Psychiatry Research, 5 I : I- 10 Elsevier

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Serum Interleukin-6 Concentration in Schizophrenia: Elevation Associated With Duration of Illness

Rohan Ganguli, Zanwei Yang, Galina Shurin, K.N. Roy Chengappa, Jaspreet S. Brar, Ananda V. Gubbi, and Bruce S. Rabin

Received November 20. 1992; revised version received July 13, 1993; accepted September 20. 1993.

Abstract. Using an enzyme immunoassay (ELBA), we measured serum interleukin-6 (IL-6) concentration in 128 schizophrenic patients (24 of whom were never medicated) and in 110 normal control subjects. Mean serum IL-6 concen- tration was significantly higher in the schizophrenic patients as compared with the control subjects (p = 0.009). Comparisons within the patient group revealed that serum IL-6 was significantly correlated with duration of illness (r = 0.32, p = 0.0004). After covariation for duration of illness, there was no relationship between IL-6 levels and the production of autoantibodies, clinical state, or medi- cation status. Thus, elevated serum IL-6 levels in schizophrenia develop during the course of illness and may be related to treatment or to disease progression.

Key Words. Autoantibodies, psychoimmunology, cytokine.

Many investigators have found immunologic alterations in schizophrenic patients,

suggesting that there is an autoimmune component to the pathogenesis of the dis-

order. As has been repeatedly pointed out, however, there are many inconsistencies in this literature (Solomon, 1981; DeLisi and Wyatt, 1982). We have proposed that some of the discrepancies between studies can be explained by the probable hetero- geneity of the biological mechanisms that underlie the production of schizophrenic symptoms (Ganguli et al., 19896). We have further proposed that one patho- physiologic subgroup of schizophrenic patients might have an autoimmune component to their disorder (Ganguli et al., 1987, 19890, 1989b). In the majority of autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythema- tosus (SLE), and Sjogren’s syndrome, specific autoantibodies have not been found. Nevertheless, the autoimmune basis of these disorders has been established by the demonstration of a characteristic non-organ-specific immunopathology that can be logically linked to autoimmunity or has been empirically found in a wide range of autoimmune diseases (Rose, 1991). Among these nonspecific findings are the increased prevalence of other autoimmune diseases, circulating autoantibodies to a number of autoantigens, and changes in the function of lymphocytes, especially T

Rohan Canguli. M.D., Zanwei Yang, Ph.D., Galina Shurin, Ph.D., K.N. Roy Chengappa, M.D., Jaspreet, S. Brar, M.D., Ananda V. Gubbi, Ph.D., and Bruce S. Rabin, M.D., Ph.D., are in the Departments of Psychiatry and Pathology, University of Pittsburgh School of Medicine. (Reprint requests to Dr. R. Ganguli, Western Psychiatric Institute and Clinic, 381 1 O’Hara St., Pittsburgh, PA 15213-2593, USA.)

0165-1781/94/$07.00 @ 1994 Elsevier Science Ireland Ltd.

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lymphocytes. These functional changes include decreased mitogenic response to phytohemagglutinin (PHA) and decreased capacity for interleukin-2 (IL-2) produc- tion by lymphocytes following stimulation with PHA.

There is considerable epidemiological evidence of a negative association between rheumatoid arthritis and schizophrenia. It has been suggested that this negative association may be the result of mutually exclusive alleles for the same gene for different disorders (Knight, 1982). Thus, inheritance of the allele for rheumatoid arthritis may prevent an individual from simultaneously possessing the allele for schizophrenia. A negative association between insulin dependent diabetes mellitus and schizophrenia has also been reported (Finney, 1989).

IL-6 is involved in the production of autoantibodies by B cells in patients with SLE (Tanaka et al., 1988). Increased production of IL-6 and increased concentra- tions of IL-6 in synovial fluid have been found in patients with RA (Hirano et al., 1988; Houssiau et al., 1988; Firestein et al., 1990). Multiple sclerosis is associated with increased serum levels of IL-6 (Frei et al., 1991). Cardiac myxoma, a tumor which secretes IL-6, is associated with a variety of paraneoplastic autoimmune mani- festations (Jourdan et al., 1990). IL-6 has been shown to be overproduced in the brains of animals with experimental autoimmune encephalomyelitis (Gijbels et al., 1990), and the introduction of the IL-6 gene into hemopoietic stem cells of mice results in a variety of autoimmune disturbances (Brandt et al., 1990). Thus, there is growing evidence that clinical and experimental autoimmune disease is associated with disordered IL-6 production.

In a pilot study of 69 schizophrenic patients and 74 normal control subjects, we found that schizophrenic patients had a higher mean concentration of IL-6 in their serum (Ganguli et al., 1991). Recently Shintani et al. (1991) also reported finding elevated serum IL-6 concentrations in a study of 90 schizophrenic patients and 90 normal control subjects in Japan. However, both our earlier report and the study of Shintani et al. used an enzyme linked immunoassay (ELISA) for IL-6 that has a low sensitivity for IL-6 detection. Consequently, many subjects in both these earlier studies had IL-6 concentrations that were not detectable.

The current study was undertaken using an improved ELISA technique that is more sensitive than previously available assays. We have attempted to replicate our earlier findings of increased serum IL-6 concentration in schizophrenic patients. We have also examined the possible correlation between the concentration of IL-6 in the serum and the production of autoantibodies, since the latter may be a trait marker for individuals who are at increased risk for developing autoimmune diseases (Rose, 1991). Both acutely ill and remitted patients were studied to examine the influence of clinical state on IL-6. We were also able to include a substantial number of patients, who were in their first episode of illness and who had never been medicated, thus allowing us also to evaluate the effects, if any, of antipsychotic medication on serum IL-6 concentration.

Methods

Subjects. The subjects were all participants in an ongoing study of immunologic dysfunction in schizophrenia. The patients were recruited from the outpatient and inpatient services of the

schizophrenia module at Western Psychiatric Institute and Clinic (WPIC). As reported elsewhere, subjects entering our research protocol do not differ in their demographic characteristics from the other patients in the clinic, and the WPIC clinical population is comparable to that of other community mental health centers (Ganguli and Brar, 1992). Healthy control subjects from the Greater Pittsburgh Metropolitan area were recruited for this study.

Subjects were interviewed with a modification of the Schedule for Affective Disorders and Schizophrenia (SADS; Spitzer and Endicott, 1978). Only patients who met Research Diagnostic Criteria (RDC; Spitzer et al., 1978) for “definite schizophrenia” or “definite schizoaffective disorder, mainly schizophrenic” were included in the study. Never-medicated first episode patients were reassessed 6-12 months later and rediagnosed based on their course of illness as determined from chart review and discussions with their treating clinicians. Only patients, who by consensus met RDC for schizophrenia or schizoaffective disorder, mainly schizophrenic, were included. A detailed description of our sample of never-medicated first- episode patients has recently been published (Ganguli and Brar, 1992).

Subjects, both patients and controls, were excluded if they met DSMIII-R criteria (American Psychiatric Association, 1987) for current substance abuse or addiction. Subjects who were receiving immunosuppressive drugs or who had a history of infection, currently or within the past week, were also excluded from the following analyses. Only control subjects without a current or past history of psychiatric or autoimmune disorder were included.

Blood Drawing. Blood for the assay was always drawn after an overnight fast between 6:30 and 8:30 a.m. in a glass vacutainer tube without anticoagulant. Before the blood draw, patients were interviewed for symptoms of intercurrent infections and excluded if any such evidence was found. The blood was allowed to clot, at room temperature, and the serum was separated by centrifugation, within 3 hours of collection, aliquotted, and stored at -70 “C until assayed.

Assays for Autoantibodies. The sera used for this study had previously been assayed for seven common autoantibodies, using standard serological methods. Tests were conducted for the following antibodies: thyroglobulin and thyroid microsomal antigen by indirect hem- agglutination; immunofluorescence for antinuclear antibody (HEp-2 cell line); smooth muscle, mitochondria and gastric parietal cell (mouse stomach and kidney); and rheumatoid factor by fluorescence immunoassay. Subjects were considered positive if the titer of any one or more of the autoantibodies exceeded the threshold established by the clinical serology labora- tory of this university hospital. A positive titer for thyroglobin is 100, 400 for thyroid microsomal, 40 for antinuclear, smooth muscle, mitochondria, and parietal cell antibodies, and > 25 IV/ ml for rheumatoid factor. The interassay variability for all these assays is under 15%.

Measurement of IL-6 Concentration in Serum. IL-6 concentration was measured with a commercially available kit (Quantikine, R & D Systems, Minneapolis, MN) which uses a quantitative “sandwich” enzyme immunoassay technique. Ninety-six well polystyrene microtiter plates were precoated by the manufacturer with murine monoclonal antibody against IL-6. For each plate, a standard curve was generated using recombinant IL-6 in a dilution series of 300 pg/ml, 100 pgiml, 50 pg/ml, 25 pg/ml, 6.25 pg/ ml, 3.13 pg/ml, and 0 pg,‘ml. All samples were run in duplicate. All the assays reported here were carried out within a 2-week period using kits from the same batch, and lOO,ol of standard or sample was added to each well. The plate was then covered and incubated for 2 hours at room tempera- ture. Each well was then aspirated and washed three times with wash buffer supplied by the manufacturer. After removal of all the buffer from the last washing, 200 ~1 of polyclonal anti-IL-6 antibody conjugated to horseradish peroxidase was added to each well. After 20 minutes of incubation, 50 ~1 of 2 N sulfuric acid was added to stop the reaction. The optical density of the color reaction in the wells was read within 30 minutes using a Titertek Multiscan (Model MCC 340, Flow Laboratory, Switzerland) microtiter plate reader set for 450 nm. Wave-length correction was used to account for possible optical imperfections in the plate by subtracting the reading at 540 nm from the reading at 4.50 nm obtained in the previous step.

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Duplicate readings were averaged and the average reading from the zero standard was subtracted from this to give the final reading for each sample or standard. The optical densities of the standards were plotted to generate a standard curve which was linear in every case. Fig. 1 shows a representative standard curve, from one of the actual assays reported here. The concentration of IL-6 in the sample was then calculated from this standard curve.

Chlorpromazine (CPZ) equivalents of neuroleptic medications were determined using established guidelines (Davis et al., 1983).

Fig. 1. Standard curve of serum interleukin-6 (IL-6) concentration and optical density

Serum IL-6 Concentration (pg/ml)

IL-6 standards provided by Quantikine, R & D System, Minneapohs. MN.

Results

Serum IL-6 concentration was below the sensitivity of the assay (0.35 pg/ml) in 30 patients (19%) and 33 control subjects (23%), proportions that are not significantly different. The distribution of measurable IL-6 concentrations in both patients and control subjects was markedly elevated toward the higher end of the curve (positive skewness). A transformation of raw serum IL-6 values using the natural logarithm of the variable resulted in a normal distribution in both patients and control subjects (Fig. 2). Consequently, we used parametric statistical tests, namely t tests, analysis of covariance (ANCOVA), and Pearson’s product-moment correlations.

The resulting sample comprised 128 schizophrenic patients and 110 healthy control subjects. Table I presents the demographic characteristics of the sample.

Demographic characteristics have been known to alter immune function. We have previously reported the effects of ethnicity and gender on certain humoral and cellular measures of the immune system in healthy control subjects (Ganguli and Rabin, 1989; Chengappa et al., 1992). A relationship between age and IL-6 has also been reported (Wei et al., 1992). Accordingly, we first ascertained whether the serum IL-6 concentration was influenced by ethnicity and gender in both patients and

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Fig. 2. Tukey’s box plots of natural logarithmic transformation of serum interleukin-6 (IL-6) concentration (pg/ml)

Mean 0

1 O 0

CONTROLS PATIENTS

In = natural logarithm.. Schlzophrenlc patients have sigmficantly higher serum IL-6 concentrations than do healthy control subjects (t = 2.6. p = 0.009).

Table 1. Demographic characteristics of study sample

Caucasian African-American Age

Patients

(n = 128)

Male

41

Female Male

29 30

Female (mean i SD)

28 34.5 f 10.0

Controls

(n = 110) 45 31 16 18 31.7 f 10.5

control subjects. Within patients, male subjects had higher serum IL-6 concen- trations than female subjects (t = 1.99. p = 0.05). Also African-American patients had higher serum IL-6 concentration levels than Caucasian patients (t = 2.44. p = 0.02) (Table 2). A significant correlation between serum IL-6 concentration and age (at the time of blood draw) was observed only in the patient group (r = 0.26, p = 0.003; in control subjects, r = 0.08, p = NS). To determine why such an association might be present only in the patient group, we examined other factors that might be associated with age. There was also a significant correlation between serum IL-6 concentration and duration of illness in this group (r = 0.32, p = 0.0004)

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Table 2. Effect of gender and ethnicity on In serum interleukin-6 (IL-6) concentration in schizophrenic patients and control subjects (mean f SD)

In Serum IL-6 concentration t D

Controls (n = 110) Male (n = 61) -0.95 + 1 .13 Female (n = 49) -1.17 f 1.07 1.04 NS

Caucasian (n = 76) -0.81 k 1.09 African-American (n = 34) -1.15 k 1.10 1.50 NS

Patients (n = 128) Male (n = 71) -0.52 + 0.95 Female (n = 57) -0.88 + 1 .l 0 1.99 0.05

Caucasian (n = 70) -0.44 + 0.95 African-American In = 58) -0.88 * 1.05 2.44 0.02

Note. All comparisons are based on independent goup t tests. In = natural logarithm

(Fig. 3). Duration of illness and current age showed marked colinearity (r = 0.85, p < 0.0000). Since duration of illness was the more significant of the two, it was used as a covariate for subsequent analysis (Table 3).

As shown in Fig. 2, patients had higher mean IL-6 concentrations than control subjects (independent group t tests: t = 2.60, p = 0.009). The effects of clinical state and autoantibody production were also examined in subgroups of patients. While there were trends for autoantibody-positive patients to have higher serum IL-6 concentrations than autoantibody-negative patients, and for remitted patients to have higher concentrations than patients experiencing an acute episode, these

Fig. 3. Relationship between duration of illness (years) and In serum IL-6 concentration (pg/ml) in schizophrenic patients

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-I 3 2 k 40 0 0 0 00

J e+ e-3 e-2 e-’ e” e’ e*

In Serum IL-6 Concentration (pg/ml)

IL-6 = mterleukln-6. In = natural logarithm. Pearson’s product-moment correlation: r = 0.32, p = 0.0004

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Table 3. Correlation matrix for In serum interleukin-6 (IL-6) concentration, age, duration of illness, and current neuroleptic dosage

In serum IL-6 concentration CPZ eauivalent Duration of illness

CPZ equivalent 0.09 (n = 104)

Duration of illness 0.32’ (n = 128) 0.21 (n = 104)

Age 0.06s (n = 128) 0.14 (n = 104) 0.8F (n = 128)

Nore. Pearson’s product-moment correlation coefficrent. Correlations with chlorpromazine (CPZ)-equivalent neuroleptic dosage. In = natural logarithm.

1. p = 0.0004. 2. p = 0.003. 3. p < 0.0000.

differences were not statistically significant after corrections for duration of illness were made (Table 4).

The effects of neuroleptic medication on serum IL-6 concentration were also examined in subgroups of schizophrenic patients. Serum IL-6 concentrations in drug-naive first episode patients did not differ from those in the previously medicated patients (Table 5). Furthermore there were no correlations between serum IL-6 concentration and current medication dosage (CPZ equivalent) (Table 3). The higher trends for serum IL-6 concentration in remitted patients as compared with those in an acute episode were not related to CPZ-equivalent neuroleptic dosage

Table 4. In serum interleukin-6 (IL-6) concentration in subgroups of schizophrenic patients and healthy control subjects

Status Mean f SD Test statistic P

Healthy controls (n = 110) -1.05 t 1 .l 1 (t test)

Schizophrenic patients (n = 128) -0.68 f 1.03 2.6 0.009

Autoantibody Sve (n = 36) -0.38 k 0.88 (ANCOVA, F)

Autoantibody -ve (n = 92) -0.79 + 1.07 2.24 NS

In acute episode (n = 35) -0.88 + 0.93 (ANCOVA, F)

In remission (n = 69) -0.45 f 1.03 1.36 NS

Note. Comparisons are based on analysis of covariance (ANCOVA) wtth duration of illness as a covariate.

Table 5. Effects of neuroleptic medication on In serum interleukin-6 (IL-6) concentration

Groups Dependent variable MeanfSD F rp

Neuroleptic-naive (n = 24) In serum IL-6 concentration -1.04 f 1.07

Previously medicated (n = 104) -0.60 + 1.02 0.002’ NS

In acute episode (n = 35) CPZ-equivalent dosage 244.01 k 462.21

In remission (n = 69) 180.38 + 249.78 0.69’ NS

Note. CPZ-equtvalent dosage = current dosage of neuroleptic medrcation (chlorpromazrne equivalent). In = natural logarithm.

1. Comparisons are based on analysis of covariance with duratron of rllness as a covariate

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(Table 5). There were no significant differences between the mean CPZ-equivalent neuroleptic dosage in these subgroups.

Discussion

There are genetic, environmental, and functional immunologic factors involved in the induction of autoimmunity (Sinha et al., 1990). Cytokines play an important role in the initiation, maintenance, modulation, and localization of normal and abnormal immune responses. Decreased in vitro levels of IL-2 produced by PHA-stimulated lymphocytes were the earliest lymphokine abnormalities to be reported in several autoimmune diseases. Similar abnormalities have also been reported in schizophrenic patients (Kolyaskina et al., 1988; Sirota et al., 1990; Ganguli et al., 1989~; Villemain et al., 1989). On the other hand, changes in baseline levels of serum IL-2 have not been commonly found in association with autoimmune disease. Gattaz et al. (1992) measured serum IL-2 levels in schizophrenic patients and found no differences as compared with levels in normal control subjects.

In this study, with the largest sample of schizophrenic patients to have serum IL-6 measured, we found that schizophrenic patients had higher serum IL-6 than control subjects. Within schizophrenic patients, however, this increase could be fully

explained by duration of illness. Once the effect of duration was covaried out, no association was found with clinical state, neuroleptic dosage, or the production of autoantibodies. Thus, the increase in serum IL-6 in schizophrenia does not appear to be present at the onset of the disorder, but rather to develop during its course. While our data indicate that the elevation in serum IL-6 is not related to current neuroleptic dosage, an effect related to duration of neuroleptic treatment cannot be ruled out. Other factors related to disease progression might also have to be considered to account for the increase in serum IL-6 in schizophrenia.

IL-6 is found in many tissues besides those of the immune system. In the brain pituitary cells, neurons and astrocytes are all affected by IL-6 (Frei et al., 1989). IL-6 has been shown to be produced by microglia and astrocytes following central nervous system (CNS) viral infections (Frei et al, 1989), and IL-6 messenger ribonucleic acid (mRNA) transcripts are induced in glial cells following IL-1 stimulation. Since abnormalities of neural development have been postulated to underlie the pathogenesis of schizophrenia (Hyde and Weinberger, 1990) it is also of interest that IL-6 has been shown to support the growth of a number of neuronal cell lines in synergy with nerve growth factor (Hama et al., 1991). In the latter experiment, IL-6 was shown to improve survival and increase the dopamine content of tyrosine hydroxylase-positive mesencephalic neurons. Thus, IL-6 is capable of playing a role in both the development and functioning of neurons and other cells of the CNS. Whether IL-6 changes in schizophrenia can be linked to the role of this lymphokine in CNS function and growth remains to be investigated. For example, could the development of elevated serum IL-6 occur in response to the development of cerebral atrophy, which has been reported to be related to duration of illness (Waddington, 1993)?

Thus, IL-6 is a cytokine that merits further study in schizophrenic patients. Since we found a correlation with duration of illness in this cross-sectional study,

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longitudinal studies, preferably with neuroleptic-naive patients, are needed to track

fluctuations in the concentration of this cytokine as the illness progresses. Since IL-6

secretion is part of an acute phase response, it may also be worthwhile to study other

acute phase proteins such as transferrin, haptoglobin, and heat-shock proteins (hsp).

It may be of relevance that some schizophrenic patients are reported to have

circulating autoantibodies against a 60 kilodalton hsp in their serum (Kilidireas et

al., 1992).

Acknowledgments. The research reported here was supported in part by a National Institute of Mental Health grant (MH-41883) and a Research Scientist Development award to R.G. (MH-00710).

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