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Plasma levels of interleukin-6 and tumor necrosis factor alpha in chronic schizophrenia: effects of clozapine treatment
Psychiatry Research 71 Ž1997. 11]17
Plasma levels of interleukin-6 and tumor necrosis factor alpha in chronic schizophrenia: effects of clozapine treatment Palmiero MonteleoneU , Michele Fabrazzo, Alfonso Tortorella, Mario Maj Institute of Psychiatry, Faculty of Medicine, Uni®ersity of Naples, Largo Madonna delle Grazie, 80138 Naples, Italy Received 15 May 1996; revised 23 December 1996; accepted 8 February 1997
Abstract Plasma levels of interleukin-6 ŽIL-6. and tumor necrosis factor alpha ŽTNFa . were assessed in 17 chronic schizophrenic patients who had been drug-free for 3 weeks and in 17 age- and sex-matched healthy subjects. Plasma concentrations of both cytokines were measured again in 12 patients after a 10-week treatment with clozapine. Compared with healthy controls, drug-free schizophrenic patients exhibited similar plasma IL-6 concentrations, but significantly higher levels of TNFa . After clozapine treatment, blood concentrations of TNFa fell to normal levels. These preliminary data support an immune activation in drug-free schizophrenic patients and an effect of clozapine on immune parameters. Q 1997 Elsevier Science Ireland Ltd. Keywords: Atypical neuroleptics; Cytokines; Immunity; Psychosis
1. Introduction Over the last 3 decades, a number of studies have suggested a dysregulation of immune system
U
Corresponding author. Tel.: q39 81 5666517; fax: q39 81 5666523.
function in patients with schizophrenia. Of all the immunological changes observed, abnormalities of cytokine physiology have been the most consistently reported. These findings include decreased in vitro production of interleukin-2 ŽIL-2. by stimulated peripheral blood mononuclear cells ŽVillemain et al., 1989; Ganguli et al., 1992, 1995. and increased levels of serum IL-2 receptors ŽsIL-
0165-1781r97r$17.00 Q 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S0165-1781Ž97.00036-X
12
P. Monteleone et al. r Psychiatry Research 71 (1997) 11]17
2Rs. ŽGanguli and Rabin, 1989; Rapaport et al., 1989., cerebrospinal fluid IL-2 ŽLicinio et al., 1993., and serum interleukin-6 ŽIL-6. and IL-6 receptors ŽsIL-6Rs. ŽShintani et al., 1991; Ganguli et al., 1994; Maes et al., 1994, 1995.. Cytokines, besides playing a key role in the peripheral immune system, also influence the activity of the central nervous system ŽRothwell and Hopkins, 1995.. Of particular interest for schizophrenia, animal studies have shown that IL-2 stimulates dopamine release from rat striatal and mesencephalic cells ŽLapchak, 1992; Alonso et al., 1993. while clinical studies have reported that cancer patients treated with high doses of recombinant IL-2 exhibited hallucinations and delusions that responded to haloperidol treatment ŽDenicoff et al., 1987.. These findings led Smith Ž1992. to formulate the macrophagerTlymphocyte hypothesis of schizophrenia, which suggests that activation of monocytesrmacrophages and T-cells is involved in the pathophysiology of the disorder. If the hypothesis was valid, antipsychotics would be expected to affect immune parameters. Indeed, typical neuroleptics have been shown to inhibit IL-2 production by activated lymphocytes in vitro ŽBoukhris et al., 1988; Schleuning et al., 1989., while subchronic treatment with haloperidol or phenothiazines has been found to decrease plasma IL-6 or sIL-6Rs in schizophrenic patients ŽMaes et al., 1995.. In addition, atypical neuroleptics, such as clozapine and risperidone, have been reported to increase sIL-2Rs ŽMaes et al., 1994, 1996; Pollmacher et ¨ al., 1995a,b.. Tumor necrosis factor alpha ŽTNFa . is a cytokine which, besides its immunological effects, promotes nerve cell growth and differentiation, and is cytotoxic to oligodendrocytes ŽMerrill, 1992.. As a consequence, TNFa may have both neurodevelopmental and neurodegenerative effects that may be relevant to the pathophysiology of schizophrenia ŽWeinberger and Lipska, 1995.. Pollmacher et al. Ž1995b. found that subchronic ¨ administration of clozapine increased plasma levels of TNFa in schizophrenic patients, but they did not provide any comparison data between drug-free patients and healthy controls. Therefore, in the present study, we measured plasma
levels of TNFa in drug-free schizophrenic patients and assessed the effects of chronic treatment with clozapine on this cytokine. Moreover, we also chose to explore plasma concentrations of IL-6 because of its direct link with other immune alterations that have been frequently reported in schizophrenia, such as increased number or percentages of T-lymphocytes and increased plasma levels of acute phase ŽAP. response proteins ŽDeLisi et al., 1982; Smidt et al., 1988.. 2. Methods Seventeen patients who met DSM-IV criteria for schizophrenia ŽAmerican Psychiatric Association, 1994. and 17 normal controls participated in the study. The presence of medical conditions known to interfere with immunity function was an exclusion criterion for both patients and controls. The patients comprised seven men and 10 women, with a mean age of 25.4 years ŽS.D.s 5.5.. The mean duration of their illness was 7.5 years ŽS.D.s 4.6.. The diagnosis of schizophrenia was ascertained by means of the Composite International Diagnostic Interview ŽCIDI. ŽWorld Health Organization, 1987.. Patients were classified as paranoid Ž n s 5., undifferentiated Ž n s 3., or disorganized Ž n s 9.; all were of the chronic or subchronic type; none met DSM-IV criteria for other Axis I psychiatric diagnoses or had previously suffered from post-psychotic depression. One patient was drug-naive; the remaining ones had been treated in the past with different typical neuroleptics, predominantly haloperidol Ž4]15 mgrday., chlorpromazine Ž300]500 mgrday., or clotiapine Ž80]120 mgrday., and they had also received different benzodiazepines Žalprazolam, 1]3 mgrday; lorazepam, 3]6 mgrday; bromazepam, 3]6 mgrday. and anticholinergic drugs Žbiperiden, 6]8 mgrday; orfenadrine, 100]150 mgrday.. None had received long-acting preparations for at least 3 months before the study. Drug-treated patients underwent a 3-week washout period. Psychopathological assessment was performed with the Comprehensive Psy˚ chopathological Rating Scale ŽCPRS. ŽAsberg et al., 1978., the Scale for the Assessment of Positive Symptoms ŽSAPS. ŽAndreasen, 1984., and the
P. Monteleone et al. r Psychiatry Research 71 (1997) 11]17
Scale for the Assessment of Negative Symptoms ŽSANS. ŽAndreasen, 1983.. Control subjects were individually matched to the patients on age Žmean " S.D.s 29.6" 7.8. and sex. They were mentally healthy as assessed by the CIDI and had no positive family history of mental disorders as assessed by the Family History Research Diagnostic Criteria ŽAndreasen et al., 1977.. Both patients and controls had normal physical examinations, normal values on routine blood and urine tests, and normal electrocardiograms. After complete description of the study to the subjects, written informed consent was obtained. Thirteen patients, who had responded insufficiently to at least three typicalneuroleptics in the past, completed a 10-week treatment with clozapine, at the end of which the daily dosage of clozapine was 175]400 mg Žmean " S.D.s 312 " 120 mg.. In these patients, the white blood cell count was monitored weekly. Of the remaining four schizophrenic patients, two underwent treatment with risperidone Ž6 mgrday. and two with haloperidol Ž6 mgrday.. Blood samples were collected between 08:00 and 09:00 h in all drug-free patients and matched controls for cytokine determination. Blood samples were also collected at the same time points in the clozapine-treated subgroup at the end of the 10-week treatment to measure cytokine concentrations as well as plasma levels of clozapine and its major metabolites. Plasma was separated, aliquoted, and stored at y808C until used. TNFa and IL-6 were determined by a sandwich enzyme-linked immunoassorbent assay ŽELISA. ŽBiotrak, Amersham, Buckinghamshire, England., based on a monoclonal]monoclonal antibody pair and a biotin]streptavidin amplification system. The sensitivities of the assays were 5 pgrml for TNFa and 1 pgrml for IL-6, respectively. Intraassay and inter-assay coefficients of variation were - 10% for both cytokines. TNFa and IL-6 assays for each patient Žbefore and after clozapine treatment . and hisrher matched control were run in the same lot. Plasma levels of clozapine, N-desmethylclozapine and clozapine-N-oxide were determined by reversed phase high pressure liq-
13
uid chromatography according to the procedure of Volpicelli et al. Ž1993.. After tests were performed for normality of distribution and for homoscedasticity, data were statistically evaluated by analysis of variance ŽANOVA., two-tailed Student’s t-test for paired data, and Pearson’s correlation test, where appropriate. 3. Results 3.1. Psychopathology and clozapine plasma le®els Mean Ž"S.D.. CPRS, SANS, and SAPS total scores of unmedicated schizophrenic patients were 37.4" 10.9, 35.2" 16.4, and 37.5" 21.5, respectively. Mean Ž"S.D.. CPRS, SANS, and SAPS total scores of the 13 patients who completed the 10-week treatment with clozapine were 39.6" 10.6, 33.9" 12.5, and 37.9" 22.2 before treatment compared with 16.0" 7.7, 20.6" 9.4, and 15.9" 9.7 after treatment. Therefore, after week 10 of clozapine therapy, there was a clear-cut improvement of psychopathology as shown by significant decreases in CPRS Ž t s 7.606, d.f.s 12, P s 0.0001., SANS Ž t s 5.769, d.f.s 12, P s 0.0001., and SAPS Ž t s 3.770, d.f.s 12, Ps 0.001. total scores. Mean Ž"S.D.. plasma levels of clozapine, Ndesmethylclozapine, and clozapine-N-oxide were 399 " 190 ngrml, 383 " 274 ngrml, and 102 " 56 ngrml, respectively. 3.2. Cytokine plasma le®els Plasma levels of IL-6 were normally distributed in both controls and patients; in the latter, both before and after clozapine treatment. The Levene test for homoscedasticity was not significant. Plasma levels of TNFa were not normally distributed in the drug-free patients Ž n s 17., because of the presence of two outliers, or in the subgroup who underwent chronic treatment with clozapine Ž n s 13., because of the presence of one outlier. After exclusion of these outliers and their matched controls, data were normally distributed. Therefore, the statistical analysis of
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P. Monteleone et al. r Psychiatry Research 71 (1997) 11]17
TNFa results refers to 15 drug-free patients and matched controls and to 12 schizophrenic patients before and after clozapine treatment. In these groups, the Levene test for homoscedasticity was not significant. In drug-free schizophrenic patients, the mean Ž"S.D.. plasma levels of IL-6Ž7.03" 4.3 pgrml; range s 1.6]16.9 pgrml. were not statistically different from those in normal controls Ž6.5" 3.6 pgrml; range s 1.9]15.1 pgrml., whereas the mean Ž"S.D.. plasma concentrations of TNFa were significantly higher Žpatients: 14.9 " 2.6, range s 11.1]21.9 pgrml; controls: 12.6 " 1.7, range s 9.2]14.8 pgrml; F s 7.89, d.f.s 1,28, Ps 0.008. ŽFig. 1.. A significant positive correlation was found between plasma IL-6 levels and illness duration Ž r s 0.54, Ps 0.01.. No correlations emerged between plasma levels of IL-6 or TNFa , on the one hand, and psychopathological and demographic indices or white blood cell counts, on the other. Plasma levels of IL-6 did not correlate with those of TNFa .
Mean Ž"S.D.. plasma IL-6 levels did not change significantly after clozapine treatment Ž6.0 " 4.2 pgrml, range s 1.7]15.7 pgrml., whereas plasma TNFa concentrations decreased to control levels ŽFig. 2.. In the 12 patients included in the analysis, mean Ž"S.D.. plasma levels of TNFa before and after clozapine treatment were 16.0" 4.2 pgrml Žrange s 11.1]25.7 pgrml. and 13.0" 2.8 pgrml Žrange s 8.9]17.5 pgrml., respectively. The difference was statistically significant Ž t s 2.45, d.f.s 11, Ps 0.03.. After clozapine treatment, no significant correlation was found between immunological parameters, on the one hand, and clinical psychopathological indices, plasma levels of clozapine, N-desmethylclozapine and clozapine-N-oxide, and white blood cell counts, on the other. 4. Discussion This study shows that drug-free schizophrenic patients, compared with matched healthy con-
Fig. 1. Plasma levels of TNFa and IL-6 in drug-free schizophrenic patients and in matched healthy subjects. Horizontal lines indicate mean values.
P. Monteleone et al. r Psychiatry Research 71 (1997) 11]17
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Fig. 2. Plasma levels of TNFa and IL-6 in schizophrenic patients when drug-free and after treatment with clozapine. Horizontal lines indicate mean values.
trols, exhibit similar plasma levels of IL-6, but increased concentrations of TNFa . In addition, chronic treatment with clozapine significantly reduces blood levels of TNFa , without affecting those of IL-6. Similar to Ganguli et al. Ž1994., we found that plasma concentrations of IL-6 in drug-free schizophrenic patients were positively correlated with the duration of the illness. Unlike Ganguli et al. Ž1994. and other research groups ŽShintani et al., 1991; Maes et al., 1994, 1995., however, we did not observe significantly increased levels of this cytokine in such subjects. Our results also contrast with the demonstration that schizophrenia is associated with an acute phase response ŽSmidt et al., 1988. where an increased production of IL-6 seems to have a pathogenetic role ŽMaes et al., 1993.. The majority of our patients had a chronic and treatment-resistant form of schizophrenia; hence, it is likely that the clinical diversity of the patient groups in the various studies may account for the discrepancies between
our data and those of other authors. Alternatively, it must be remembered that measurement of plasma cytokines, including IL-6, is often hampered by their short half-lives and their instability over time, so that the sensitivity of the assay methods and the different freezing times may be further factors contributing to the inconsistencies among the studies. Finally, we cannot exclude the possibility that the small size of our patient group may have led to false-negative results. To our knowledge, this is the first study exploring plasma levels of TNFa in drug-free schizophrenic patients compared with matched healthy controls. Our results showing an increase of blood levels of this cytokine in schizophrenia are in line with previous data supporting an activation of the macrophagermonocyte cell lineage in the disorder, such as the enhanced number of activated peripheral T-lymphocytes and the increased blood levels of their secretory products ŽMuller, 1995.. ¨ At present, the significance of the increased
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P. Monteleone et al. r Psychiatry Research 71 (1997) 11]17
production of TNFa in relation to the pathophysiology of schizophrenia is not clear. It has been shown that TNFa is produced not only by peripheral monocytes, but also by astrocytes and neurons ŽLieberman et al., 1989; Hunt et al., 1992., and specific receptors for this cytokine have been found in the brain ŽKinouchi et al., 1991.. Moreover, TNFa has been implicated in the regulation of certain cerebral functions, and it has been shown to affect neural growth, differentiation, and survival ŽRothwell and Hopkins, 1995.. Hence, a direct link between this cytokine and the pathophysiological process underlying the production of at least certain schizophrenic symptoms might be supposed. Furthermore, after 10 weeks of treatment with clozapine, patients showed a decrease of plasma TNFa concentrations. In contrast to our findings, Pollmacher et al. Ž1995b. reported an increase in ¨ plasma levels of TNFa in schizophrenic patients after 6 weeks of clozapine administration. The difference in the duration of the treatment periods Ž10 weeks in our study vs. 6 weeks in that of Polmacher et al.. may be the major determinant ¨ of this discrepancy, although methodological differences may also be involved. The clozapine-induced decrease of plasma TNFa levels found in the present study suggests that the immune activation of drug-free schizophrenic subjects is reduced by subchronic administration of the atypical neuroleptic. Consistent with these results, clozapine has been shown to increase serum IL2Rs in schizophrenic patients ŽMaes et al., 1994, 1996; Pollmacher et al., 1995b., which may repre¨ sent an immunosuppressant effect. Indeed, although the increase in sIL-2Rs has been associated with an immunoactivation in medical disorders such as T-cell leukemias, rheumatoid arthritis, and Sjogren syndrome ŽEaton et al., ¨ 1992., it has been suggested that sIL-2Rs may bind to their ligand, thereby competing for the binding of IL-2 to IL-2Rs on the responding cells ŽCaruso et al., 1993.. As a consequence, increased concentrations of sIL-2Rs may lead to an immunosuppressant effect. On the basis of these considerations, the present data suggest that cloza-pine may normalize an enhanced immune function in schizophrenia, although the relevance
of this action to its therapeutic effects remains to be determined. The mechanism by which clozapine decreases the levels of TNFa in the blood is not clear. Schleuning et al. Ž1989. showed that chlorpromazine, a typical neuroleptic, inhibits the production of mRNA for TNFa in human T-cells and thymocytes. Whether such a mechanism may also apply to the atypical neuroleptic clozapine remains to be elucidated. Since, in our study, pretreatment plasma samples were frozen 10 weeks longer than post-treatment samples, we cannot exclude the possibility that the higher baseline values of TNFa may be an artifact of these different storage times. However, in non-schizophrenic psychiatric patients, we did not find any difference in plasma TNFa levels in samples collected before and after 12 weeks of specific drug therapies and stored under the same conditions as those of the present study Žpersonal data.. Finally, due to the small size of our patient sample, the possibility of a type II statistical error cannot be excluded. As a consequence, the present results should be regarded as preliminary. Future studies involving a larger number of patients and, possibly, different patient groups, treated with both typical and atypical neuroleptics, are warranted. References Alonso, R., Chaudieu, I., Diorio, J., Krishnamurthy, A., Quirion, R., Boksa, B., 1993. Interleukin-2 modulates evoked release of w 3 Hxdopamine in rat cultured mesencephalic cells. J. Neurochem. 61, 1284]1290. American Psychiatric Association, 1994. DSM-IV: Diagnostic and Statistical Manual of Mental Disorders, 4th edn. American Psychiatric Press, Washington, DC. Andreasen, N.C., 1983. The Scale for the Assessment of Negative Symptoms ŽSANS.. University of Iowa, Iowa City, IA. Andreasen, N.C., 1984. The Scale for the Assessment of Positive Symptoms ŽSAPS.. University of Iowa, Iowa City, IA. Andreasen, N.C., Endicott, J., Spitzer, R.L., Winokur, G., 1977. The family history method using diagnostic criteria: reliability and validity. Arch. Gen. Psychiatry 34, 1229]1235. ˚ sberg, M., Montgomery, S.A., Perris, C., Schalling, D., SedA vall, G., 1978. A comprehensive psychopathological rating scale. Acta Psychiatr. Scand. 271 ŽSuppl.., 63]69. Boukhris, W., Kouassi, E., Revillard, J.P., 1988. Differential effect of mixed D1rD2 and selective D2 dopaminergic
P. Monteleone et al. r Psychiatry Research 71 (1997) 11]17 antagonists on mouse T and B lymphocyte proliferation and interleukin production in vitro. Immunopharm. Immunotoxicol. 10, 501]512. Caruso, C., Candore, G., Cigna, D., Colucci, A.T., Modica, M.A., 1993. Biological significance of soluble IL-2 receptor. Med. Inflamm. 2, 3]21. DeLisi, L.E., Goodman, S., Neckers, L.M., Wyatt, R.J., 1982. An analysis of lymphocyte subpopulations in schizophrenic patients. Biol. Psychiatry 17, 1003]1009. Denicoff, K.D., Rubinow, D.R., Papa, M.Z. et al., 1987. The neuropsychiatric effects of treatment with interleukin-2 and lymphokine activated killer cells. Ann. Intern. Med. 107, 293]300. Eaton, W.W., Hayard, C., Ram, R., 1992. Schizophrenia and rheumatoid arthritis: a review. Schizophr. Res. 6, 181]192. Ganguli, R., Brar, J.S., Chengappa, K.N.R. et al., 1995. Mitogen-stimulated interleukin-2 production in never-medicated, first-episode schizophrenic patients. Arch. Gen. Psychiatry 52, 668]672. Ganguli, R., Brar, J.S., Solomon, W., Chengappa, K.N.R., Rabin, B.S., 1992. Altered interleukin-2 production in schizophrenia. Psychiatry Res. 44, 113]123. Ganguli, R., Rabin, B.S., 1989. Increased serum interleukin-2 receptor concentration in schizophrenic and brain-damaged subjects. Arch. Gen. Psychiatry 46, 292. Ganguli, R., Yang, Z., Shurin, G. et al., 1994. Serum interleukin-6 concentration in schizophrenia: elevation associated with duration of the illness. Psychiatry Res. 51, 1]10. Hunt, J.S., Chen, H.L., Hu, X.L., Chen, T.T., Morrison, D.C., 1992. Tumor necrosis factor-alpha gene expression in the tissues of normal mice. Cytokine 4, 340]346. Kinouchi, K., Brown, G., Pasternak, G., Donner, D.B., 1991. Identification and characterization of receptors for tumor necrosis factor-alpha in the brain. Biochem. Biophys. Res. Comm. 181, 1532]1538. Lapchak, P.A., 1992. A role for interleukin-2 in the regulation of striatal dopaminergic function. Neuroreport 3, 165]168. Licinio, J., Seibyl, J.P., Altemus, M., Charney, D.S., Krystal, J.H., 1993. Elevated CSF levels of interleukin-2 in neuroleptic-free schizophrenic patients. Am. J. Psychiatry 150, 1408]1410. Lieberman, A.P., Pitha, P.M., Shin, H.S., Shin, M.L., 1989. Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or neurotropic viruses. Proc. Natl. Acad. Sci. USA 399, 608]612. Maes, M., Bosmans, E., Calabrese, J., Smith, R., Meltzer, H.Y., 1995. Interleukin-2 and interleukin-6 in schizophrenia and mania: effects of neuroleptics and mood stabilizers. Psychiatry Res. 29, 141]152. Maes, M., Bosmans, E., Ranjan, R. et al., 1996. Lower plasma CC16, a natural anti-inflammatory protein, and increased plasma interleukin-1 receptor antagonist in schizophrenia: effects of antipsychotic drugs. Schizophr. Res. 21, 39]50.
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Maes, M., Meltzer, H.Y., Bosmans, E., 1994. Immune-inflammatory markers in schizophrenia: comparison to normal controls and effects of clozapine. Acta Psychiatr. Scand. 89, 346]351. Maes, M., Scharpe, ´ S., Meltzer, H.Y. et al., 1993. Relationships between interleukin-6 activity, acute phase proteins and HPA-axis function in severe depression. Psychiatry Res. 49, 11]27. Merrill, J.E., 1992. Tumor necrosis factor alpha, interleukin 1 and related cytokines in brain development: normal and pathological. Dev. Neurosci. 14, 1]10. Muller, N., 1995. Psychoneuroimmunology. Implications for ¨ the drug treatment of psychiatric disorders. CNS Drugs 4, 125]140. Pollmacher, T., Hinze-Selch, D., Mullington, J., 1995a. Cy¨ tokines and antipsychotic drugs. 26th Congress of the ISPNE, Munich, September 11]15, Abstracts, 86. Pollmacher, T., Hinze-Selch, D., Mullington, J., Holsboer, F., ¨ 1995. Clozapine-induced increase in plasma levels of soluble interleukin-2 receptors. Arch. Gen. Psychiatry 52, 877]878. Rapaport, M.H., McAllister, C.G., Pickar, D., Nelson, D.L., Paul, S.M., 1989. Elevated levels of soluble interleukin 2 receptors in schizophrenia. Arch. Gen. Psychiatry 46, 291]292. Rothwell, N.J., Hopkins, S.J., 1995. Cytokines and the nervous system II: Actions and mechanisms of action. Trends Neurosci. 18, 130]135. Schleuning, M.J., Duggan, A., Reem, G.H., 1989. Inhibition by chlorpromazine of lymphokine-specific RNA expression in human thymocytes. Eur. J. Immunol. 19, 1491]1496. Shintani, F., Kanba, S., Maruo, N. et al., 1991. Serum interleukin-6 in schizophrenic patients. Life Sci. 49, 661]664.. Smidt, E., Axelsson, R., Steen, G., 1988. Treatment of chronic schizophrenia with glucocorticoids in combination with neuroleptic drugs: a pilot study. Curr. Ther. Res. 43, 842]850. Smith, R.S., 1992. A comprehensive macrophage-T-lymphocyte theory of schizophrenia. Med. Hypoth. 39, 248]257. Villemain, F., Chatenoud, L., Galinowski, A. et al., 1989. Aberrant T cell-mediated immunity in untreated schizophrenic patients: deficient interleukin-2 production. Am. J. Psychiatry 146, 609]616. Volpicelli, S.A., Centorrino, F., Puopolo, P.R. et al., 1993. Determination of clozapine, norclozapine and clozapineN-oxide in serum by liquid chromatography. Clin. Chem. 39, 1656]1659. Weinberger, D.R., Lipska, B.K., 1995. Cortical maldevelopment, antipsychotic drugs, and schizophrenia: a search for common ground. Schizophr. Res. 16, 87]110. World Health Organization, 1987. Composite International Diagnostic Interview. WHO, Geneva, Switzerland.
Plasma levels of interleukin-6 and tumor necrosis factor alpha in chronic schizophrenia: effects of clozapine treatment Palmiero MonteleoneU , Michele Fabrazzo, Alfonso Tortorella, Mario Maj Institute of Psychiatry, Faculty of Medicine, Uni®ersity of Naples, Largo Madonna delle Grazie, 80138 Naples, Italy Received 15 May 1996; revised 23 December 1996; accepted 8 February 1997
Abstract Plasma levels of interleukin-6 ŽIL-6. and tumor necrosis factor alpha ŽTNFa . were assessed in 17 chronic schizophrenic patients who had been drug-free for 3 weeks and in 17 age- and sex-matched healthy subjects. Plasma concentrations of both cytokines were measured again in 12 patients after a 10-week treatment with clozapine. Compared with healthy controls, drug-free schizophrenic patients exhibited similar plasma IL-6 concentrations, but significantly higher levels of TNFa . After clozapine treatment, blood concentrations of TNFa fell to normal levels. These preliminary data support an immune activation in drug-free schizophrenic patients and an effect of clozapine on immune parameters. Q 1997 Elsevier Science Ireland Ltd. Keywords: Atypical neuroleptics; Cytokines; Immunity; Psychosis
1. Introduction Over the last 3 decades, a number of studies have suggested a dysregulation of immune system
U
Corresponding author. Tel.: q39 81 5666517; fax: q39 81 5666523.
function in patients with schizophrenia. Of all the immunological changes observed, abnormalities of cytokine physiology have been the most consistently reported. These findings include decreased in vitro production of interleukin-2 ŽIL-2. by stimulated peripheral blood mononuclear cells ŽVillemain et al., 1989; Ganguli et al., 1992, 1995. and increased levels of serum IL-2 receptors ŽsIL-
0165-1781r97r$17.00 Q 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S0165-1781Ž97.00036-X
12
P. Monteleone et al. r Psychiatry Research 71 (1997) 11]17
2Rs. ŽGanguli and Rabin, 1989; Rapaport et al., 1989., cerebrospinal fluid IL-2 ŽLicinio et al., 1993., and serum interleukin-6 ŽIL-6. and IL-6 receptors ŽsIL-6Rs. ŽShintani et al., 1991; Ganguli et al., 1994; Maes et al., 1994, 1995.. Cytokines, besides playing a key role in the peripheral immune system, also influence the activity of the central nervous system ŽRothwell and Hopkins, 1995.. Of particular interest for schizophrenia, animal studies have shown that IL-2 stimulates dopamine release from rat striatal and mesencephalic cells ŽLapchak, 1992; Alonso et al., 1993. while clinical studies have reported that cancer patients treated with high doses of recombinant IL-2 exhibited hallucinations and delusions that responded to haloperidol treatment ŽDenicoff et al., 1987.. These findings led Smith Ž1992. to formulate the macrophagerTlymphocyte hypothesis of schizophrenia, which suggests that activation of monocytesrmacrophages and T-cells is involved in the pathophysiology of the disorder. If the hypothesis was valid, antipsychotics would be expected to affect immune parameters. Indeed, typical neuroleptics have been shown to inhibit IL-2 production by activated lymphocytes in vitro ŽBoukhris et al., 1988; Schleuning et al., 1989., while subchronic treatment with haloperidol or phenothiazines has been found to decrease plasma IL-6 or sIL-6Rs in schizophrenic patients ŽMaes et al., 1995.. In addition, atypical neuroleptics, such as clozapine and risperidone, have been reported to increase sIL-2Rs ŽMaes et al., 1994, 1996; Pollmacher et ¨ al., 1995a,b.. Tumor necrosis factor alpha ŽTNFa . is a cytokine which, besides its immunological effects, promotes nerve cell growth and differentiation, and is cytotoxic to oligodendrocytes ŽMerrill, 1992.. As a consequence, TNFa may have both neurodevelopmental and neurodegenerative effects that may be relevant to the pathophysiology of schizophrenia ŽWeinberger and Lipska, 1995.. Pollmacher et al. Ž1995b. found that subchronic ¨ administration of clozapine increased plasma levels of TNFa in schizophrenic patients, but they did not provide any comparison data between drug-free patients and healthy controls. Therefore, in the present study, we measured plasma
levels of TNFa in drug-free schizophrenic patients and assessed the effects of chronic treatment with clozapine on this cytokine. Moreover, we also chose to explore plasma concentrations of IL-6 because of its direct link with other immune alterations that have been frequently reported in schizophrenia, such as increased number or percentages of T-lymphocytes and increased plasma levels of acute phase ŽAP. response proteins ŽDeLisi et al., 1982; Smidt et al., 1988.. 2. Methods Seventeen patients who met DSM-IV criteria for schizophrenia ŽAmerican Psychiatric Association, 1994. and 17 normal controls participated in the study. The presence of medical conditions known to interfere with immunity function was an exclusion criterion for both patients and controls. The patients comprised seven men and 10 women, with a mean age of 25.4 years ŽS.D.s 5.5.. The mean duration of their illness was 7.5 years ŽS.D.s 4.6.. The diagnosis of schizophrenia was ascertained by means of the Composite International Diagnostic Interview ŽCIDI. ŽWorld Health Organization, 1987.. Patients were classified as paranoid Ž n s 5., undifferentiated Ž n s 3., or disorganized Ž n s 9.; all were of the chronic or subchronic type; none met DSM-IV criteria for other Axis I psychiatric diagnoses or had previously suffered from post-psychotic depression. One patient was drug-naive; the remaining ones had been treated in the past with different typical neuroleptics, predominantly haloperidol Ž4]15 mgrday., chlorpromazine Ž300]500 mgrday., or clotiapine Ž80]120 mgrday., and they had also received different benzodiazepines Žalprazolam, 1]3 mgrday; lorazepam, 3]6 mgrday; bromazepam, 3]6 mgrday. and anticholinergic drugs Žbiperiden, 6]8 mgrday; orfenadrine, 100]150 mgrday.. None had received long-acting preparations for at least 3 months before the study. Drug-treated patients underwent a 3-week washout period. Psychopathological assessment was performed with the Comprehensive Psy˚ chopathological Rating Scale ŽCPRS. ŽAsberg et al., 1978., the Scale for the Assessment of Positive Symptoms ŽSAPS. ŽAndreasen, 1984., and the
P. Monteleone et al. r Psychiatry Research 71 (1997) 11]17
Scale for the Assessment of Negative Symptoms ŽSANS. ŽAndreasen, 1983.. Control subjects were individually matched to the patients on age Žmean " S.D.s 29.6" 7.8. and sex. They were mentally healthy as assessed by the CIDI and had no positive family history of mental disorders as assessed by the Family History Research Diagnostic Criteria ŽAndreasen et al., 1977.. Both patients and controls had normal physical examinations, normal values on routine blood and urine tests, and normal electrocardiograms. After complete description of the study to the subjects, written informed consent was obtained. Thirteen patients, who had responded insufficiently to at least three typicalneuroleptics in the past, completed a 10-week treatment with clozapine, at the end of which the daily dosage of clozapine was 175]400 mg Žmean " S.D.s 312 " 120 mg.. In these patients, the white blood cell count was monitored weekly. Of the remaining four schizophrenic patients, two underwent treatment with risperidone Ž6 mgrday. and two with haloperidol Ž6 mgrday.. Blood samples were collected between 08:00 and 09:00 h in all drug-free patients and matched controls for cytokine determination. Blood samples were also collected at the same time points in the clozapine-treated subgroup at the end of the 10-week treatment to measure cytokine concentrations as well as plasma levels of clozapine and its major metabolites. Plasma was separated, aliquoted, and stored at y808C until used. TNFa and IL-6 were determined by a sandwich enzyme-linked immunoassorbent assay ŽELISA. ŽBiotrak, Amersham, Buckinghamshire, England., based on a monoclonal]monoclonal antibody pair and a biotin]streptavidin amplification system. The sensitivities of the assays were 5 pgrml for TNFa and 1 pgrml for IL-6, respectively. Intraassay and inter-assay coefficients of variation were - 10% for both cytokines. TNFa and IL-6 assays for each patient Žbefore and after clozapine treatment . and hisrher matched control were run in the same lot. Plasma levels of clozapine, N-desmethylclozapine and clozapine-N-oxide were determined by reversed phase high pressure liq-
13
uid chromatography according to the procedure of Volpicelli et al. Ž1993.. After tests were performed for normality of distribution and for homoscedasticity, data were statistically evaluated by analysis of variance ŽANOVA., two-tailed Student’s t-test for paired data, and Pearson’s correlation test, where appropriate. 3. Results 3.1. Psychopathology and clozapine plasma le®els Mean Ž"S.D.. CPRS, SANS, and SAPS total scores of unmedicated schizophrenic patients were 37.4" 10.9, 35.2" 16.4, and 37.5" 21.5, respectively. Mean Ž"S.D.. CPRS, SANS, and SAPS total scores of the 13 patients who completed the 10-week treatment with clozapine were 39.6" 10.6, 33.9" 12.5, and 37.9" 22.2 before treatment compared with 16.0" 7.7, 20.6" 9.4, and 15.9" 9.7 after treatment. Therefore, after week 10 of clozapine therapy, there was a clear-cut improvement of psychopathology as shown by significant decreases in CPRS Ž t s 7.606, d.f.s 12, P s 0.0001., SANS Ž t s 5.769, d.f.s 12, P s 0.0001., and SAPS Ž t s 3.770, d.f.s 12, Ps 0.001. total scores. Mean Ž"S.D.. plasma levels of clozapine, Ndesmethylclozapine, and clozapine-N-oxide were 399 " 190 ngrml, 383 " 274 ngrml, and 102 " 56 ngrml, respectively. 3.2. Cytokine plasma le®els Plasma levels of IL-6 were normally distributed in both controls and patients; in the latter, both before and after clozapine treatment. The Levene test for homoscedasticity was not significant. Plasma levels of TNFa were not normally distributed in the drug-free patients Ž n s 17., because of the presence of two outliers, or in the subgroup who underwent chronic treatment with clozapine Ž n s 13., because of the presence of one outlier. After exclusion of these outliers and their matched controls, data were normally distributed. Therefore, the statistical analysis of
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P. Monteleone et al. r Psychiatry Research 71 (1997) 11]17
TNFa results refers to 15 drug-free patients and matched controls and to 12 schizophrenic patients before and after clozapine treatment. In these groups, the Levene test for homoscedasticity was not significant. In drug-free schizophrenic patients, the mean Ž"S.D.. plasma levels of IL-6Ž7.03" 4.3 pgrml; range s 1.6]16.9 pgrml. were not statistically different from those in normal controls Ž6.5" 3.6 pgrml; range s 1.9]15.1 pgrml., whereas the mean Ž"S.D.. plasma concentrations of TNFa were significantly higher Žpatients: 14.9 " 2.6, range s 11.1]21.9 pgrml; controls: 12.6 " 1.7, range s 9.2]14.8 pgrml; F s 7.89, d.f.s 1,28, Ps 0.008. ŽFig. 1.. A significant positive correlation was found between plasma IL-6 levels and illness duration Ž r s 0.54, Ps 0.01.. No correlations emerged between plasma levels of IL-6 or TNFa , on the one hand, and psychopathological and demographic indices or white blood cell counts, on the other. Plasma levels of IL-6 did not correlate with those of TNFa .
Mean Ž"S.D.. plasma IL-6 levels did not change significantly after clozapine treatment Ž6.0 " 4.2 pgrml, range s 1.7]15.7 pgrml., whereas plasma TNFa concentrations decreased to control levels ŽFig. 2.. In the 12 patients included in the analysis, mean Ž"S.D.. plasma levels of TNFa before and after clozapine treatment were 16.0" 4.2 pgrml Žrange s 11.1]25.7 pgrml. and 13.0" 2.8 pgrml Žrange s 8.9]17.5 pgrml., respectively. The difference was statistically significant Ž t s 2.45, d.f.s 11, Ps 0.03.. After clozapine treatment, no significant correlation was found between immunological parameters, on the one hand, and clinical psychopathological indices, plasma levels of clozapine, N-desmethylclozapine and clozapine-N-oxide, and white blood cell counts, on the other. 4. Discussion This study shows that drug-free schizophrenic patients, compared with matched healthy con-
Fig. 1. Plasma levels of TNFa and IL-6 in drug-free schizophrenic patients and in matched healthy subjects. Horizontal lines indicate mean values.
P. Monteleone et al. r Psychiatry Research 71 (1997) 11]17
15
Fig. 2. Plasma levels of TNFa and IL-6 in schizophrenic patients when drug-free and after treatment with clozapine. Horizontal lines indicate mean values.
trols, exhibit similar plasma levels of IL-6, but increased concentrations of TNFa . In addition, chronic treatment with clozapine significantly reduces blood levels of TNFa , without affecting those of IL-6. Similar to Ganguli et al. Ž1994., we found that plasma concentrations of IL-6 in drug-free schizophrenic patients were positively correlated with the duration of the illness. Unlike Ganguli et al. Ž1994. and other research groups ŽShintani et al., 1991; Maes et al., 1994, 1995., however, we did not observe significantly increased levels of this cytokine in such subjects. Our results also contrast with the demonstration that schizophrenia is associated with an acute phase response ŽSmidt et al., 1988. where an increased production of IL-6 seems to have a pathogenetic role ŽMaes et al., 1993.. The majority of our patients had a chronic and treatment-resistant form of schizophrenia; hence, it is likely that the clinical diversity of the patient groups in the various studies may account for the discrepancies between
our data and those of other authors. Alternatively, it must be remembered that measurement of plasma cytokines, including IL-6, is often hampered by their short half-lives and their instability over time, so that the sensitivity of the assay methods and the different freezing times may be further factors contributing to the inconsistencies among the studies. Finally, we cannot exclude the possibility that the small size of our patient group may have led to false-negative results. To our knowledge, this is the first study exploring plasma levels of TNFa in drug-free schizophrenic patients compared with matched healthy controls. Our results showing an increase of blood levels of this cytokine in schizophrenia are in line with previous data supporting an activation of the macrophagermonocyte cell lineage in the disorder, such as the enhanced number of activated peripheral T-lymphocytes and the increased blood levels of their secretory products ŽMuller, 1995.. ¨ At present, the significance of the increased
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production of TNFa in relation to the pathophysiology of schizophrenia is not clear. It has been shown that TNFa is produced not only by peripheral monocytes, but also by astrocytes and neurons ŽLieberman et al., 1989; Hunt et al., 1992., and specific receptors for this cytokine have been found in the brain ŽKinouchi et al., 1991.. Moreover, TNFa has been implicated in the regulation of certain cerebral functions, and it has been shown to affect neural growth, differentiation, and survival ŽRothwell and Hopkins, 1995.. Hence, a direct link between this cytokine and the pathophysiological process underlying the production of at least certain schizophrenic symptoms might be supposed. Furthermore, after 10 weeks of treatment with clozapine, patients showed a decrease of plasma TNFa concentrations. In contrast to our findings, Pollmacher et al. Ž1995b. reported an increase in ¨ plasma levels of TNFa in schizophrenic patients after 6 weeks of clozapine administration. The difference in the duration of the treatment periods Ž10 weeks in our study vs. 6 weeks in that of Polmacher et al.. may be the major determinant ¨ of this discrepancy, although methodological differences may also be involved. The clozapine-induced decrease of plasma TNFa levels found in the present study suggests that the immune activation of drug-free schizophrenic subjects is reduced by subchronic administration of the atypical neuroleptic. Consistent with these results, clozapine has been shown to increase serum IL2Rs in schizophrenic patients ŽMaes et al., 1994, 1996; Pollmacher et al., 1995b., which may repre¨ sent an immunosuppressant effect. Indeed, although the increase in sIL-2Rs has been associated with an immunoactivation in medical disorders such as T-cell leukemias, rheumatoid arthritis, and Sjogren syndrome ŽEaton et al., ¨ 1992., it has been suggested that sIL-2Rs may bind to their ligand, thereby competing for the binding of IL-2 to IL-2Rs on the responding cells ŽCaruso et al., 1993.. As a consequence, increased concentrations of sIL-2Rs may lead to an immunosuppressant effect. On the basis of these considerations, the present data suggest that cloza-pine may normalize an enhanced immune function in schizophrenia, although the relevance
of this action to its therapeutic effects remains to be determined. The mechanism by which clozapine decreases the levels of TNFa in the blood is not clear. Schleuning et al. Ž1989. showed that chlorpromazine, a typical neuroleptic, inhibits the production of mRNA for TNFa in human T-cells and thymocytes. Whether such a mechanism may also apply to the atypical neuroleptic clozapine remains to be elucidated. Since, in our study, pretreatment plasma samples were frozen 10 weeks longer than post-treatment samples, we cannot exclude the possibility that the higher baseline values of TNFa may be an artifact of these different storage times. However, in non-schizophrenic psychiatric patients, we did not find any difference in plasma TNFa levels in samples collected before and after 12 weeks of specific drug therapies and stored under the same conditions as those of the present study Žpersonal data.. Finally, due to the small size of our patient sample, the possibility of a type II statistical error cannot be excluded. As a consequence, the present results should be regarded as preliminary. Future studies involving a larger number of patients and, possibly, different patient groups, treated with both typical and atypical neuroleptics, are warranted. References Alonso, R., Chaudieu, I., Diorio, J., Krishnamurthy, A., Quirion, R., Boksa, B., 1993. Interleukin-2 modulates evoked release of w 3 Hxdopamine in rat cultured mesencephalic cells. J. Neurochem. 61, 1284]1290. American Psychiatric Association, 1994. DSM-IV: Diagnostic and Statistical Manual of Mental Disorders, 4th edn. American Psychiatric Press, Washington, DC. Andreasen, N.C., 1983. The Scale for the Assessment of Negative Symptoms ŽSANS.. University of Iowa, Iowa City, IA. Andreasen, N.C., 1984. The Scale for the Assessment of Positive Symptoms ŽSAPS.. University of Iowa, Iowa City, IA. Andreasen, N.C., Endicott, J., Spitzer, R.L., Winokur, G., 1977. The family history method using diagnostic criteria: reliability and validity. Arch. Gen. Psychiatry 34, 1229]1235. ˚ sberg, M., Montgomery, S.A., Perris, C., Schalling, D., SedA vall, G., 1978. A comprehensive psychopathological rating scale. Acta Psychiatr. Scand. 271 ŽSuppl.., 63]69. Boukhris, W., Kouassi, E., Revillard, J.P., 1988. Differential effect of mixed D1rD2 and selective D2 dopaminergic
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Maes, M., Meltzer, H.Y., Bosmans, E., 1994. Immune-inflammatory markers in schizophrenia: comparison to normal controls and effects of clozapine. Acta Psychiatr. Scand. 89, 346]351. Maes, M., Scharpe, ´ S., Meltzer, H.Y. et al., 1993. Relationships between interleukin-6 activity, acute phase proteins and HPA-axis function in severe depression. Psychiatry Res. 49, 11]27. Merrill, J.E., 1992. Tumor necrosis factor alpha, interleukin 1 and related cytokines in brain development: normal and pathological. Dev. Neurosci. 14, 1]10. Muller, N., 1995. Psychoneuroimmunology. Implications for ¨ the drug treatment of psychiatric disorders. CNS Drugs 4, 125]140. Pollmacher, T., Hinze-Selch, D., Mullington, J., 1995a. Cy¨ tokines and antipsychotic drugs. 26th Congress of the ISPNE, Munich, September 11]15, Abstracts, 86. Pollmacher, T., Hinze-Selch, D., Mullington, J., Holsboer, F., ¨ 1995. Clozapine-induced increase in plasma levels of soluble interleukin-2 receptors. Arch. Gen. Psychiatry 52, 877]878. Rapaport, M.H., McAllister, C.G., Pickar, D., Nelson, D.L., Paul, S.M., 1989. Elevated levels of soluble interleukin 2 receptors in schizophrenia. Arch. Gen. Psychiatry 46, 291]292. Rothwell, N.J., Hopkins, S.J., 1995. Cytokines and the nervous system II: Actions and mechanisms of action. Trends Neurosci. 18, 130]135. Schleuning, M.J., Duggan, A., Reem, G.H., 1989. Inhibition by chlorpromazine of lymphokine-specific RNA expression in human thymocytes. Eur. J. Immunol. 19, 1491]1496. Shintani, F., Kanba, S., Maruo, N. et al., 1991. Serum interleukin-6 in schizophrenic patients. Life Sci. 49, 661]664.. Smidt, E., Axelsson, R., Steen, G., 1988. Treatment of chronic schizophrenia with glucocorticoids in combination with neuroleptic drugs: a pilot study. Curr. Ther. Res. 43, 842]850. Smith, R.S., 1992. A comprehensive macrophage-T-lymphocyte theory of schizophrenia. Med. Hypoth. 39, 248]257. Villemain, F., Chatenoud, L., Galinowski, A. et al., 1989. Aberrant T cell-mediated immunity in untreated schizophrenic patients: deficient interleukin-2 production. Am. J. Psychiatry 146, 609]616. Volpicelli, S.A., Centorrino, F., Puopolo, P.R. et al., 1993. Determination of clozapine, norclozapine and clozapineN-oxide in serum by liquid chromatography. Clin. Chem. 39, 1656]1659. Weinberger, D.R., Lipska, B.K., 1995. Cortical maldevelopment, antipsychotic drugs, and schizophrenia: a search for common ground. Schizophr. Res. 16, 87]110. World Health Organization, 1987. Composite International Diagnostic Interview. WHO, Geneva, Switzerland.