CSF IL-1 and IL-2 in medicated schizophrenic patients and normal volunteers

SCHIZOPHRENIA RESEARCH ELSEVIER

Schizophrenia Research 25 (1997) 123-129

CSF IL-l and IL-2 in medicated schizophrenic patients and normal volunteers Mark Hyman Rapaport a,b,*, Cathy G. McAllister ‘, David Pickar d, Lawrence Tamarkin e, Darrell G. Kirch f, Stephen M. Paul g a Department of Psychiatry, University of California, San Diego, CA, USA b Psychiatric Service, San Diego Veterans Affairs Medical Center, San Diego, CA, USA ’ Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA, USA d Experimental Therapeutics Branch, National Institute of Mental Health (NIMH). Bethesda, MD, USA e Cytoimmune Incorporated, USA ’ Medical College of Georgia, USA g Lilly Research institute, Indiana, USA

Received9 January 199.5;receivedin revisedform 6 December1996;accepted28 December1996

Abstract It is clear that cytokines exert a variety of modulatory actionson the central nervoussystem.As part of our work exploring the relationshipbetweenimmuneactivation and psychosis,we measuredcerebrospinalfluid (CSF) IL-la and IL-2 levels in 60 medicatedschizophrenicpatients and in 21 normal volunteers using a competitive enzyme immunoassay.The two groupsdid not differ significantly in their meancytokine levels: 1.01(0.149) rig/ml (patients) vs. 1.28 (0.150) rig/ml (controls) for IL-la and 0.970 (0.038) rig/ml (patients) vs. 1.25 (0.086)rig/ml (controls) for IL-2. There was a significantpositive correlation betweenCSF IL-la and IL-2 levelsfor all subjects(r=0.50, n=44, p=0.0001). Keywords:

Biological psychiatry; CSF; IL-l; IL-2; Immunology; Schizophrenia

1. Introduction Schizophrenia is a complex disorder currently phenomenologically described (American Psychiatric Association (APA, 1994)). This suggests that schizophrenia is a syndrome rather than one specific disease;therefore, research to elucidate the * Corresponding author. Psychopharmacology Research Program, Department of Psychiatry (0985), UCSD Schoolof Medicine, 8950 Villa La Jolla Drive Suite 2243, La Jolla, CA 92037-0985. Tel.: + 1619 6226160; fax: + 1 619 4501491. 0920-9964/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO920-9964(97)00008-X

pathophysiological mechanisms which cause schizophrenia has been hampered by the heterogeneity of patients. There have been some recent investigations which report reproducible biological

correlates in some schizophrenic patients (Bogerts et al., 1990; Casanova et al., 1990; Goldberg et al., 1990; Suddath et al., 1990; Gur et al., 1994). One of these findings is the presence of peripheral immune activation in some patients with schizophrenia (Ganguli et al., 1987, 1989; McAllister et al., 1989; Heath, et al., 1989; Villemain 1990). Our group consistently has found that

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schizophrenic patients have elevated serum-soluble interleukin-2 receptors (SIL-2Rs) (Rapaport et al., 1989, 1993, 1994; Rapaport and Lohr, 1994). The next logical step is to assesswhether similar measures of immune activation can be found in the central nervous system. In a small study of 28 medicated patients with schizophrenia, El-Mallakh et al. (1993) reported that CSF levels IL-la and IL-2 were not different from those of matched controls, while Licinio et al. (1993) reported elevated CSF levels of IL-2 in neuroleptic-free schizophrenic patients, but similar IL-la levels for schizophrenic patients and controls. In the most comprehensive study to date, McAllister et al. (1995) evaluated CSF levels of IL-le, IL-2, and IL- 1 b in 79, male, schizophrenic patients who were stabilized on haloperidol and then participated in a B-week medication-free trial. McAllister and colleagues reported that plasma levels of IL-la and IL-2 and CSF levels of IL-la and IL-2 were not correlated. They also determined that only four subjects had detectable levels of plasma IL-l B and only two subjects had detectable CSF levels of IL-B. McAllister and colleagues found no relationship between Brief Psychiatric Rating Scale (BPRS), Bunney-Hamburg Scale scores, measures of negative symptoms or measures of depression and plasma or CSF cytokine levels. However, they did find that medicated schizophrenia patients with higher baseline CSF IL-2 levels were more likely to relapse during the 6-week medication-free interval than medicated patients with lower baseline CSF IL-2 levels. (McAllister et al., 1995). In a recent small study, Barak et al. (1995), evaluated CSF and serum of 16 schizophrenic patients and 10 healthy controls. This group measured tumor necrosis factor-a (TNF-a) IL-lB, IL-2, IL-6, and SIL-2Rs. No significant differences were found in CSF or serum levels of TNF-ct, IL-2, or IL-6. IL-1B was significantly decreased in patient CSF and serum as compared to controls. SIL-2Rs were decreased in the CSF of patients but were markedly elevated in the serum of the schizophrenic patients as compared to normal controls (Barak et al., 1995). These findings suggest that there may be disregulation of CSF IL-2 in some patients with schizophrenia, and further investigation of CSF IL-2 and its modulators is warranted.

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IL-2 is of particular interest to researchers because it has been shown to modulate dopaminergic, opioid, and choline& systems within the CNS (Lapchak, 1992; Alonso et al., 1993; Awatsuji et al., 1993; Hanisch et al., 1993; Lapchak and Araujo, 1993; Zalcman et al., 1994). Studies have demonstrated that IL-2 can increase dopamine metabolism in the prefrontal cortex without affecting serotonin metabolism (Zalcman et al., 1994). IL-2 also inhibits acetylcholine in the hippocampus and in the frontal cortex (Awatsuji et al., 1993; Han&h et al., 1993). There is preliminary evidence from animal studies suggesting that chronic exogenous IL-2 may cause a loss of neurons in the hippocampus and may decrease mnestic functioning (Nemni et al., 1992). Denicoff et al. (1987) demonstrated that peripheral treatment with IL-2 and lymphokine-activated natural killer cells induced psychotic symptoms in some cancer patients and these symptoms were ameliorated by haloperidol (Haldol) treatment. Because these findings from preclinical and clinical research suggest that IL-2 may be an important modulator of CNS function, it is important to determine what factors regulate IL-2 production in the central nervous system. There are data from studies in the periphery and from animal work which suggest that IL-l is a potent positive modulator of IL-2 (Durum et al., 1986; Durum and Oppenheim, 1989). To our knowledge no one has investigated this relationship in the CSF of humans. Based on our findings of peripheral immune activation, we hypothesized that some patients with schizophrenia would have marked elevations of IL-la and IL-2. We further postulated that there would be a significant positive correlation between IL-l CL and IL-2 in the CSF.

2. Methods

Schizophrenic subjects in this study were medically healthy inpatients who were hospitalized in the two inpatient units of the National Institutes of Mental Health (NIMH) which are devoted to schizophrenia research: the Clinical Therapeutics Branch located at the Clinical Center in Bethesda, Maryland (n = 19), and the Neuropsychiatric

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Research Hospital located at St. Elizabeth’s Hospital in Washington, DC (n=41). These two groups of patients could not be differentiated clinically nor by CSF IL-la and IL-2 levels. All patients had complete physical and laboratory examinations and gave informed written consent. Subjects were diagnosed as meeting the Diagnostic and Statistical Manual of Mental Disorders (DSM-III-R) criteria for schizophrenia using either a modified Schedule of Affective Disorders and Schizophrenia (SADS) interview or the Structured Clinical Interview for DSM-III-R (SCID) (Spitzer and Endicott, 1975; Spitzer et al., 1988). The subjects were medicated with either fluphenazine (Prolixin), haloperidol, thioridazine (Mellaril), or molindone (Moban) for a minimum of 4 weeks. ( We found no differences in CSF IL- 1u and IL-2 in subjects taking different neuroleptics.) The mean age of the patients was 29+5; 34 were male and 26 were female. Fifty-six (93%) of the patients were Caucasian, two patients were African American (3.2%), and two patients were Asian American (3.2%). The 21 normal volunteers were medically and psychiatrically healthy research subjects who had responded to advertisements for normal subjects and gave informed written consent. They had complete medical histories, physical examinations, laboratory analyses, and SADS interviews. These volunteers had no significant physical findings and received no psychiatric diagnoses. The normal volunteers had a mean age of 27 f4 years; 11 were male and 10 were female. Nineteen (90%) of the subjects were Caucasian, one (5%) was African American, and one (5%) was oriental. Samples were stored at - 70°C without preservatives prior to assaying, and 90% of the these samples had not been thawed prior to analysis. The samples were thawed at 37°C in a water bath and were mixed at room temperature. IL-161 and IL-2 were measured blindly using commercially available, competitive enzyme immunoassays (Cytoimmune, Inc.; College Park, MD). Samples were split into duplicate fractions and run simultaneously. The assay relies on the competition of biotinylated cytokine with specific cytokines in standards or subject samples for a limited number of specific antibody-binding sites. Primary anticy-

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tokine antibody (100 ~1) is plated into each of 96 wells of the Elisa plate and incubated for 2 h at room temperature. The unbound antibody is removed by washing 5 times in buffer. Either standard or sample (50 ~1) is pipetted into the wells, and then 50 pl of conjugated IL-l% or IL-2 is added. (Concentrations for the standard curve range from 0.005 to 5 ng, and are prepared in a synthetic fluid which is designed to approximate normal cerebral spinal fluid (El-Mallakh et al., 1993; Licinio et al., 1993; McAllister et al., 1995).) The mixture of biotinylated cytokine and standard/sample is incubated with the antibody for 2 h and then washed 5 times with buffer. A 1:2000 dilution streptavidin-conjugated alkaline phosphatase is added and incubated with the sample at room temperature for 45 min. The streptavidin alkaline phosphatase is removed by 5 washes with buffer, and then 100 pl of substrate (p-nitrophenyl phosphate disodium; 2 mg/ml in 10 mM NaHO,, 12 mM Na,CO,, 1 mM MgCl,) is added to each well. The resultant color is read at 405 nm after 24 h. The data are analysed by computer-assisted logit-log transformation of the resultant optical densities (microtiter plate manager@ bio-RAD; Richmond, CA). The sensitivity limit of each assay is 0.1 rig/ml with intra- and inter-assay coefficients of variation of 7 and 15%, respectively (Paciotti et al., 1992). The data were tested for homogeneity of variance and normal distribution and the IL-2 data were transformed using a log transfoundation. The data were then analyzed using ANOVA, X2-test, f-tests, and Pearson’s correlations.

3. Results

The mean BPRS for the patients was 54.95+ 10.89 (SD). There were no clinically or statistically significant relationships between either mean BPRS scores or BPRS subscales scores and CSF IL-la or IL-2. The patients and the normal volunteers did not differ significantly in age, race, or sex. Fourteen of 21 controls (66%) and 32 of 60 (53%) of the schizophrenic patients had detectable CSF levels of IL-lu (x2: df= 1, p =0.23). Schizophrenic

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patients and normal volunteers also had similar IL-la levels (0.96f.64 vs. 1.31+.56ng/ml; f= 1.37, df=1,44, p=O.195). Twenty of 21 (95%) normal volunteers and 46 of 60 (77%) schizophrenic patients had detectable CSF levels of IL-2 (x2: df= 1 p=O.O7). The mean CSF IL-2 levels were not significantly different: 1.03 + 0.64 rig/ml for schizophrenic patients vs. 1.25 + 0.76 rig/ml for the normal volunteers (f=2.73, df = 1,65 p = 0.103) (Fig. 1). One control and 11 schizophrenic patients had undetectable levels of both IL-la and IL-2. When normal volunteers and patients were stratified on the basis of the presence of CSF IL-2, significant differences emerged. Normal volunteers who had detectable levels of both IL-la and IL-2 had mean CSF IL-2 levels of 1.60+0&l rig/ml vs. mean CSF IL-2 levels of 0.41 f0.19 rig/ml for CSF IL-la and IL-2 Levels for Schizophrenic Patients and Mormal Controts 3.5 -

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normal volunteers without detectable IL-la levels (t=6.38, df= 1,17, p
There was a significant, positive correlation between IL-la and IL-2 CSF levels (r=0.050, n = 44, p =0.0001) (Fig. 2). This correlation persisted when the patients and controls were evaluated separately: r =0.49, n = 30, p = 0.0006 for the schizophrenic patients and r-=0.52, n= 14, p= 0.059 for the normal volunteers.

4. Discussion

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Fig. 1. This figure presents a scattergram of CSF IL-ln and IL-2 levels for schizophrenic patients and normal volunteers. The sample means are indicated by the black bars.

We found that medicated schizophrenic patients and normal volunteers had similar mean CSF IL- 1CLlevels. This finding is consistent with previous studies which measure CSF IL-la levels in schizophrenic patients and normal volunteers. Our preliminary analysis also determined that normal volunteers and schizophrenic patients had similar CSF IL-2 levels. This agrees with reports by El-Mallakh et al. (1993) and Barak et al. (1995). Licinio et al. (1993) found that unmedicated schizophrenic patients had higher CSF IL-2 levels than their matched normal volunteers, but the total sample size was 18 in this analysis. They postulated that neuroleptic treatment might help to normalize CSF IL-2 levels. McAllister et al. (1995) reached different conclusions. They found that CSF IL-2 levels did not change significantly when patients were studied while stabilized on haloperidol and during a 6-week interval on placebo. They reported that medicated patients with higher CSF IL-2 levels were more likely to relapse during the placebo interval. One explanation which would account for both of these findings is that CSF IL-2 dysregulation occurs in a subset of

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The Correlation Between CSF IL-la and IL-2 Levels Relationship

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patients with schizophrenia, and that the studies by Licinio et al. (1993) and McAllister et al. (1995) had more patients with elevated CSF IL-2 levels. We found a significant positive correlation between CSF IL-la and IL-2 concentrations. The correlation held even when patients and normal volunteers were evaluated separately. This suggests that CSF levels of these two cytokines do seem to be related. Our secondary analyses where we compared and contrasted CSF IL-2 levels in patients and normal volunteers with and without detectable levels of IL-la, found that individuals with detectable levels of IL-la had significantly higher CSF levels of IL-2. These analyses support our belief that IL-la may be a positive modulator of IL-2 in the central nervous system. An additional post hoc finding from this analysis was that normal volunteers with detectable levels of both cytokines had higher CSF IL-2 levels than schizophrenic patients with detectable levels of both IL-la and IL-2. This finding needs confirmation and careful scrutiny in future studies because it appears to contradict Licinio et al. (1993). It is important to recognize methodological limitations that may affect the generalizability of these results. One limitation is our observation that some patients and controls had undetectable cytokine levels. Other groups which investigated CSF cytokines in normal volunteers, psychiatric patients, and nonpsychiatric patients have reported similar observations (Maimone et al., 1991; Licinio et al., 1993; Barak et al., 1995; McAllister et al., 1995). These observations might be accounted for by: (1) CSF cytokines being present but at levels beneath the sensitivity of the assays; (2) CSF cytokines being present at a regional site distal to the lumbar spine and not measurable in lumbar spinal fluid; (3) cytokines not always being present in human CSF and detectable only in response to specific stimuli. Further work is needed to explain this technical concern. Cytokine measurement might also be complicated by the presence of proteins in the periphery and the CSF which bind to them and may inhibit their detection by conventional assays.

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One approach to overcome this problem which has been employed in studies of the peripheral immune system is to study the production of cytokines by lymphocytes in culture (Ganguli et al., 1989; Villemain et al., 1989). However, this model is not useful for CSF studies. In conclusion, we did not find significant differences between schizophrenic patients and normal volunteers in measures of CSF IL-lcr and IL-2. However, we found significant positive correlations between CSF IL-la and IL-2 levels. In a secondary analysis we found that patients and normal volunteers with detectable levels of IL-la had significantly higher levels of IL-2 than patients and controls without detectable IL-la levels.

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