan, Italy g Department of Psychiatry, Vanderbilt University, Nashville, USA Received 15 October 1997; accepted 23 March 1998
Abstract There is some evidence that the pathophysiology of schizophrenia is related to activation of the inflammatory response system (IRS ), as indicated by increased serum concentrations of interleukin-6 (IL-6), IL-6 receptor (IL-6R), IL-1R antagonist (IL-1RA) and IL-2R and lower serum concentrations of CC16, an endogenous anti-inflammatory protein with immunosuppressive and anti-inflammatory effects. The aims of the present study were to examine serum CC16 in relation to IL-6, IL-6R and gp130, the IL-6 transducing signal protein, in schizophrenia and in treatmentresistant schizophrenia (TRS ). Serum IL-6 and sIL-6R were significantly higher in medicated schizophrenic patients than in normal controls. Serum IL-6 was significantly higher in TRS than in normal volunteers, whereas schizophrenic patients without TRS showed intermediate values. Serum CC16 was significantly lower in schizophrenic patients with a positive family history for psychoses than in normal volunteers and patients without a positive family history. There was a significant inverse relationship between serum CC16 and serum IL-6 or sIL-6R in schizophrenic patients, but not in normal volunteers. The results suggest that the inflammatory response in schizophrenia, as indicated by increased serum IL-6 and sIL-6R, may be causally related to lower serum CC16 and that the latter might be a trait marker for schizophrenia. © 1998 Elsevier Science B.V. All rights reserved. Keywords: CC16; Cytokines; Inflammation; Interleukin-6; Neuroleptics; Treatment-resistant schizophrenia
* Corresponding author. Fax: +32 3 4483265; e-mail: [email protected] 0920-9964/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 9 20 - 9 96 4 ( 98 ) 00 0 3 4 -6
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1. Introduction There is now some evidence that schizophrenia is related to activation of the inflammatory response system (IRS ) (Smith, 1991, 1992; Smith and Maes, 1995; Maes et al., 1997b). Plasma concentrations of interleukin-6 (IL-6) and the soluble IL-6 receptor (sIL-6R) are higher in schizophrenic patients than in normal volunteers (Maes et al., 1994a, 1995, 1996; Naudin et al., 1996). IL-6 is a pleiotropic cytokine that plays an important role in the immune and acute phase response, hematopoiesis, and the function of the (central ) nervous system [for a review, see Maes et al. (1993)]. IL-6 exerts its biological effects by binding to a cell surface receptor complex consisting of two subunits, i.e. the IL-6R and gp130, a signaltransducing molecule ( Yamasaki et al., 1988). Gp130 is a common signal transducer for IL-6, leukemia inhibitory factor, oncostatin M, IL-11 and ciliary neurotrophic factor (Gearing et al., 1992; Ip et al., 1992; Yin et al., 1993). The sIL-6R in serum has the potential to mediate IL-6 signals (even in IL-6-insensitive cells) by forming a complex with IL-6, which, in turn, associates with gp130 on the responding cells (Saito et al., 1993; Benigni et al., 1996). Serum sgp130 may compete with its membrane-bound counterpart for binding to the sIL-6/IL-6R complex, which may result in an inhibition of IL-6 signalling in some responding cells (Murakami-Mori et al., 1996). Additional evidence for the IRS activation hypothesis of schizophrenia is that the plasma levels of interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1RA) are increased in patients with schizophrenia ( Katila et al., 1994; Sirota et al., 1995; Maes et al., 1996, 1997a). Some studies have demonstrated that the plasma concentrations of the soluble interleukin 2 receptor (sIL-2R) are significantly increased in schizophrenia (Rapaport et al., 1989; Maes et al., 1994a), indicating a moderate T-cell activation in that illness (Caruso et al., 1993). Plasma concentrations of CC16 are significantly lower in unmedicated schizophrenic patients than in normal controls (Maes et al., 1996). CC16 or Clara cell 16KD protein is a product of the Clara cells lining the bronchiolar epithelium (Singh and Katyal, 1984) and cells of
the urogenital tract (Bernard et al., 1992). CC16 has anti-inflammatory and immunosuppressive functions (Dierynck et al., 1995). Since CC16 is an endogenous anti-inflammatory protein, lower serum CC16 concentrations may be causally related to activation of the IRS in schizophrenia. Thus, it could be hypothesized that lower serum CC16 concentrations in schizophrenic patients might be related to increased plasma IL-6 and sIL-6R concentrations. There is now evidence that typical and atypical antipsychotic drugs have complex in-vivo immunomodulatory effects in schizophrenic subjects (Maes et al., 1994a, 1995, 1996; Pollmacher et al., 1996). Repeated administration of typical antipsychotic drugs may suppress plasma IL-6 and sIL-6R, whereas repeated administration of these drugs does not significantly alter plasma sIL-2R concentrations. These findings suggest that typical antipsychotic drugs have immunosuppressive effects in vivo through suppression of IL-6 related mechanisms. The aims of the present study were to examine (1) whether serum IL-6, sIL-6R and gp130 are increased and serum CC16 is decreased in medicated schizophrenic patients, divided into these with and without treatment resistance (TRS ) or with and without a positive family history for schizophrenia; and (2) whether there are any inverse relationships between serum CC16 and IL-6 or sIL-6R in schizophrenic patients and normal controls.
2. Subjects and methods 2.1. Subjects Forty-two subjects participated in this study: 15 healthy volunteers and 27 schizophrenic subjects. Table 1 shows the demographic data. Schizophrenic patients were admitted to the Psychiatric Institute Fatebenefratelli (Brescia, Italy). Patients were diagnosed according to DSMIII-R criteria (American Psychiatric Association, 1987). All schizophrenic patients were of the chronic or subchronic type. The patients were classified as being treatment-resistant on the basis
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A. Lin et al. / Schizophrenia Research 32 (1998) 9–15 Table 1 Demographic data of the 15 normal volunteers and 27 schizophrenic patients included in this study Groups
Age (years)
Men: women
BPRS
Duration of illness (years)
Haloperidol dose (mg/day)
Length of treatment (days)
Normal controls Schizophrenia
45.1 (11.4) 47.1 (11.6)
8:7 18:9
— 71.0 (17.5)
— 24.5 (11.4)
— 26.4 (16.7)
— 162 (201)
All results are shown as mean±s.d.
of Kane’s criteria ( Kane et al., 1988). Fifteen TRS patients and 12 non-TRS patients were included. Patients were divided into those with a positive family history for schizophrenia in first- or seconddegree relatives (n=7) and those with a negative family history of schizophrenia (n=20). Severity of illness was assessed by means of the Brief Psychiatric Rating Scale (BPRS; 1–7 version; Rhoades and Overall, 1988). Exclusionary criteria for patients were other axis I diagnoses, such as organic mental disorder and substance use disorder (6 months before participating in the studies). The healthy volunteers were free of any medication for at least 1 month prior to blood sampling. None of the volunteers was a regular drinker or had ever taken psychotropic drugs. Controls were excluded for a present, past and family history (first-degree relatives) of psychiatric disorders. Exclusionary criteria for patients and controls were: (1) use of non-psychotropic drugs, which are known to interfere with immune or endocrine function, e.g. corticosteroids and cyclosporin A; (2) chronic illnesses known to affect the immune system; and (3) acute infectious or inflammatory reactions for at least 2 weeks prior to the study. All subjects had a normal physical examination, normal values of blood and urine tests, such as SGOT, SGPT, cGT, serum electrolytes, thyroid function tests and renal tests (blood urea and creatinine). 2.2. Methods Following an overnight fast, plasma for assay of the IRS variables was sampled at 8.00 a.m. and stored at −70°C until thawed for assay. All blood samples in normal volunteers and schizophrenic subjects were collected in October 1996 in order to control for seasonal any variations in immune
variables, such as serum IL-6 and sIL-6R (Maes et al., 1994b). Serum concentrations of IL-6, sIL-6R, CC16 and sgp130 were measured by quantitative enzyme-linked immunosorbent assay ( ELISA) techniques. Monoclonal antibodies specific for each component have been pre-coated on to 96-well microtiter plates. Standards and samples were pipetted into the wells and then incubated at 37°C. Each cytokine receptor or soluble protein was bound by the immobilized antibody and incubated at 37°C. After washing away all unbound substances, an enzyme-linked polyclonal antibody specific for each of these components was added to the wells and incubated at 37°C. Following a wash to remove all of the unbound antibody–enzyme reagent, a substrate solution was added to the wells for 10 min for colour to develop in proportion to the amount of receptor or protein bound in the initial step. The colour development was stopped by sulphuric acid and the intensity of the colour measured by a microtiter plate reader (absorbency at 450 nm). Each assay was carried out at the same time and the same run by the same investigator (AL). The intra-assay coefficients were less than 8% for all variables. 2.3. Statistics Group mean differences were checked by means of analysis of variance (ANOVA) or covariance (ANCOVA). Multiple a priori comparisons among treatment means were checked with the Dunn test, taking into account Bonferroni’s p correction (Howell, 1982). The independence of classification systems was ascertained by means of an analysis of contingence (x2 test). Relationships between variables were assessed with Pearson’s product
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moment correlations or with multiple regression analyses. The normality of the distribution was checked using the Smirnov–Kolmogorov test. Box–Cox transformations were used in order to normalize the distribution of the data or to adjust for heterogeneity of variance. The diagnostic performance of the IRS variables for schizophrenia was assessed by means of ROC (receiver operating characteristics) analysis and computation of sensitivity, specificity and predictive value of a positive test result (PV+) with kappa statistics.
3. Results 3.1. Demographic data Table 1 shows the demographic data of the subjects in the present study. There were no significant differences in age (F=0.3, df=1/40, p= 0.6) or men/women ratio (x2=0.7, df=1, p=0.4) between normal volunteers and schizophrenic patients. There were no significant correlations between age and serum IL-6 (r=0.18, p=0.2), sIL-6R (r=0.12, p=0.5) or sgp130 (r=−0.18, p= 0.3) (all results of regression analyses pooled over the study groups of the normal controls and schizophrenic subjects). In normal volunteers (r=−0.51, p=0.049), but not in schizophrenic patients (r= 0.02, p=0.9), there was a significant and inverse relationship between age and serum sgp130. The regression analysis, pooled over both study groups, showed a significantly negative correlation between age and serum CC16 (r=−0.32, p=0.04). By means of ANOVAs, no significant differences between men and women could be found in serum IL-6 (F=0.9, df=1/37, p=0.7), sIL-6R (F=0.4, df=1/37, p=0.5), sgp130 (F=2.2, df=1/37, p= 0.1) or CC16 (F=0.00, df=1/37, p=0.96). In any case, we have adjusted subsequent statistical analyses for possible effects of age and gender by entering these variables as covariates in regression analyses. Table 1 shows the mean BPRS, duration of illness, and mean dose and length of treatment with haloperidol.
3.2. IRS variables and schizophrenia Table 2 shows that serum IL-6 and sIL-6R were significantly higher in schizophrenic patients than in normal controls. The area under the ROC curve for IL-6 was 82.0%; at the optimal cut-off value, i.e. IL-6≥1 pg/ml, the diagnostic performance was: sensitivity, 65.4%; specificity, 86.7%; and PV+89.5% (k=0.47, t=3.49, p=0.002). There was a trend toward a higher serum sgp130 and lower CC16 in schizophrenic patients. Regression analyses showed significant and positive relationships between serum IL-6 and sIL-6R (r=0.35, p=0.02) in the two study groups combined. There were no significant correlations between serum sgp130 and either IL-6 (r=−0.10, p=0.5) or sIL-6R (r=0.11, p=0.5). There were significant and inverse relationships between serum CC16 and either IL-6 (r=−0.50, p=0.001) or sIL-6R (r=−0.42, p=0.007). The inverse relationship between serum CC16 and IL-6 was significant in schizophrenic patients (r=−0.43, p= 0.03), but not in normal volunteers (r=−0.31, p=0.2). The inverse relationship between serum CC16 and sIL-6R was significant in schizophrenic subjects (r=−0.44, p=0.02), but not in normal controls (r=−0.07, p=0.8). No significant correlations were found between dose of haloperidol, length of treatment with haloperidol or duration of illness, on the one hand, and serum IL-6, sIL-6R or sgp130, on the other. There was a significant and inverse relationship between serum CC16 and duration of illness (r= −0.40, p=0.03). There were no significant relationships between serum CC16 and dose or length of treatment with haloperidol. There were no significant correlations between the total BPRS score and serum IL-6, sIL-6R, sgp130 or CC16. By means of ANCOVAs (with age and gender as covariates), no significant differences could be found in serum IL-6, sIL-6R or sgp130 between schizophrenic subjects with (n=7) and without (n=20) a positive family history for psychoses. By means of ANCOVA (with age and gender as covariates), significantly lower serum CC16 values were found in schizophrenic patients with a positive family history for psychoses (mean= 25.3±13.6 ng/ml ) than in patients without a pos-
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A. Lin et al. / Schizophrenia Research 32 (1998) 9–15 Table 2 Measurements of serum IL-6, sIL-6R, gp130 and CC16 in normal controls and schizophrenic patients Groups
IL-6 (pg/ml )
sIL-6R (ng/ml )
gp130 (ng/ml )
CC16 (ng/ml )
Normal controls Schizophrenia Fa df p
0.38 (0.65) 4.85 (6.13) 12.7 1/37 0.001
188 (43) 279 (199) 10.1 1/37 0.003
1030 (206) 1157 (210) 3.3 1/37 0.07
41.5 (13.8) 34.6 (19.4) 2.7 1/37 0.1
All results are shown as mean±s.d. aAll results of ANCOVAS with age and sex as covariates. All variables were processed in the Box–Cox transformation.
itive family history (mean=37.9±20.4 ng/ml ) or normal controls (mean=41.5±13.8 ng/ml ) (ANCOVA: F=3.5, df=2/36, p=0.03). In schizophrenic patients, it was found that 33.1% of the variance in serum CC16 could be explained by the multiple regression on age (F=7.1, p=0.01) and a positive family history for psychoses (F=4.3, p=0.04) (overall regression: F=5.7, df=2/23, p=0.009). By means of ANCOVAs (with age and gender as covariates), no significant differences in serum sIL-6R, sgp130 or CC16 could be found between TRS and non-TRS patients. Serum IL-6 was significantly higher (mean=6.68±7.31 pg/ml ) in TRS patients (t=4.0, p=0.0006) than in normal volunteers (mean=0.39±0.65 pg/ml ), whereas non-TRS patients (mean=2.72±3.62 pg/ml ) took up an intermediate position. The latter had serum IL-6 values that were not significantly different from normal volunteers (t=2.1, p=0.03) or patients with TRS (t=1.6, p=0.1) (ANCOVA: F=7.8, df=2/36, p=0.002).
4. Discussion Our finding that serum IL-6 is significantly higher in medicated schizophrenic patients than in normal controls is in accordance with previous studies (Shintani et al., 1991; Maes et al., 1995). Increased serum IL-6 (≥1 pg/ml ) had a good diagnostic performance for schizophrenia and detected 65.4% of the patients with a specificity of 86.7%. Increased serum IL-6 and sIL-6R have also been observed in unmedicated schizophrenic patients (Maes et al., 1994a, 1995; Naudin et al., 1996). Subchronic treatment with typical antipsy-
chotic agents may, in part, suppress elevated serum concentrations of IL-6 and sIL-6R in schizophrenic patients (Maes et al., 1995). The above results suggest that (sub)chronic treatment with typical antipsychotics only partially suppresses the initial elevations in serum IL-6 and sIL-6R in schizophrenia. A major finding of this study is that patients with TRS had significantly higher serum IL-6 levels than healthy volunteers, whereas patients with non-TRS did not differ in serum IL-6 levels from controls. In this study, we did not find a correlation between the dose of haloperidol, length of treatment with haloperidol or duration of illness and serum IL-6, sIL-6R or gp130, whereas there were no differences in dose of haloperidol or treatment duration between TRS and non-TRS patients. Taken together, these findings corroborate the hypothesis that schizophrenia is accompanied by IRS activation and that the latter is related to treatment resistance to typical antipsychotic agents. Another major finding of this study is the significant inverse relationship between serum CC16 and serum IL-6 or sIL-6R in schizophrenic patients, but not in healthy volunteers. CC16 can inhibit the biological activity of interferon-c (IFNc) (Dierynck et al., 1995). IFN-c is produced by activated T cells and natural killer cells, and is one of the important activators of monocyte/ macrophage functions and IL-6 secretion. Thus, lower concentrations of CC16 in schizophrenia may be causally related to IRS activation, as indicated by increased serum IL-6 and sIL-6R concentrations. In a previous study, the age at the onset of schizophrenia was positively and significantly related to plasma CC16 (Maes et al., 1996).
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In the present study, we found a significant and inverse relationship between serum CC16 and duration of illness. Lowered serum CC16 was a characteristic of schizophrenic patients with a positive family history for schizophrenia, whereas patients without a family history for schizophrenia had values that were not significantly different from those of normal volunteers. Moreover, in the present study, no significant relationships were found between serum CC16 and the dose of haloperidol or length of treatment. Therefore, it is possible that lower plasma CC16 is a trait marker for schizophrenia. Whether activation of the IRS is related to the pathophysiology rather than to the pathoetiology of schizophrenia remains elusive. In any case, the activation of the IRS found in schizophrenia could be (1) involved in the development of autoimmune responses in schizophrenia (DeLisi and Crow, 1986); (2) related to a viral infection or reactivation occurring in schizophrenia ( Kirch, 1993); or (3) an aspecific response to, for example, psychological stress (Maes et al., in press). In summary, the results of the present study suggest that TRS is accompanied by increased serum IL-6 concentrations and that lower CC16 levels in schizophrenia may constitute a trait marker for schizophrenia and may predispose these patients to IRS activation, such as increased production of IL-6 and IL-6R.
Acknowledgment The research reported was supported in part by the Staglin Investigator Award (NARSAD) to Dr M. Maes, the Clinical Research Center for Mental Health, Antwerp, Belgium, and the Clinical Research Center for Mental Health, Brescia, Italy. The secretarial assistance of Mrs M. Maes is greatly appreciated.
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