147

Psychiatry Research, 37: 147-160 Elsevier

Cellular Immunity in Schizophrenic During Neuroleptic Treatment Norbert Miiller, Manfred Mempel, and Reinhold

Ackenheil, Eckstein

Patients

Elisabeth

Received July 24, 1990; revised version received September 1990.

Before

Hofschuster,

4, 1990; accepted

and

Wolfgang

November

10,

Abstract. A possible connection between immunological alterations and schizohas been discussed for many years. We studied 55 schizophrenic patients in an acute stage of illness before they began neuroleptic treatment, 35 patients who showed clinical improvement on neuroleptics,and 51 healthy controls. Our interest was focused on parameters of cellular immunity. We found an increased lymphocyte response to stimulation with pokeweed mitogen (PWM) and phytohemagglutinin (PHA) in patients before neuroleptic therapy and also an increased response to stimulation with PWM and PHA during treatment compared to controls. Stimulation with antigens generally showed a lower lymphocyte response phrenia

in patients than in controls, but the difference was only significant after stimulation with tuberculin before neuroleptic treatment and after stimulation with varidase, diphtena-toxoid, tuberculin, vaccinia, and rubella during neuroleptic treatment. The number of CD3+ and CD4+ cells, but not the number of CD8’ cells, was increased before and during treatment in comparison to controls. Suppressor-cell activity was reduced in three different suppressor cell assays before and during neuroleptic medication compared to controls. We therefore conclude that alterations of the immunological system which are, as has been demonstrated, not due to treatment with neuroleptics might play a role in schizophrenia. Key Words. Immunology,

suppressor

cell activity,

T cells, mitogen

stimulation.

Several studies provide hints of a connection between immunological alterations and schizophrenia. Some authors have suggested that immunological factors may play a role in the pathogenesis of schizophrenia (Heath and Krupp, 1967; Goldstein et al., 1980; Hong et al., 1988). Speculations about the possible role of an autoimmunological process (Knight, 1982) were based on findings of antibrain antibodies (Fessel, 1963; Heath and Krupp, 1967; Pandey et al., 1981; DeLisiet al., 1985; Kelly et al., 1987). To date, however, these antibodies have not been characterized in detail. New immunological examination techniques have led to numerous interesting observations concerning the cellular and humoral immunological systems of schizo-

This artxle is dedicated to Prof. H. Hlppius for his 65th birthday. Norbert Miiller, M.D., is Senior Psychiatrist; Manfred Ackenhed. M.D., is Professor and Head of Department of Neurochemistry; and Ehsabeth Hofschuster, M.D., is Psychiatrist. Psychiatric Hospital. Umversity of Munich, Germany. Wolfgang Mempel, M.D., is Professor and Senior Hematologist, Department of Internal Medicme III, Klinikum Grosshadern, University of Munich. Reinhold Eckstein, M.D., is Professor and Senior Hematologist, Department of Internal Medicine, Klinikum Charlottenburg. Free University of Berlin, Berlin. (Reprint requests to Dr. N Miiller. Psychiatnsche Klinik der Universittit Miinchen. Nussbaumstr. 7, D-8000 Mtinchen 2, Germany.)

OI65-1781/91/$03.50 @ I991 Elsevier Scientific Publishers Ireland Ltd

148

phrenic patients. Studies using monoclonal antibodies have found increased numbers of CD3+ and CD8+ cells (DeLisi et al., 1982), CD4+ cells (Henneberg et al., 1990), and “activated” CD4+ cells (Ganguli et al., 1987) in schizophrenic patients. Recently, elevated numbers of CD5’ B-lymphocytes, which are present in patients with autoimmune disorders, have been observed in a subgroup of schizophrenic patients (Rapaport et al., 1989). Interleukin-2 (lL-2), a factor that is produced by activated T-lymphocytes and is usually reduced in patients with autoimmunological disorders (Kay et al., 1986), has been reported to be reduced in schizophrenic patients (Villemain et al., 1987; Rabinet al.. 1988). Contrary to these results, increased serum levels of soluble IL-2 receptors have been observed in patients with autoimmunological disorders (Huang et al., 1988) and there have been recent reports about increased IL-2 receptors in schizophrenic patients (Rapaport et al., 1989; Ganguli and Rabin, 1989). Immunological alterations in schizophrenia have been described from another point of view: Ganguli et al. ( 1989) found a history of atopic disorders or diseases with an autoimmunological basis in 20 out of 103 schizophrenic patients, but in only 3 out of 100 controls. The present study attempted to shed further light on the involvement, if any, of the cellular immunological system in the course of schizophrenia. Toward this end, we studied schizophrenic patients before they received neuroleptic treatment and schizophrenic patients who showed clinical improvement in response to neuroleptic therapy. Methods Subjects. Fifty-five patients (28 females. 27 males). aged 18-58 years (mean q 32), suffering from an acute exacerbation of a schizophrenic (n q 48; ICD 295. I. 295.3, 295.4. and 295.6) or schizoaffectlve psychosis (n 1 7; ICD 295.7) who had not been treated with neuroleptlcs for at least 4 weeks, were admitted to the study. In the patients suffering from schizoaffective psychosis, the schizophrenic symptomatology was predommant. Seven patients were given the diagnosis of a schizoaffective psychosls before being discharged. A few of these patients had never been treated with neuroleptics before, and the majority had not been treated with neuroleptics for at least some months. With respect to other drugs taken before the admission to our hospital, one patient received propranolol, one patlent received trazodone, and one patient received I -tryptophan, but there was a drug-free interval of 3 days before blood was drawn from these patients. The rest of the patients had not received any medication forat least 4 weeks. After admlssion. 39 patients were not given any drug therapy before the blood tests; the rest of the patients were given benzodiazepines for a short time. Twenty-four of these patients (I 2 females. I2 males), aged 21-57 years (mean = 32 years), who showed a definite improvement on neuroleptic medication, were reexamined (4-86 weeks; mean = 20 weeks) before being discharged. Eleven patients (3 females, 8 males), aged 21-57 years (mean=30 years). who were also receiving neuroleptic medication and who were about to be discharged. were added to our study to increase the number of medicated schlzophremc patients. Fifty-one healthy volunteers (22 females, 29 males), aged 22-55 years (mean q 29). were examined for our study and served as controls. Neither the schizophrenic patients nor the controls suffered from an acute or chronic infection or from any other somatic disease. The psychopathological status of the patients was assessed using the Brief Psychiatric Rating Scale (BPRS; Overall and Gorham. 1976). The BPRS total score was 60 (SD = I I) on admission and 48 (SD = 14) on reexamination. The breakdown for duration of illness was as follows: G 3 months (n = IO), 4-12 months (n= 8). l-5 years (n= 22). 5-10 years (n = 5). and IO years (n q IO).

149 Procedure. Blood (100 ml) was drawn by venous puncture from patients and controls at about 9 a.m. The immunological examinations included mitogen stimulation, antigen stimulation, typing of T-cell subgroups, and measurement of suppressor cell activity. Peripheral blood lymphoid (PBL) cells were separated from fresh heparinized venous blood by Ficoll-isopaque density gradient centrifugation, and were then washed and resuspended in RPMT- 1640 culture medium with Hepes buffer containing 1% penicillin-streptomycin, 1% L-glutamine (Gibco Europe), and 20% human AB-serum (BSD-BRK). These suspensions contained lymphocytesand monocytes at the ratio of approximately 4 to 1. We incubated for each test 1 x 106 PBL cells in 200 IJI medium. The mitogen tests included 12.5 pg protein A (Pharmacia Fine Chemicals, Uppsala), 2.5 c(g pokeweed mitogen (PWM; Gibco Europe, Glasgow), and 1 pg phytohemagglutinin (PHA; Wellcome); the incubation time was 72 hours. Our antigen tests included the incubation of the cells with 2 ~1 varidase (Lederle, Wolfratshausen), 2.5 ~1 tetanus toxoid, 2.5 ~1 diphtheria toxoid, 1 p1 tuberculin, 0.5 ~1 measles antigen, 0.5 ~1 vaccinia antigen, and 16 ~1 rubella antigen for 144 hours (all: Behring, Marburg). The doses of the antigens and the mitogens (see Nowell, 1960; Janossyand Doenhoff, 1974; Gelfaud et al., 1985) were chosen after performing dose-response curves, and the optimal concentrations have been used in various other studies (Eckstein et al., 1982, 1984a, 19846,1985). Ineachcase, six cultures of every mitogen and antigen were examined in parallel , and the average results were taken to avoid artifacts, The 3H-thymidine uptake into the DNA of responder blasts, which reflects the proliferative activity of the cells, was measured by liquid scintillation. The background counts were substracted in all experiments. T-cell subpopulations were measured by monoclonal antibodies (Ortho Diagnostic Systems; OKT 3 = all T-cells = CD3+; OKT 4 q T-helperi inducer-cells = CD4+; OKT8= T-suppressor/T-cells= CD8+) in an immunofluorescent assay from cells that were stored at -80 “C until the beginning of the tests. In addition, we performed a differentiation of white blood cells. For technical reasons, however, it was not possible to perform each immunological test on each patient’s blood, either because not enough blood could be obtained from each patient or because not enough PBL could be isolated from the available blood. The suppressor cell activity was stimulated by adding concanavahn A (Sigma Chemicals, Munich), at a concentration of 60 pg/ ml. to the cell suspensrons consisting of 1 x 106cellsi2.5 ml culture medium. This mixture was afterwards incubated for 48 hours in a humidified CO, atmosphere at 37 “C. The bystander cells were prepared in the same way, except that concanavahn A was not added. The bystander cells and the responder cells were stored in liquid nitrogen for 48 hours (Rich and Pierce, 1973) to preserve their biological reactivity. To stimulate the responder cells in the mixed lymphocyte culture (MLC), we used a pool of PBL cells taken from five healthy unrelated donors with drfferent HLA-D alleles (Shearer et al., 1986); in mitogen cultures, we used PWM and PHA. To inhibit their proliferation activity, bystander, suppressor, and stimulator cells were irradiated with 40 GY from a cesium source (Wllischmiller). The responder cells were not irradiated. We stimulated the responder cells, in the presence of the suppressor cells, with the stimulator cells (in the ratio of I:l:I) to enable a study of the suppressor cell actrvity (Rich and Rich, 1975; Shou et al., 1976; Eckstein et al., 1982). The responder cells and the suppressor cells were stimulated wtth mitogens in separate experiments (bl). Analogous stimulations of responder, bystander, and stimulator cells were performed separately as controls (al). Medium controls were carried out simultaneously (b2, a2). Each single experiment was again performed six times, and the average of these data was used. The ‘H-thymidine uptake into the DNA of responder blasts was measured by liquid scintillatron. Counts per minute (cpm) were used to calculate an inhibition index for the suppression of the responder cell blastogenesis (expressed in %) by concanavalin-A (con A) activated PBL cells according to the formula: Suppression

(%) = (1 - (bl - b2,al

- a2)) x 100

150 Our calculations are based on the fact that a biological function like suppressor cell activity must have a positive value. On this premise, we calculated arithmetically negative suppression values as 0 (Eckstein et al., 19846). In an additional experiment performed to exclude a serum effect, we took the sera of three schizophrenic patients and added them to the suppressor cell assays of healthy controls. Student’s t test for unpaired data was used to calculate the differences beween the groups. The data of the 24 schizophremc patients who had been examined twice were compared by the paired t test. Because we did not get data from every assay in every patient twice, the number of subjects IS lower than 24 in the paired I test.

Results Lymphocyte Response to Stimulation With Mitogens. We did not observe a significant difference between schizophrenic patients and healthy controls after having stimulated their cells with Protein A. Schizophrenic patients before and during neuroleptic treatment showed a significantly higher response to PWM and PHA than healthy controls. Among the schizophrenic patients, a significant increase of the lymphocyte response to PWM was associated with neuroleptic treatment as compared to the unmedicated state (see Table I). The background counts of the stimulation (mean f SD) were as follows: acute schizophrenic patients, 9 16 + 523; medicated schizophrenic patients, 793 + 301; controls, 1140 + 720. Table

1. Lvmohocvte

resDonse to stimulation

Cont. (cpm)

Acute schiz. (cpm)

Med. schiz. (cpm)

Mean

4598

4622

5936

SD Ifll

4565

4953

5693

Mitogen Prot.

A

with different .A

A

mitoaens 1

Cont./ Cont./ Acute/ Acute/ Acute schiz. Med. schiz. Med. schiz.1 Med. schiz.2

A

I51

155

1261

NS

NS

NS

NS

NS

NS

NS

NS

PWM Mean

27895

46659

62110

t=3.63

t=5.5

SD

19674

32842

28651

p

Cellular immunity in schizophrenic patients before and during neuroleptic treatment.

A possible connection between immunological alterations and schizophrenia has been discussed for many years. We studied 55 schizophrenic patients in a...
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