Psychiatry Research. 44:113-I 23 Elsevier

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Altered Interleukin-2 Production in Schizophrenia: Association Between Clinical State and Autoantibody Production

Rohan Ganguli, Jaspreet S. Brar, Wendy Solomon, K.N. Roy Chengappa, and Bruce S. Rabin

Received April 9, 1992; revised version received August 3. 1992; accepted November 7. 1992.

Abstract. Mitogen-stimulated interleukin-2 (IL-2) production was measured in 122 patients who met Research Diagnostic Criteria for schizophrenia and 98 normal control subjects. The presence of autoantibodies against seven common antigens was also determined. There was no relationship between the presence of circulating autoantibodies and IL-2 production in control subjects. In patients, however, autoantibody-positive, acutely ill patients had significantly lower IL-2 production as compared with other patients and control subjects. Never- medicated patients showed the same trends for decreased IL-2 production in association with autoantibodies. These data suggest that decreased IL-2 production is associated with acute illness in schizophrenic patients who produce autoantibodies, a trait known to be associated with increased vulnerability to autoimmune disease.

Key Words. Interleukin-2, immunology, lymphokine, acute psychosis, neuroleptic- naive, autoimmunity, cytokine.

The etiology and pathophysiology of schizophrenia remain unknown. It has been postulated that an autoimmune process may play a role in the pathogenesis of symptoms in some patients (Heath, 1954; Knight, 1982; Ganguii et al., 1987). Early studies of this hypothesis, which were predicated on finding specific antibodies directed against the brain, produced inconsistent results and much skepticism about the role of autoimmunity in schizophrenia. Early studies of other autoimmune dis- eases were also initially based on searching for organ-specific autoantibodies. Such specific antibodies appear to cause disease in some disorders such as myasthenia gravis, hemolytic anemia, pemphigoid disease, and Graves’ disease. In the vast

majority of autoimmune diseases, however, including rheumatoid arthritis, systemic

Rohan Ganguli, M.D., is Associate Professor of Psychiatry and Pathology, Department of Psychiatry, and Medical and Research Director, Schizophrenia Treatment and Research Center (STRC), Western Psychiatric Institute and Clinic (WPIC), University of Pittsburgh School of Medicine. Jaspreet S. Brar, M.D., is Senior Clinician, Department of Psychiatry, STRC, WPIC, University of Pittsburgh School of Medicine. Wendy Solomon, M.P.M., is Senior Research Associate, STRC, WPIC, University of Pittsburgh School of Medicine. K.N. Roy Chengappa, M.D., is Assistant Professor of Psychiatry, Department of Psychiatry, STRC, WPIC, University of Pittsburgh School of Medicine. Bruce S. Rabin, M.D., Ph.D., is Professor of Pathology and Psychiatry, Department of Pathology, and Director of Immunopathology, University of Pittsburgh School of Medicine. (Reprint requests to Dr. R. Gang&, Western Psychiatric Institute and Clinic, 3811 O’Hara St., Pittsburgh, PA 15213-2593, USA.)

0165-1781/92/SO5.00 @ 1992 Elsevier Scientific Publishers Ireland Ltd

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lupus erythematosus, and Sjogren’s syndrome, no specific or pathogenic antibody has been identified. Even when antibodies are associated with an autoimmune disease, they are frequently a result of the disease process rather than the cause. Despite the lack of evidence of organ-specific antibodies, there is a well-defined immunopathology that characterizes these latter disorders.

The presence of autoantibodies in an individual indicates an increased risk for the development of autoimmune disease (Rose, 1991). Several studies have reported finding an increased prevalence of autoantibodies in schizophrenic patients (Delisi and Wyatt, 1982; Ganguli et al., 1987; Chengappa et al., 1991) and their relatives (Sirota et al., 1991). On the basis of this evidence, we have postulated that the presence of autoantibodies might serve to identify schizophrenic patients who are more likely to have an autoimmune component to their illness (Ganguli et al., 1987).

Interleukin-2 (IL-2) is the best characterized lymphokine and plays a pivotal role in initiating and regulating the immune response (Smith, 1988). Decreased in vitro IL-2 production, following stimulation with mitogens such as phytohemagglutinin (PHA) and concanavalin A has been consistently reported in a variety of auto- immune diseases in both human and animal studies (Kroemer et al., 1991). Diseases associated with lowered IL-2 production include systemic lupus erythematosus (Alcocer-Varela and Alarcon-Segovia, 1982), insulin-dependent diabetes mellitus (Zier et al., 1984), rheumatoid arthritis (Cathely et al., 1986; Kitas et al., 1988), Sjogren’s syndrome (Miyasaka et al., 1984; Cathely et al., 1986; Kitas et al., 1988), Graves’ disease (Eisenstein et al., 1988), rheumatic fever (Alarcon-Riquelme et al., 1990), and active multiple sclerosis (Selmaj et al., 1988). The degree in reduction in IL-2 production is reported to correlate with disease activity in rheumatoid arthritis and multiple sclerosis. It is thus suspected that low IL-2 production is an important feature in the pathophysiology of autoimmunity. In twins who are discordant for insulin-dependent diabetes mellitus, it has been shown that only the twin with the disorder shows a reduction in IL-2 production. In mice that spontaneously develop autoimmune disease, the capacity to secrete IL-2 decreases before the disease manifests itself. Thus, decreased IL-2 production is regarded as a key feature in the pathophysiology of autoimmune disease.

In a study of 70 schizophrenic patients and 51 control subjects, we previously reported that acutely ill schizophrenic patients had significantly lower IL-2 production than either control subjects or remitted patients (Ganguli et al., 1989~). Villemain et al. (1989) also reported finding lowered IL-2 production in a sample of 16 never-medicated, acutely ill patients with schizophrenia. There have been at least two further studies that report finding lowered IL-2 production in schizophrenia (Kolyaskina et al., 1988; Sirota et al., 1990). In all these studies, however, not all patients even among the acutely ill have had lowered IL-2 production. We therefore undertook this study to see whether autoantibody production might serve as a means of identifying a subset of patients with immunologic abnormalities.

Methods

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

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and inpatient services of the schizophrenia module at Western Psychiatric Institute and Clinic (WPIC). WPIC provides all the psychiatric services for an urban catchment area within the city of Pittsburgh. We have previously shown that subjects entering our research protocol do not differ in their demographic characteristics from the other patients in the clinic and that the WPIC clinical population is comparable to that of other Community Mental Health Centers (Ganguli and Brar, 1992). Controls were specifically recruited for this study and came from the Greater Pittsburgh Metropolitan area.

Subjects were interviewed with a modification of the Schedule for Affective Disorders and Schizophrenia (Endicott and Spitzer, 1978). The severity of symptomatology was assessed with the Brief Psychiatric Rating Scale (BPRS; Overall and Gorham, 1962). The results of the diagnostic interview and information from all other available sources were presented at a diagnostic conference attended by at least one experienced research psychiatrist and a consensus final diagnosis was determined. At the diagnostic conference, the clinical state of the patients was also discussed and they were assigned to “acutely ill” or “remitted” groups on the basis of their psychotic symptoms at the time of blood sampling. Patients met Research Diagnostic Criteria (RDC; Spitzer et al., 1978) for “definite schizophrenia” or “definite schizoaffective disorder, mainly schizophrenic.” Patients who were recruited during a first episode were reassessed 6-12 months later and their course of illness examined from the chart and discussions with the treating clinicians. Only patients who by consensus met RDC for schizophrenia or schizoaffective disorder, mainly schizophrenic, qualified for study. Only control subjects without a current or past history of psychiatric or autoimmune disorder were included.

Subjects were excluded if they met criteria for current substance abuse or addiction. Subjects who were on immunosuppressant drugs or who had history of infection, currently or within the past week, were also excluded from the following analyses.

Blood Drawing. Blood for the assays was always drawn after an overnight fast between 6:30 and 8:00 a.m. Before the blood drawing, patients were interviewed for symptoms of intercurrent infections and excluded if any such evidence was found. For serologic studies, the blood was allowed to clot and the serum separated by centrifugation and stored at -70 ‘C until assayed. For IL-2 generation, blood was collected in heparinized tubes and incubated with PHA within 2 hours of being drawn.

Assays for Autoantibodies. Seven common autoantibodies were tested using standard serological methods: thyroglobulin and thyroid microsomal antigen by indirect hemagglutin- ation; immunofluorescence for antinuclear antibody (HEp-2 cell line), smooth muscle, mi- tochondria, and parietal cells (mouse stomach and kidney); and rheumatoid factor by fluorescence immunoassay. All these assays were conducted in the serology section of the Division of Immunopathology of the University of Pittsburgh Medical Center. The results of each test were reviewed by one of the authors (B.S.R.), an experienced immunopathologist. Positive titers in this laboratory are: 1:lOO for thyroglobulin, 1400 for thyroid microsmes, 1:40 for antinuclear, smooth muscle, mitochondrial, and parietal cell antibodies, and > 25 IV/ ml for rheumatoid factor. Subjects were considered autoantibody positive if they had at least one titer above that used by clinical serology for the same purpose. The interassay variability for all these assays is under 15%.

Measurement of PHA-stimulated IL-2 Production. A whole blood technique, modified in our laboratory, was used to measure the IL-2 generating capacity of lymphocytes. A brief description of the technique, which has been described in detail elsewhere (Lyte, 1987, 1988), is as follows. Heparinized whole blood was diluted 1:5 with RPMI-1640 (GIBCO, Grand Island, NY) + 10 mM Wpes (GIBCO) + 2 mM glutamine (GIBCO) i- 50 ng/ ml gentamicin (GIBCO). Five hundred microliters of the above were added to wells of a 12-well multicluster plate containing either 500 ~1 of media or 500 ~1 of media containing 10 ng/ml of PHA (Welcome Diagnostics, Research Triangle Park, NC). Plates were incubated at 37 ‘C in air + 5% CO,.

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The complete well contents were transferred to polypropylene tubes and immediately freeze- thawed twice in liquid nitrogen. Supernatants containing IL-2 were stored at -70 ‘C until they were assayed.

The concentration of IL-2 in the supernatants was measured by a standard bioassay (Gillis et al., 1978) based on its ability to support the proliferation of an IL-2-dependent murine cytotoxic T cell line (CTLL-2). Briefly, supernatants were serially diluted with RPM1 and added in 0.1 ml volumes, in duplicate, to 96-well microtiter plates (Costar). Next, I X 104 CTLL-2 cells (American Type Tissue Culture Collection, TIB-214) in 0.1 ml volumes of RPM1 + 10% fetal calf serum (FCS, GIBCO) were added to all wells. Plates were incubated at 37 “C in 5% CO,. Cells were harvested at 24 hours after a 5-hour pulse with I @Zi[JH]thymidine (ICN 24066) per well. The mean CPM for duplicate wells was calculated and compared to a standard curve. A computer program, kindly provided by P. Marder of Eli Lilly laboratories (Marder, 1984), was used to calculate IL-2 concentration in multiples of the dose that induced half-maximal stimulation (ED,,).

Statistical Analyses. Comparisons were first made between patients and control subjects. Then patients were divided into those who were positive for any one of the tested autoantibodies versus those who were negative for these antibodies (Autoab i-ve and Autoab -ve, respectively, in Figs. 2 and 3). The patients were further divided, on the basis of their clinical state at the time of study, into “acutely ill” and “remitted” groups (Fig. 3). Group means were compared by Student’s t tests and analysis of variance procedures. The nonparametric Mann-Whitney U test and Kruskal-Wallis test were used to compare mean ranks between smaller data sets. To make post hoc comparisons between groups, t tests and Bonferroni tests were used. Since the final groups were not matched for age, race, or sex, analysis of covariance was used to correct for the possible confounding effect of these variables on IL-2 production.

Results

As shown in the box plots for IL-2 production in patients and control subjects (Fig. l), 80% of the patients had IL-2 production that was lower than the mean value for

Fig. 1. Tukey’s box plot of interleukin-2 (IL-2) production I

PAlTENTS CONTROLS (n-122) (n=9a

Upper and lower margcns of the box represent the 75th and 25th percentiles for each goup. The whiskers are at the 5th and 95th Dercentiles.

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control subjects. The box plots also show that the distribution of lL-2 production values in both groups was slightly skewed toward the right. Most of the IL-2 produc- tion values in both groups were observed to be toward the lower end of the range.

There were significant differences in the distribution of gender and ethnicity between patient and control groups (Table 1). Therefore, we first examined the effect of age, race, and sex on IL-2 production. Age and gender did not have any significant relationship to IL-2 production in control subjects or patients. On the other hand, black subjects produced significantly lower IL-2 than white subjects, as previously reported (Ganguli and Rabin, 1989). Race was thus used as a covariate in all the subsequent analyses.

Table 1. Demographics of the sample

White Black be (yr)

Total Male Female Male Female Mean SD

Patients (n = 122) 33 24 35 30 33.88 11.59

Controls (n = 98) 29 35 14 20 30.52 8.78

Acutely ill patients tended to have lower IL-2 production than remitted patients, but there was considerable variance even within the acutely ill group. When patients were grouped according to both clinical state and autoantibody status, however, it became apparent that only acutely ill, autoantibody-positive patients showed significantly lower IL-2 production (Fig. 2). An overall analysis of covariance with race as a covariate produced a significant result (F = 2.91; df= 4, 175; p = 0.023). Post hoc Bonferroni tests and t tests showed that IL-2 production in acutely ill, autoantibody-positive patients was significantly lower than that in control subjects or in any of the other patient groups. Surprisingly, autoantibody-positive patients

who were in a state of remission had the highest mean IL-2 production of all the groups, but this difference was not statistically significant.

Fig. 2. Interleukin-2 (IL-2) production in patients grouped by clinical and autoantibody status

30 ‘Ei 4 2s N

$ 10

1 l5

II 10

0 Autab+w Aufmb-w

PAlTENTS CONTROLS

See iext (Methods section) for description of assignment of patients to “acutely ill” vs. “remitted” or “autoab fve” vs. “autoab -ve” groups.

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Since medications may have played a role in altering IL-2 production, we examined the results for the 29 neuroleptic-naive patients separately. Within this group of patients, all of whom were acutely ill, the relationship between IL-2 production and autoantibody production was the same as in medicated patients (Fig. 3). The autoantibody-positive patients (n = 7) had a mean IL-2 production of 7.56 (SD = 9.11) CTLL-2 units as compared with a mean of 15.87 (SD = 22.68) CTLL-2 units in antibody-negative patients (n = 22). Due to the small absolute numbers of antibody-positive patients in this subgroup, these trends did not, however, reach statistical significance.

Fig. 3. Interleukin-2 (IL-2) production in never-medicated patients grouped by autoantibody status

(22)

- Amab +ve .4utmb -yL

^-_. PATENTS UAVTROIS

(98)

I

See text (Methods section) for description of assignment of patients to “autoab +ve” vs. “autoab -ve” groups.

Could decreased IL-2 production simply be an epiphenomenon of increased psychopathology? If this were so, then we would expect the BPRS scores to be higher in the group with the lowest IL-2 production. However, the mean BPRS scores in the acutely ill patients with the lowest IL-2 production (Autoab +ve) was 66.53 (SD = 14.15), while the acutely ill Autoab -ve group had a mean BPRS score of 65.26 (SD = 15.51). There was also no significant correlation between IL-2 production and BPRS score when all acutely ill patients were analyzed together.

Discussion

As in our previous report (Ganguli et al., 1989~) we found decreased IL-2

production in acutely ill schizophrenic patients. However, our current results show that, when patients are divided based on whether they have or do not have circulating autoantibodies, low IL-2 production is largely confined to autoantibody- positive patients. Remitted patients had normal IL-2 production, but autoantibody- positive remitted patients had a tendency toward the production of greater than normal levels of IL-2. Thus, as we had hypothesized, the production of autoantibodies, a stable trait, appeared to be a useful marker for identifying individuals with a higher likelihood of showing functional immunologic

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abnormalities, such as altered IL-2 production, which are linked to clinical state. Our finding of a trend for remitted patients with autoantibodies to have abnormally high levels of IL-2 further suggested an abnormality of lymphokine regulation in patients with this trait. While we are inclined to interpret our findings as providing further support for the role of autoimmunity in some patients with schizophrenia, some of the following points must be considered.

When any biological variable is found to differ in schizophrenic patients as compared with control subjects, the possibility always exists that the difference is an effect of medication. Finding identical trends in previously medicated and never- medicated patients would appear to rule this out. In the study by Villemain et al. (1989), some never-medicated patients also showed decreased IL-2 production. Another possible clinical confounding factor might be that the differences between schizophrenic patients and control subjects were nonspecific effects of stress. We believe this to be unlikely since there is no reason to suspect that autoantibody- negative patients who had normal IL-2 levels were experiencing less stress as compared with equally psychotic autoantibody-positive patients. Furthermore, Glaser et al. (1990) found that stress was associated with an increase rather than a decrease in IL-2 production.

The bioassay used in this study measures the concentration of IL-2 by its effect on the growth of an IL-2 dependent murine T cell line. It has been suggested that these cells may also respond to IL4 We assayed the concentration of IL-2 in 83 of the supernatants tested in the bioassay using an enzyme immunoassay (ELISA) that is specific for IL-2 and found a high correlation (r = 0.8) between the concentrations measured by the two methods. Thus, it seems highly likely that our findings do relate to IL-2 rather than another growth factor.

A recent report by Gattaz et al. (1992) failed to find any difference in the concentration of circulating IL-2 between 16 schizophrenic patients and 15 control subjects. It is extremely important to distinguish between what is being measured in their study as opposed to the study by Villemain et al. (1989) and the present study. Mitogen-stimulated IL-2 production, the measure reported here and by Villemain et al. (1989), is a test of the functional capacity of T cells to secrete IL-2 under conditions of maximal stimulation in vitro. Decreased production in this test may arise in two ways: The T cells may be deficient in their ability to generate and secrete IL-2 or may have been overstimulated in vivo and thus have a reduced response to the mitogen. On the other hand, serum IL-2, a reflection of overall in vivo production, as measured by Gattaz et al. (1992), is difficult to interpret because IL-2 acts primarily in an autocrine or pericrine manner (Smith, 1988) and thus disease- associated changes in levels of in vivo IL-2 production tend to be localized (Kroemer et al., 1991). As stated earlier, decreased in vitro mitogen-stimulated production is the most frequently reported IL-2 abnormality in a number of autoimmune disorders. Changes in serum IL-2, which have been less frequently reported, take the form of increased concentration (Kroemer et al., 1991). Localized increases at the site of the autoimmune reaction are frequently found, for example, in the salivary gland in Sjiigren’s syndrome (Fox et al., 19853, in the synovial fluid of patients with rheumatoid arthritis (Lemm and Warnatz, 1986), in the eye in autoimmune uveitis

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(Hooks et al., 1988), and in the plaques of patients with multiple sclerosis (Hofman et al., 1989).

Why IL-2 production is lowered in autoimmune disease is not clear. A simple explanation might be that patients with low IL-2 may simply have fewer T cells in their blood. However, T-cell percentage and numbers did not differ in autoantibody- positive versus autoantibody-negative patients nor was there a correlation between IL-2 production and T-cell indices (data not shown). In other autoimmune diseases, like systemic lupus erythematosus, in which low IL-2 production has been found, no correlation has been observed with T-cell indices (Kroemer et al., 1991).

At least two further possibilities exist to account for low IL-2 production. The more likely of these is that lowered in vitro IL-2 production is a consequence of overproduction of IL-2 in vivo. Support for this theory of T-cell exhaustion comes from the finding that when T cells from patients with autoimmune disease are “rested” in culture, before being stimulated with mitogen, the normal capacity for IL-2 production is restored (Huang and Mclaren, 1976). Alternatively, auto- immunity may be accompanied by an intrinsic defect in the capacity of T cells to secrete IL-2. There is some evidence that T cells from patients with autoimmune disease may have such a defect as well (Kroemer et al., 1991). Experiments to investigate these mechanisms in schizophrenic patients have yet to be conducted.

The exact mechanism whereby autoimmunity might alter brain function and cause symptoms characteristic of schizophrenia is not known. It is even possible that altered IL-2 production may directly affect brain function independently of the immune system. Other cvtokines like IL-l and IL-6, which are lymphokines, also appear to have physiological functions in the brain (Nistico and De Sarro 1991; Hall and Rao, 1992). There has been recent speculation that IL-2 might be a neuro- modulator (Nisticb and De Sarro, 1991). At least one recent study has reported that schizophrenic patients have higher mean IL-2 concentration in their cerebrospinal fluid (Licinio et al., 1991). However, another group that measured CSF IL-2 by the same method failed to find any difference in IL-2 concentration between schizo- phrenic patients and control subjects (Rapaport et al., 1990). Thus, the definitive study of lymphokines in the central nervous system (CNS) remains to be done.

It should also be noted that whereas we have found the prevalence of some autoimmune diseases to be increased in schizophrenic patients, at least one auto- immune disease, rheumatoid arthritis, has been found to have a decreased prevalence in schizophrenia (for reviews, see Vinogradov et al., 1991; Eaton et al., 1992). There is also some evidence that juvenile diabetes mellitus is less prevalent among schizophrenic patients (Finney, 1989). These findings are interesting but not necessarily incompatible with the possibility of an autoimmune component to schizo- phrenia. For example, Knight (1982) has hypothesized that different alleles of the same gene may be responsible for susceptibility to both schizophrenia and rheumatoid arthritis. If this were true, an individual inheriting the gene for one disorder would not inherit the gene for the other. We have found that the allele of the major histocompatibility complex HLA DQ PI gene that confers protection from diabetes is also negatively associated with schizophrenia in black subjects (Nimgaonkar et al., submitted for publication).

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Conclusions

This study confirms that lymphocytes from a subgroup of acutely ill schizophrenic patients produce less IL-2 upon mitogen stimulation, an abnormality that is highly characteristic of autoimmune diseases. Acutely ill patients are further characterized by the presence of circulating autoantibodies. Normal or higher than normal IL-2 in remitted patients suggests that, as is found in some autoimmune diseases, IL-2 production might covary with symptoms of the illness. However, a longitudinal study of IL-2 production measured in the same patients in different states and stages of illness is needed. The mechanism for lowered IL-2 production in this subgroup remains to be determined as does the possible pathophysiologic role of altered lymphokine production. Studies of IL-2 in the cerebrospinal fluid and of IL-2 production by lymphocytes within the CNS should also be pursued. Since immune regulation depends on the concerted and appropriate action of a number of different cytokines, studies, in schizophrenic patients, of other lymphokines such as IL-6 and tumor necrosis factor might also be productive.

Acknowledgments. The studies reported in this article were partially supported by a grant from the National Institute of Mental Health (MH-41883) and a National Institute of Mental Health Research Scientist Development Award. The authors are grateful to Marcia DeLeo for data collection, and JoAnn Miller and Rosemarie Perla for expert administrative assistance.

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