Cellular immunity, HLA-class I antigens, and family history of psychiatric disorder in endogenous psychoses

PsychiatryResearch, 48:201-217

201

Elsevier

Cellular Immunity, HLA-Class I Antigens, and Family History of Psychiatric Disorder in Endogenous Psychoses Norbert Miiller, Manfred Ackenheil, Elisabeth Hofschuster, Wolfgang Mempel, and Reinhold Eckstein Received February 12,1992; revised version received March 8,1993; accepted March 31,1993. Abstract. We found an increased lymphocyte proliferation after stimulation with an antigen “cocktail” in 49 schizophrenic patients and 37 patients suffering from affective psychosis, compared with 45 healthy control subjects. On the basis of this and other findings such as increased numbers of CD3+ and CD4+ cells, an increased ratio of CD4+/ CD8+ cells, and a reduced level of suppressor cell activity in schizophrenia and endogenous depression, we investigated the influence of the human leukocyte antigen-Class I (HLA-A, HLA-B, HLA-C) system on the altered immune function and evaluated the relationship to immune function of a family history of psychiatric disorders. A cluster analysis of cases with regard to the HLA-Class I antigens was first performed in a group of 133 healthy control subjects, and two immunogenetically different clusters were found; then 86 patients (49 schizophrenics, 37 affective psychoses) for whom immune functional data were available were assigned to the two HLA-I clusters that had been determined in the control subjects. Analyses of variance (ANOVAs) showed no differences in immune function between the two clusters. With respect to the cluster assignment and the family history of psychiatric diseases, a two-way ANOVA revealed significant differences in the lymphocyte response to the antigen cocktail, in the number of CD8+ cells, and in one suppressor cell assay. When patients were compared by ANOVA on the basis of family history of psychiatric disorder, patients with a positive family history showed a significantly higher number of CD4+ cells and a higher CD4+/CD8+ ratio. Moreover, certain HLA genes, especially HLA-Al, HLA-B8, HLA-B16, and HLA-C2 seemed to be related to the immune function and/or to the immune function and the family history. Key Words. Immune system, affective disorder, cytes, human leukocyte antigens.

schizophrenia,

genetics,

lympho-

An involvement of the immune system in psychiatric diseases was proposed long before the era of neuroleptics (Dameshek, 1930; Lehmann-Facius, 1939). In recent years, with the development of new immunological methods, especially monoclonal antibodies, a growing number of studies have found evidence that alterations of the immune system may play a role in the pathogenesis of endogenous psychoses.

Norbert Muller, M.D., Dipl. Psych., is Senior Psychiatrist; Elisabeth Hofschuster, M.D., is Psychiatrist; and Manfred Ackenheil, M.D., is Professor and Head, Department of Neurochemistry, Psychiatric Hospital, University of Munich, Germany. Wolfgang Mempel, M.D., is Professor and Senior Hematologist, Department of Internal Medicine 111, Klinikum Grosshadern, University of Munich. Reinhold Eckstein, M.D., is Professor and Senior Hematologist, Department of Internal Medicine, Klinikum Charlottenburg, Free University of Berlin. (Reprint requests to Dr. N. Miiller, Psychiatrische Klinik der Universitlt Munchen, Nussbaumstr. 7, D-80336 Munchen, Germany.) 0165-l 781/93/$06.00

@ 1993 Elsevier Scientific

Publishers

Ireland

Ltd.

202 Recent studies in schizophrenic patients point to an activation of the immune system-for example, an increased number of interleukin-2 receptors (Ganguli and Rabin, 1989; Rapaport et al., 1989), an increased number of CD4+ cells (Henneberg et al., 1990) and the presence of heat-shock protein antibodies (Kilidireas et al., 1992). Moreover, an increased percentage of schizophrenic patients showed CDS+ B cells (McAllister et al., 1989), and patients with negative symptoms showed autoantibodies in the serum (Nimgaonkar, 1991). Our own studies (Miiller et al., 1991) also point to an immune activation in schizophrenic patients. In affective disorders, the psychoneuroimmunological findings are more inconsistent: Some authors observed in vitro a reduced lymphocyte response to different mitogens in major depression (Kronfol et al., 1983; Schleifer et al., 1984; Cosyns et al., 1989), but an enhanced lymphocyte response to mitogens was described as well (Altshuler et al., 1989). Studies of in vivo parameters of the immune system show different results: Krueger et al. (1984) observed a decreased number of CD4+ cells in major depression, whereas Darko et al. (1988) reported an increased number. An increased CD4+/CD8+ ratio has been observed by many investigators (Syvalahti et al., 1985; Irwin and Gillin, 1987; Darko et al., 1988; Maes et al., 199la, 1992). The increased CD4+/ CD8+ ratio points to an immune activation as do reports of increased human leukocyte antigen (HLA)-DR receptorand increased interleukin-2 receptor-bearing cells (Maes et al., 1992). An increased CD4+/CD8+ ratio, however, has also been observed in the sera (Compstone, 1983) and the cerebrospinal fluid (Matsui et al., 1988) of patients with multiple sclerosis, as well as enhanced levels of soluble interleukin-2 receptors (Hartung et al., 1990). Some of the in vitro immune parameters in depression seem to be affected by age, sex, hospitalization, and severity of illness (Schleifer et al., 1989), although other authors could not replica.te these findings (Cosyns et al., 1989). In depressive patients, a greater variance in the in vitro immune parameters has been described (Altshuler et al., 1989). Some authors have suggested that different diagnostic subgroups in depressive disorders may contribute to the inconsistent findings (Maes et al., 1991h), especially the proportion of patients with the endogenous versus the nonendogenous subtype (Ader et al., 1990). Our own findings in patients with endogenous depression show a non-state-dependent reduction in levels of suppressor cell activity, elevated numbers of CD3+ and CD4+ cells, and an elevated CD4+/CD8+ ratio in this subgroup of depression (Miiller et al., 1989). The HLA system is localized on chromosome 6 and is related to the immune response (Mempel et al., 1973a, 1973b; Wank, 1989). Until now, two different systems, HLA-Class I (HLA-A, HLA-B, HLA-C) and HLA-Class II (HLA-D, HLA-DR, HLA-PQ, HLA-DQ), have been defined. Many associations with both schizophrenia and affective disorders have been described (Tiwari and Terasaki, 1986), particularly with the HLA-Class I system. Since there is a great variety in the results of these HLA-Class I investigations, depending on ethnic and local genetic differences, but also on different psychiatric diagnostic criteria and possibly on different laboratory methods, meta-analytical studies have been performed. Weak positive associations of HLA-A9 and HLA-A28 with schizophrenia and of HLAB16 with bipolar affective disorder emerged when meta-analytical criteria were applied (Tiwari and Terasaki, 1986). Bersani et al. (1985) and Propert et al. (1981),

203 however, found that HLA-B16 was only associated with unipolar depressive disorder. The weak associations that have been found suggest that these HLA genes may play a role in endogenous psychosis. Perhaps because the associations described above were relatively weak, the results of different studies were contradictory, and no relation to functional disturbances of endogenous psychoses could be found, HLA-I investigations were not pursued further. Moreover, since family studies of the HLA-Class I system in schizophrenia and affective disorder did not show a linkage with HLA-Class I (Tiwari and Terasaki, 1986), other genetic mechanisms must play a role in these diseases. Nevertheless, if the immune activation plays a role in the pathogenesis of endogenous psychosis, the altered immune parameters should be related to genetic mechanisms on the one hand and to HLA-Class I on the other. The combined evaluation of both types of endogenous psychoses is suggested by the fact that immune functional findings-as is true of findings from other disciplines of psychiatric research--show similar results in both types of disorders. An integrated evaluation of the determinants of the disturbed immune function thus seemed to be indicated (Wexler, 1992). The aim of the present study was to investigate the relationships to immune function of (1) the HLA-Class I system and (2) a family history of psychiatric disorder.

Methods Subjects. Eighty-six patients suffering from endogenous psychosis and 133 control subjects were admitted to the study. Forty-nine patients (26 women, 23 men; mean age = 32 years, range = 18-58 years) suffered from an acute exacerbation of a schizophrenic (n = 42; ICD-9: 295.1, 295.3, 295.4, 295.6) or schizoaffective (n = 7; ICD-9: 295.7) psychosis (the latter showing predominantly schizophrenic symptoms) and had not been treated with neuroleptics for at least 4 weeks. Thirty-seven patients (15 women, 22 men; mean age = 41 years, range = 24-61 years) were diagnosed as suffering from affective psychosis (ICD-9: 296.1, 296.3). Twenty-three of these patients were studied during an acute depressive episode, while 14 of them were studied during remission. Fifteen of the depressive patients had been drug free for at least 2 weeks, while eight patients were receiving antidepressant medication at the time of study. Among the patients who were studied during remission, five patients were receiving lithium treatment and eight were not. The schizophrenic and schizoaffective patients had a mean total score of 60 (SD = I I) on the Brief Psychiatric Rating Scale (BPRS; Overall and Gorham, 1976) and a mean total score of 63 (SD = 35) on the German version of Andreasen’s Scale for the Assessment of Negative Symptoms (SANS; Dieterle et al., 1986). The depressed patients had a mean total score of 28 (SD = 6) on the Hamilton Rating Scale for Depression (HRSD; Hamilton, 1960), while the remitted patients had a mean total score of 1.5 (SD = 1.7). All diagnoses were performed according to the Research Diagnostic Criteria (RDC; Spitzer et al., 1982). Seventy-two patients were hospitalized (schizophrenic, schizoaffective, and depressive patients); the 14 remitted patients were studied as outpatients. Serological typing of the HLA-Class I antigens was performed in 86 patients and in 141 healthy control subjects (80 women, 61 men; mean age = 31 years; range = 17-57 years), as described below. In addition, studies of immune function were carried out in these 86 patients and in 45 of the healthy volunteers (21 women, 24 men: mean age = 29 years; range = 22 - 55 years), who served as controls. lnformed consent was obtained from all patients and control subjects according to the declaration of Helsinki. None of the patients and none of the control subjects suffered from an acute or chronic infection or from any immunological disease. The control subjects had no

204

psychiatric disorder.

disorder,

no history

of psychiatric

disorder,

and no family history

of psychiatric

Family History. The family history method (Weissman et al., 1986) was used to determine the presence or absence of psychiatric disorder in the relatives of the patients. After basic information was obtained about first- and second-degree relatives, the patient and-as far as possible-each of the patient’s first- and second-degree relatives were asked about the psychiatric status of family members systematically. Moreover, data were collected from the records of hospitals and private practitioners. The family history was rated “positive” if at least one first- or second-degree relative had a certain diagnosis or a high probability of a diagnosis of major depression, schizophrenia, alcoholism, or a clinically relevant personality disorder. Procedure. Blood (100 ml) was drawn by venous puncture from patients and control subjects between 9 and I I a.m. The immunological examinations included stimulation with an antigen “cocktail,” typing of T-cells and T-cell subgroups, measurement of suppressor cell activity, and serological typing of HLA-A, HLA-B, and HLA-C loci. Peripheral blood lymphoid (PBL) cells were separated from fresh heparinized venous blood by Fiqoll-isopaque density gradient centrifugation, and were then washed and resuspended in McCoy’s 5A Medium (Gibco. Europe). For HLA testing, the lymphocyte number was 4 X IOh per I ml medium. Antigen typing was performed with the Microtoxicity Test Method (Terasaki and McClelland, 1964) by quadruplicate analysis in microtiter plates (Nunc Intermed, Roskilde, Denmark). One microliter of lymphocyte supension was incubated with HLA antisera (Biotest, Marburg, Germany) for 3rum sample and a negative control serum sample were used for comparison. For the antigen-cocktail assay, I X IO5 PBL cells were incubated in 200 l_rl RPMT-1640 culture medium with Hepes buffer containing 1% penicillin-streptomycin, 17~~t_-glutamine (Gibco, Europe), and 20% human AB-serum (BSD-BRK, Munich). The cells were incubated for 120 hours in a humified CO, atmosphere at 37 “C with a 2.75 ~1 antigen cocktail containing purified protein derivate, tetanus toxoid, streptolysine, and mumps and vaccinia antigen (Behringwerke, Marburg) and afterwards for 24 hours with 3H-thymidin. The dose of the antigen cocktail was chosen after dose-response curves were obtained, and the optimal concentrations have been used in various other studies (Eckstein et al., 1984, 1985). The ‘H-thymidine uptake into the DNA of responder blasts, which reflects the proliferative activity of the cells, was measured by liquid scintillation. To avoid artifacts, six cultures of the antigen cocktail were performed in parallel, and the data were averaged. The background counts were subtracted. T-cells and T-cell subpopulations were measured with monoclonal antibodies (Ortho Diagnostic Systems, Neckargmiind, Germany; OKT 3 = all T-cells = CD3+; OKT 4 = Thelper/inducer cells = CD4+; OKT 8 = T-suppressor/cytotoxic T-cells = CD8+) in an immunofluorescent assay. Two hundred microliters of a suspension of 5 X 10” cells,‘ml culture medium were incubated for 30 minutes (0 ‘C; dark) with the three different antibody solutions, washed, centrifuged, and incubated with 100 ~1 fluorescent marked rabbit-antimouse-immunogammaglobulin again for 30 minutes. As controls, 2 X 200~1 cell suspensions were prepared and incubated in the same way, but without antibodies. After another washing, the cells were put on slides, dried, fixed, and evaluated by fluorescent microscopy (Miiller et al., 1989, 1991). In addition, white blood cells were differentiated. The suppressor cell activity was induced by adding 150 1.181’0.5ml concanavalin A (Con-A; Sigma Chemicals, Munich) to the cell suspensions consisting of IO X 106 cellsi2.5 ml culture medium. This mixture (IO X I06 cells/ 2.5 1.11;150 pg Con-A/2.5 ml) was afterwards incubated for 48 hours in a humified CO,-atmosphere at 37 “C. The bystander cells were prepared in the same way except that Con-A was not added. The bystander cells and the responder cells were stored in liquid nitrogen for 48 hours to preserve their biological reactivity. To stimulate the responder cells in the mixed lymphocyte culture (MLC). we used a pool of PBL cells taken

205 from five healthy unrelated donors with different HLA-D alleles; in mitogen cultures, we used pokeweed mitogen (PWM) and phytohemagglutinin (PHA). To inhibit their proliferative activity, bystander, suppressor, and stimulator cells were irradiated with 40 GY from a cesium source (Wiilischmiller). 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 1: 1:l) to enable a study of the suppressor cell activity. The responder cells and the suppressor cells were stimulated with mitogens in separate experiments. Analogous stimulations of responder, bystander, and stimulator cells were performed separately as controls. Medium controls were carried out simultaneously. Each experiment was performed six times, and the average of these data was used. The 3Hthymidine uptake into the DNA of responder blasts was measured by liquid scintillation. An inhibition index for the suppression of the responder cell blastogenesis (expressed in ‘?Jc)was calculated (Miiller et al., 1991). For technical reasons, however, it was not possible to perform each immunological test on each patient’s blood, for neither enough blood could be obtained from each patient nor enough PBL could be isolated from the available blood. All experiments were performed without knowledge of diagnostic group by the same assistant. Statistical Analysis. Statistical analyses were performed in several stages as follows: First, one group of 88 healthy control subjects was divided into immunogenetically different subgroups by cluster analysis of cases according to Ward’s (1963) hierarchical method, using squared euclidean distance. The agglomeration schedule of Ward’s method combines in each step those clusters with the least squared euclidean cluster center distance to minimize the error sum of squares, and additionally takes account of the cluster sizes. The aim of this procedure was to distinguish at least two subgroups of control subjects that differ immunogenetically but are immunogenetically homogeneous inside them. The nature of the clusters and the number of clusters chosen for further analysis were not of crucial importance for the aim of the study. The Scree test (Cattell, 1966) for clusters I to 11, which were found by the cluster analysis, tended to suggest a two-cluster solution after cluster analysis of 133 control subjects by Ward’s method; therefore, the two-cluster model was chosen for further statistical analysis. Moreover, a two-cluster model had the advantage of easier statistical handling for further analyses. In a second step, the stability/reliability of the two-cluster model identified by Ward’s method was examined by a subsequent K-means procedure using the calculated Ward cluster centers as starting centers. The iterative nonhierarchical K-means algorithm allocates each object to that cluster that yields minimal euclidean distance between object and cluster center, recalculates the new cluster centers, and checks again for minimal object-cluster center distances (MacQueen, 1967). As only five control subjects changed cluster membership, the chosen two-cluster solution appeared to be very stable. The K-means clusters and cluster centers, respectively, were used for further analysis. In the next step, the 83 psychiatric patients were divided into two subgroups by a K-means procedure using the patients’ immunogenetic data and the cluster centers found from the control subjects. The cluster centers of the control clusters were kept fixed while assorting the patients (see Fig. 3). The immune functional differences between the HLA clusters were analyzed by analysis of variance (ANOVA) to evaluate the influence of HLA-Class I distribution on the immune function. The x2 test was used to calculate differences in the distribution of patients and control subjects to the clusters, with respect to the family history of psychiatric diseases. To evaluate the interrelationship of the cluster affilation and the family history of psychiatric diseases on the immune functional variables, a two-way ANOVA was performed. Moreover, the relation to immune function of certain HLA antigens that contributed to the cluster differentiation (HLA-Al, HLA-A2, HLA-B8, HLA-B12, HLA-B27, HLA-C2, HLAC7) or had been reported in the literature to be associated with endogenous psychoses (HLAA 1, HLA-A9, HLA-B8, HLA-B 16) was analyzed by ANOVA and the interrelations of these

206 HLA antigens, the family history of psychiatric disorder, and the immune functional variables were assessed in a two-way ANOVA. Student’s t tests were used to assess differences in immune function between patients and control subjects as well as group differences regarding age and sex. Some calculations were performed after finding the logarithms of the variables. Differences in immune function between depressive patients who were and were not being treated with antidepressants, and between remitted patients who were and were not being treated with lithium, were evaluated by Student’s t test. The statistical calculations were performed with SPSS/ PC.

Results The following differences in immune function between healthy control subjects and patients suffering from endogenous psychosis were previously described: a significantly reduced level of suppressor cell activity, an increased number of CD3+ and CD4+ cells, and an increased ratio of CD4+/CD8+ cells (see Table 1; Miiller et al., 1989, 1991).

Table 1. Differences of cell numbers and suppressor cell activity between patients and control subjects (mean f SD) Control CD3+ 03’ CD4+ 04’ CD8’ CD8’

[absolute) (%) (absolute) (%) (absolute) (%) CD’ICDB’ ratio

Schizophrenia

1016+457

1324 t 551*-

45 f 12% 6975312 31 *

1l%'*'*

882 + 409* 9%

517+248 22 I!

54 *

36 t 1 O%* 498*218

7%

21+

Affective psychosis 1234 F 465 56 + 1l%'*'* 905 k 362’ 42 +

1l%'*'*

437 t 229 6%

1.5 + 0.6

1 .a * 0s

20 +

7%

2.4 t

1.4’**’

SCA

PHA

37 * 30%

23 _+ 20%‘*”

18 * 20%‘“‘*

SCA

PWM

40 +_ 31 %

17 f 22%*’

17 + 22%**‘*

19k27%

10 f 23%’

9 f 23%*

MLC

Note. SCA = suppressor cell activity. PHA = phytohemagglutinln.

PWM = pokeweed

mitogen. MLC = mixed

lymphocyte culture. ‘p c 0.05; “p 5 0.01; ***p ?A0.005;

““p 5

0.001 compared with control subjects.

The present study focuses on an analysis of determinants of these immune functional alterations. Fig. 1 illustrates the lymphocyte proliferation after stimulation with the antigen cocktail. The ANOVA showed significantly higher stimulation both in schizophrenic patients (p < 0.05) and patients with affective psychosis (p < 0.05) than in control subjects. Table 2 summarizes the HLA antigens that have been tested in control subjects, schizophrenic patients, and patients with affective psychosis. A cluster analysis of cases was performed with 133 control cases according to Ward’s method. Because cluster analysis showed a slight break after two clusters according to the Scree test, a two-cluster solution was suggested for further analysis. The two clusters were reevaluated with the K-means method. On the basis of Ward’s method, 104 control subjects (78%) were assigned to cluster I, and 29 control subjects (22%) to cluster 2. When the K-means method was used, only a slight change in cluster assignment was noted: 99 control subjects (74$+) were

207

.1.t r

Fig. 1, Lymphocyte proliferation after stimulation with the antigen cocktail

-*

1

.

-*

:

.

,

& .

1ooooc

r . .7 5 . 8. . :.

. .. .

r

50000

. f’

l-R-L

3

.l): 01 f . f

fi3% cxnlws n-46 ii-23480

Schlm phrenics

n=53

3

; DepressIves n - 34 ii = 55890

z - 55580

assigned to cluster 1, and 34 control subjects (26%) to cluster 2. Therefore, the distribution of the control subjects into these two clusters was accepted as valid and reliable. Significant differences between the two clusters were observed in HLA-Al (p < O.OOOOOl), HLA-A2 (p < O.OOOOOl), HLA-A3 (p < O.OOS), HLA-A28 (p B 0.0006), HLA-BS (p < 0.014), HLA-B8 (p d O.OOOOOl),HLA-B27 (p < 0.04), and HLA-C2 (p < 0.02) (see Fig. 2). The differences in immune function between cluster 1 and cluster 2 were compared by ANOVA to evaluate the influence of HLA-Class I on immune function. A significant difference was only found in the MLC. Cluster 2 showed a significantly lower level of suppressor cell activity (p < 0.04) than cluster 1 (cluster 1: mean = 24oj0, SD = 30%; cluster 2: mean = 5%, SD = 16%). In a second step, all patients were assigned to one or the other of the two clusters on the basis of the criteria used to assign the control subjects. Accordingly, the differences between cluster 1 and cluster 2 in the patients corresponded with those in the control subjects. Significant differences in the patient clusters were found in HLA-AI (p < O.OOl), HLA-A2 (p < O.Ol),HLA-B8 (p G O.OOl),HLA-B12 (p < 0.03), and HLA-C2 (p < 0.01). Slight differences (NS) were observed in HLA-B27 (p < 0.09) and HLA-C7 (p ~2 0.09) (see Fig. 3). Our first hypothakwas that the differences in immune function between pati.ents and control subjects would be related to quantitative differences in the distribution of the patients to the HLA clusters (e.g., patients with disturbed immune function

208 Table 2. Frequency of HLA antigens schizophrenia and affective psychosis

HLA antigen

in controls

and in patients

Control (n= 133)

Schizophrenia (n= 49)

Affective psychosis -(n= 37)

%

%

%

Al

la.4

13.1

17.6

A2

34.0

26.2

35.3

A3

27.0

17.9

17.6

A9

15.6

13.1

17.6

A10

5.0

a.3

a.8

All

7.1

10.7

11.8

A28

7.1

10.7

5.9

A29

5.0

0.0

0.0

Aw30

2.8

0.0

2.9

Aw32

0.7

0.0

5.9

85

12.8

11.9

a.8

87

24.2

16.7

a.8

BE

9.2

7.1

8.8

B12

14.2

19.0

14.7

813

4.3

9.5

0.0

814

5.7

4.8

20.6

Bw15

6.4

7.1

8.8

Bw16

5.7

0.0

8.8 5.9

Bw17

1.4

1.2

Bw18

5.0

2.4

0.0

Bw21

2.8

0.0

0.0 0.0

Bw22

1.4

1.2

827

3.5

3.6

5.9

Bw35

14.2

14.3

ii.8

Bw39

2.1

2.4

8.8

Bw40

12.1

9.5

5.9

Bw51

0.7

3.6

0.0

Bw53

0.7

2.4

0.0

Cwl

1.4

3.6

2.9

cw2

3.5

a.3

11.8

cw3

4.3

1.2

14.7

cw4

2.8

4.8

8.8

cw5

0.0

3.6

5.9

Cw6

1.4

4.8

0.0

cw7

0.7

0.0

2.9

Note. No statistically significant difference in the human leukocyte patients and control subjects after a correction.

with

antigen (HLAJ distribution was found between

209

Fig. 2. Distribution of control subjects (n = 133) to cluster 1 and cluster 2 with respect to the HLA antigens frequencyof~genes

0%

20%

40%

60%

100%

'*'p< 0.001; "p < 0.01; *p< 0.05. +p < 0.1.

would be overrepresented in one cluster). To evaluate this hypothesis, differences in the distribution to the two clusters between patients and control subjects were computed. As described, 74Y0 of the control subjects were assigned to cluster 1 and 26% to cluster 2. There was no difference in the distribution of the patients: 74% (61 patients) were assigned to cluster 1 and 26% (22 patients) to cluster 2. For further analysis, the family history of psychiatric disorder was investigated as an additional variable. Thirty-eight patients (46%) were rated as having a positive family history (schizophrenia, 34%; affective psychosis, 59%). Thirty-four patients (42%) were rated as having a negative family history (schizophrenia, 54%; affective psychosis, 29%). For 10 patients (12%), there was not enough information available to make a determination about the presence or absence of a family history of psychiatric disorder.

210

Fig. 3. Distribution of patients (n = 89) to clusters 1 and 2 (clusters defined on the basis of the control subjects) with respect to the HLA antigens frequeflcy of the genes

+

,

0%

***p<

0.001:

20%

40%

,

60%

,

80%

c 100%

‘p < 0.01; *p < 0.05;+p< 0.1.

The patients were divided into two groups on the basis of a positive versus a negative family history of psychiatric disorder, and the immune functional variables in the two groups were compared by ANOVA. A significant difference was observed in the CD4+/CD8+ ratio: patients with a positive family history showed a higher number of CD4+ cells (mean = 42%, SD = 10%; n = 34) and a higher CD4+/CD8+ ratio (mean = 2.5, SD = 1.3; n = 34) than patients with a negative family history (CD4’: mean = 37%, SD = 7%; n = 3; p < 0.02; ratio: mean = 1.9, SD = 0.07; n=31;pd0.05).

The influence of family history on cluster assignment was analyzed by x2 test, with the following significant results emerging: cluster 1 contained more patients with a negative family history (48%) than were found in cluster 2 (23%); cluster 1 contained fewer patients with a positive family history (43%) than were found in cluster 2 (54%; y < 0.05); not enough information could be obtained on the rest of the patients (cluster 1: 8%; cluster 2: 23%). Because HLA cluster assignment appeared to be

211 related to the family history of psychiatric diseases, we hypothesized that the HLA genes and the family history may influence the immune function in patients suffering from endogenous psychoses. We therefore performed a two-way ANOVA to evaluate the immune functional data with respect to the cluster affilation and the family history. An ANOVA was performed comparing the two clusters with immune function as the first variable and family history as the second variable. Significant results were found for the lymphocyte response to the antigen cocktail @ d 0.02), the number of CD8+ cells (p Q 0.03), and the suppressor cell assay after stimulation with PHA (p Q 0.01). The number of CD3+ and CD4+ cells and the level of suppressor cell activity after stimulation with PWM also showed slight (NS) differences (CD3’: p < 0.06; CD4+: p < 0.07; suppressor-cell activity PWM: p < 0.09). With respect to the distribution to the two clusters, elevated lymphocyte response to stimulation with the antigen cocktail and increased numbers of CD3+ and CD4+ cells characterized patients with a negative family history in cluster 1 (n = 27) and patients with a positive family history in cluster 2 (n = If). A reduced level of suppressor cell activity and a low number of suppressor cells, however, were observed in patients with a positive family history in HLA cluster 1 (n = 25) and with a negative family history in HLA cluster 2 (n = 5). Not regarding the cluster analysis but certain HLA antigens, significantly higher CD4+ cells (p < 0.02) and a higher ratio CD4+/CD8+ (p < 0.03) were found in HLA-B16 positive patients. HLA-C2 positive patients had higher CD3+ cells (p < 0.004). With respect to the interaction of family history and certain HLA antigens, we found significant results in HLA-A1 and CD3’ (p < 0.03), and CD4+ cells @ < O.Ol), and the lymphocyte response to stimulation with the antigen cocktail (p < 0.05). Moreover, the two-way ANOVA showed significant interactions between HLA-B8, family history, and CD3’ (p < 0.02) and CD4’ cells (p < 0.002), and of HLA-B16, family history, and suppressor-cell activity after stimulation with PWM (p < 0.05). An influence of age and sex on immune function could not be determined; moreover, no difference between patients who were and who were not receiving antidepressant therapy could be found. With regard to lithium, lithium-treated patients showed even lower inhibitory effects in the MLC (mean = O.O%, SD = 0.0%) than did patients who were not being treated with lithium (mean = 19%, SD = 34%). No other lithium-related difference was found.

Discussion HLA investigations, a cluster ‘analysis of cases, and a two-way ANOVA were performed to evaluate the influence of immune genetics and family history of psychiatric diseases on the disturbed immune function in endogenous psychoses. Other studies have also used cluster analysis to compare immune function and clusters immune genetics (Eckstein et al., 1985). In this study, the immunogenetic were defined on the basis of data for 133 healthy control subjects according to the “scree test,” though the latter is not a crucial point in this methodological approach. The two-cluster solution was very stable, as confirmed by the K means procedure, and was easy to handle for further statistical analyses. So far, the two clusters seem

212 to reflect two immunogenetically different groups in healthy control subjects, and no bias of former HLA findings in psychiatric patients influences the distribution of HLA antigens to the clusters. This calculation was performed to evaluate whether assignment to one of the HLA clusters conferred a higher immunogenetic risk for the type of immune alteration that has been described in endogenous psychoses. It was expected that one of the clusters would be associated with an alteration of immune function and that the majority of the patients would be assigned to this immunogenetic cluster. Such was not found to be the case. A statistically significant difference in the immune function between the two clusters was only found in the MLC, which is known to be influenced by HLA antigens (Mempel et al., 1973a, 19736) whereas no other immune functional test found to be altered in patients showed a difference between the two clusters. In addition, no difference between control subjects and patients or between the two different diagnostic groups was found with respect to the distributions to the two clusters. These findings indicate that the HLA clusters do not explain the immune functional alterations in patients with endogenous psychosis. On the other hand, HLA-Al, HLA-B8, HLA-B16, and HLA-C2 seem to be related to both the enhanced number of CD3’ and CD4+ cells, and the family history of psychiatric diseases. HLA-B16 positive patients showed a higher number of CD4’ cells and a higher CD4+/CD8+ ratio; the frequency of HLA-B16 has been described to be elevated in affective psychosis (Shapiro et al., 1976, 1977) HLA-C2 contributes to our cluster difference, and HLA-C2 positive patients show higher CD3’ cells. HLAAl and HLA-B8 also contribute to our cluster differences; HLA-Al and HLA-B8 by themselves are not significantly related to the cell number, but HLA-Al and HLAB8 and the family history are significantly related to the cell number. Moreover, HLA-A 1 has been reported to be reduced in schizophrenic patients (Mendlewicz and Linkowski, 1980) and HLA-BX, often associated with autoimmune diseases, has been found to be reduced in affective psychosis (Shapiro et al., 1976). The literature shows that associations between certain HLA antigens and endogenous psychoses are different in different studies, different countries, and different ethnic groups (see: Tiwari and Terasaki, 1986) and that the linkage imbalance (disequilibrium) probably also contributes to the different findings. Perhaps for these reasons, HLA associations have not been further pursued in studies of endogenous psychoses. although slight associations have been confirmed in the literature. From our point of view, these data take on new significance in view of the interactions of these HLA genes with disturbances in immune function and the presence of a family history of endogenous psychoses, It is important, however. for these findings to be confirmed by other studies, since a corrections were not used in the present exploratory analyses. HLA genes and family history seem to play a role in the enhanced proliferation rate of lymphocytes after stimulation with the antigen cocktail. in the increased numbers of CD3’ and CD4’ cells, and in the reduced level of suppressor cell activity. The lymphocyte response to the antigen cocktail and the CD3’ and CD4’ cell numbers were found to be highest in cluster 2 with a positive family history and in cluster 1 without a family history. The same significant effect was found in CD8’

213 cells, which were not elevated in patients r%nplrred with levels in control subjects. This result seems to suggest that the number of CDS+ cells, which is not altered in the total group of patients, may play a role in compensation effects of an altered immune system. This is underlined by the results of the suppressor cell activity: the suppressor cell activity is higher in patients who show high CD8’ cells and the suppressor cell activity, which was reduced in all four groups, was lowest in patients of cluster 2 without a family history and in cluster 1 with a positive family history (i.e., in those groups who show low CD8+ and CD4+ cells). As the suppressor cell activity is higher in these groups, in which CD4+ and CD8’ cells are higher, it could be suggested that a compensatory mechanism takes place which enhances the suppressor cell activity via the number of CD8’ cells and via raised CD4+ cells; possibly the immune alteration can be partially compensated for in these groups. The data do not reveal whether the different groups showing enhanced CD3+/ CD4+/ CD8+ cells and increased lymphocyte proliferation after stimulation with the antigen cocktail (cluster 1, family history negative; cluster 2, family history positive) as well as reduced suppressor cell activity (cluster 1, family history positive; cluster 2, family history negative), reflect different immunological groups of risk for endogenous psychosis and/ or different states of compensation by cell numbers of a defect in the suppressor cell function. Nevertheless, the results show that HLA-I and the family history influence the altered immune function. The suppressor cell assays and the MLC show a very low activity in all groups of patients. Therefore, the calculation of significant differences regarding the variables HLA antigens or family history within the groups may only be significant in the PHA assay. An increased CD4+/CD8+ ratio has been described previously both in schizophrenic patients (Rabin et al., 1988; Miiller et al., 1991) and in patients suffering from affective psychosis (SyvUahti et al., 1985; Irwin et al., 1987; Darko et al., 1988; Miiller et al., 1989; Maes et al., 1991~) as well as in multiple, sclerosis, a central nervous system disease with a possible autoimmune pathogenesis (Wekerle, 1991) and in which a genetic influence is known. The influence of a family history of psychiatric diseases has not been ruled out until now in psychoneuroimmunological studies of endogenous psychosis, but our data show that the family history is involved in the immune function. Patients with a positive family history present significantly higher CD4+ cells and a significantly higher CD4+/CD8+ ratio. On the other hand, there are results showing that high CD4+ cells correlate with psychopathology, as measured by rating scales. In depressive patients, the CD4+/CD8+ ratio was reported to correlate positively with the HRSD score (Irwin et al., 1987), and CD4+ cells, which are suggested to be due to the high CD4+/CD8+ ratio in depressive patients (Syvllahti et al., 1985; Miiller et al., 1989) show a positive correlation with the HRSD score as well (Levy et al., 1991). In schizophrenic patients, a relationship between high CD4+ cells, the severity of the symptoms in the course of schizophrenia, and the outcome of neuroleptic therapy could be demonstrated (Miiller et al., 1993). These data underline the interaction between elevated CD4+ cells, a high CD4+/CD8+ ratio, psychopathology, and the family history. Elevated CD4+ cells and an increased CD4+/CD8+ ratio are nonspecific markers of an immune alteration, suggesting an activation, but the relationship to

214

the level of psychopathology and to the presence of a family history of psychiatric diseases points to a relationship in the pathogenesis, too. A possible function of certain HLA genes and another genetic factor as cofactors for a disturbed immune function has to be taken into consideration. The “family history” variable seems to represent a genetic vulnerability which is described both in schizophrenia and in affective psychoses (Ertl et al., 1990). 1Jntil now, genetic investigations in psychiatric diseases could not describe the genetic marker predisposing to an enhanced vulnerability for endogenous psychoses. In the context of the finding that genetic vulnerability may be related to immune function, beyond the HLA-I system, the HLA-II system should be taken into consideration. The HLA-Class II system is known to determine the immune function and mostly shows stronger associations with diseases of a possible autoimmune origin than HLA-I antigens (Wank, 1989). As yet, there are only a few reports of associations of HLA-II antigens and endogenous psychoses (Miyanaga et al., 1984; Riemann et al., 1988; Miiller et al., 1992), and family studies of HLA-II antigens are lacking. Our study, which used statistical methods to compare immune functional, immunogenetic, and family history data, has shown the influence of the HLA-1 system and of the family history of psychiatric diseases on the altered immune function in endogenous psychoses. Although the significance of this relationship is unclear, our results show the relevance of combined analysis of HLA and immune function for further analysis of different immune system variables on the pathogenesis and the course of psychiatric diseases. References Ader, R.; Felten, D.; and Cohen, N. Interactions Annual

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