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Lymphocyte subsets in schizophrenic disorders
Schizophrenia Elsevier
SCHIZO
Research,
00123
Lymphocyte Relationship Cinzia ‘Institute
269
3 (1990) 269-215
subsets in schizophrenic
with clinical, neuromorphological
Masserini’, Peruzzi’, of Psychiatry,
disorders
and treatment
variables
Antonio Vita i, Roberto Basile’, Raffaele Morselli’, Paolo Boato’, and Carlo Lorenzo Cazzullo’ Luigi Pugnetti 2, Pasquale Ferrante’
Cinzia
Milan University School of Medicine, Policlinico,Pad. Guardia II, via F. Sforza 35, 20122 Milan, Italy, and ‘Don Gnocchi Multiple Sclerosis Center, via Capecelatro 60. Milan, Italy. (Received
4 August
1989, accepted
6 March
1990)
Peripheral blood lymphocyte subsets and serum immunoglobulin levels were assessed in 42 patients and 37 healthy controls. 24 patients were free from neuroleptic medication and 15 had never been treated with neuroleptics. 31 patients had a diagnosis of schizophrenia (DSM-III criteria) and 11 a diagnosis of a disorder of the schizophrenic spectrum. As compared to healthy subjects, the drug naive schizophrenic patient group showed an increase of T suppressor lymphocytes, while the drug-treated schizophrenic group showed an increase of T helper lymphocytes. The drug-treated schizophrenic group differed from the drug naive one relative to a decrease ofT suppressor lymphocytes. As compared to healthy subjects, the drug naive spectrum disorder patients showed an increase of absolute number of lymphocytes, while the drug treated spectrum group showed an increase of B lymphocytes. These findings did not correlate with any clinical or neuromorphological variables taken into account. Key words: Schizophrenic
disorder;
Lymphocyte
subset;
Immunoglobulin;
INTRODUCTION
Immune system abnormalities in schizophrenic patients have been described previously. Evidence for the presence of antibrain antibodies in serum (Lehmann-Facius, 1937), morphological changes of lymphocytes (Hirata-Hibi et al., 1982), abnormal serum and CSF immunoglobulin distribution (Zarrabi et al., 1979; DeLisi et al., 1984), decreased cellular in vitro function of lymphocytes (Vartanian et al., 1978) and of natural killer cells (DeLisi et al., 1983) and abnormal distribution of lymphocyte subsets in peripheral blood have been reported. With regard to the last problem, there is evidence of T suppressor cell decrease (Kolyaskina et al., 1987; Muller et al., 1987), T helper increase (Ganguli et al., 1987), T total, T suppressor and B lymphocyte increase (DeLisi and Wyatt, 1982) Correspondence sitaria, via Besta
0920-9964/90/$03.50
to: C. Masserini, 1, 20161 Milan,
0
Clinica Psichiatrica Italy.
1990 Elsevier
Science
Univer-
Publishers
Neuroleptic
side effect
and/or reports of no changes at all (Kaufman et al., 1987; Villemain et al., 1987, 1989). These contradictory results have been found in chronic schizophrenic patients with a long history of disease and of neuroleptic treatment. Even when the patients were described as free from drugs, the withdrawal period did not exceed 4 weeks. To obviate this, we employed a population of patients affected by schizophrenia or schizophrenic spectrum disorders, a third of whom had never been treated with neuroleptic drugs, and studied the lymphocyte subsets and the immunoglobulin distribution in peripheral blood, dividing the patient group according to the presence or absence of neuroleptic medication. PATIENTS
AND METHODS
Patients 42 patients, referred to the Institute of Psychiatry
of the University
B.V. (Biomedical
Division)
of Milan both as in- or out-
270
patients over a period of 2 years, were included in the study. None of the patients had clinical evidence of a medical or neurological illness that could be responsible for psychiatric symptoms, nor any history of alcohol or drug abuse, head trauma with loss of consciousness, neurologic disorder or electroconvulsive therapy. No recent infections or diseases associated with immunological abnormalities were reported. 31 patients (24 males and seven females) fulfilled the DSM-III criteria for the diagnosis of schizophrenia or schizophreniform disorder and 11 patients (eight males and three females) had a diagnosis of schizophrenic spectrum personality disorder such as schizotypal (seven patients) or of schizoid (four patients) disorder. The study included 12 patients with disorganized, eight undifferentiated, seven paranoid subtypes of schizophrenia and four schizophreniform disorders. 15 subjects (nine schizophrenics and six personality disorders) had never been treated with neuroleptic medication (drug naives), and nine subjects (seven schizophrenics and two personality disorders) had been free of neuroleptic medication for at least 3 months (six patients for 3, one for 4, one for 6 months and one for 2 years). 18 subjects (15 schizophrenics and three personality disorders) had been on neuroleptic medication for a period lasting from 1 month to 3 years, with daily doses ranging from 2 to 13 mg of haloperidol. 37 healthy volunteer blood donors (22 males and 15 females, mean age+ SD, 30 + 10 years), screened for a history of psychiatric and immune disorders, served as a control group for the immunological parameters. There were no significant differences among controls and patients, as far as age was concerned. Methods The clinical evaluation of patients included a psychiatric interview and the Brief Psychiatric Rating Scale (Overall and Gorham, 1962). The immunological evaluation of subjects consisted in the assessment of peripheral blood lymphocyte subsets and serum immunoglobulins. Lymphocytes were separated from whole blood by the Bdyum method (Boyurn, 1968). Subsets of T and B lymphocytes and interleukin-2 receptor positive cells were determined by the immunoperoxidase staining method described by Hofman (Hofman et al., 1982), on cytocentrifugated smears previously coated with antibodies avaible monoclonal commercially
(OKT3, 0KT4, OKT8 from Ortho and anti-leu12 and anti-IL-2 receptor from Becton Dickinson). The percentage of lymphocytes positive to staining, i.e., having receptors for each monoclonal antibody, was determined by microscopical examination: 600 cells for each smear were counted. T total, helper, suppressor, B lymphocytes and IL-2 receptor bearing cells per microliter were calculated by multiplying the percentage of positive cells by the total number of lymphocytes/p1 obtained by a total and differential white cell blood count. The values of IgA, IgG and IgM serum immunoglobulins were assessed by the radial immunodiffusion technique on Nor-partigen plates (Behringwerke, Marburg). 28 patients underwent computerized tomographic (CT) scan examination of the brain. CT scans were performed with a model 1010 EM1 scanner to assess the presence of neuromorphological alterations. Ventricular size was measured on the tomographic slice which showed the greatest extension of the lateral ventricles by a manual planimetric method (Sacchetti et al., 1987) and expressed as the ventricular brain ratio (VBR) (Synek and Reuben, 1976). Cortical atrophy was measured using a fourpoint visual scale, assessing increasing degrees of atrophy (0 = absent, 1 = uncertain/mild, 2 = moderate, 3=severe) (Vita et al., 1988). The variables taken into account for correlation with immunological findings were: (a) age at the time of immunological evaluation; (b) sex; (c) age at disease onset, defined as the age of the patient’s first psychiatric referral; (d) duration of illness; (e) family history of schizophrenia in first degree relatives, assessed by means of interviews with the patient and at least one of his relatives; (f) intellectual functioning, expressed as the patient’s total IQ and percent of deterioration on the Wechsler Adult Intelligence Scale (WAIS); (g) ventricular brain ratio; (h) presence or absence of cortical atrophy. Statistical analysis of the results was performed by analysis of variance (ANOVA) followed by the Student’s t test, using Bonferroni’s correction, and the x2 test, using Yates’ correction when necessary.
RESULTS
Schizophrenic patients and those with a schizophrenic spectrum disorder did not differ signifi-
271
cantly on immunological results. Nevertheless the results of schizophrenic patients and of those with a schizophrenic spectrum disorder were analyzed and presented separately. The lymphocyte subset counts in the schizophrenic group are listed in Table 1. The drug naive schizophrenic patient group showed an increase of the percentage of T suppressor (PC 0.05) lymphocytes as compared with the control values. This difference also was evident by comparing the absolute number of T suppressor (PC 0.05) lymphocytes/p1 of blood. The percentage of T suppressor lymphocytes was significantly (P~0.05) higher than that present in the drugtreated patient group. The drug-free schizophrenic patients showed lymphocytes/pl of blood to be significantly higher than in healthy subjects (PcO.05) and an increase, as TABLE
absolute number, of T total, T suppressor and B lymphocytes (P-C 0.05). The drug-treated schizophrenic patient group differed from normal controls relative to a T helper cell absolute number increase (P < 0.006) but the T helper percentage did not reach statistical significance (P~0.10). No difference was detected in the distribution of IL-2 receptor positive cells among the four groups of subjects. Table 2 shows the immunoglobulin values found in the serum in the schizophrenic group. No significant difference of immunoglobulin values could be found between patients and healthy subjects. We observed a trend for a decrease of IgM values in drug-treated schizophrenic patients vs. drug-free ones (P~0.10). The lymphocyte subset counts in the spectrum personality disorder group are listed in Table 3.
1
Lymphocyte subsets in schizophrenic patients and healthy subjects Values expressed as mean f SD. Statistical analysis by Student’s t test, using Bonferroni’s healthy subjects. *P
Lymphocytes/p1 T total (%) (cells/PI) T helper (%) (cells/PI) T suppressor (%) (cells/PI) B (%) (cells/pi) IL2-r positive (X) (cells/pi)
TABLE
1,958+62 64k9 1,214+406 43&6 833 &274 31*6 619+208 14+3 268kll2 5&2 90+47
correction.
Bold typed values P < 0.05 versus
Schizophrenic subjects Drug naive (n= 7)
Drug-free (n=9)
On drug (n= 15)
2,251*717 69klO 1,587f696 44+9 1,200f432 38*8* 867 f 351 17*3 368kll6 7&3 145k66
2,789 + 714 73&6 1,987 f 605 41+5 1,068* 175 35&9 983k417 16+4 503 + 136 4+2 104+48
2,319*748 65+12 1,591+581 49&7 1,200*441 29+5 691+215 16+8 352+ 174 6k3 138+71
2
Serum immunoglobulins in schizophrenic palients and healthy subjects Values expressed as mean k SD. Statistical analysis by Student’s f test, using Bonferroni’s versus drug-free patients. Healthy subjects
IgA (mg/lOO ml) IgG (mg/lOO ml) IgM (mg/lOO ml)
231 f87 1,348 k 336 209 f 80
correction.
PC 0.10 IgM of patients
Schizophrenic subjects Drug naive
Drug-free
On drug
202 f 56 1,300&349 227 k 87
240&-111 1,344*252 283 k98
206 k 75 1,266 f 305 162k37
on drugs
212 TABLE
3
Lymphocyte subsets in spectrum disorders and healthy subjects Values expressed as mean + SD. Statistical analysis by Student’s t test using Bonferroni’s correction. healthy subjects. *PC 0.02 versus drug free patients and **PC 0.05 versus patients on drug. Healthy subjects (n=37)
Lymphocytes/p1 T total (%) (cells/pi) T helper (%) (cellslpl) T suppressor (X) (cells/pi) B (%) (cells/$) IL-2-r positive (%) (cells/fil)
Schizophrenic
1,958*62 64&9 1,214&406 43k6 833 + 274 31 k6 619+208 14_+3 268k112 5+2 90&47
spectrum personality
disorder subjects
Drug naive (n=6)
Drug-free (n=2)
On drug (n=4)
3,359 & 623 71&6 2,387*467*.** 46kl 1,524+211* 37+12 1,263 * 540 16&4 531+198 6+3 193+115
2,268*314 66k2 1,498*209 41+6 929k272 37&l 837&86 19*1 433 +93 7&4 165k118
2,838 + 1,255 63&6 1,687 + 565 43k2 1,258 + 593 29&9 865+ 147 22+9 801& 552 5*1 134*20
The drug naive spectrum patient group showed the number of lymphocytes/p1 of blood was higher than in healthy subjects (PC 0.005) while an increase of absolute number of T total, T helper and T suppressor lymphocytes was also present (PC 0.005). The drug naive patients also had an increase of the absolute number of T total and T helper lymphocytes vs. drug-free spectrum patients and of T total lymphocytes vs. drug-treated patients (PC 0.05). The drug-free spectrum patient group did not show any difference from normal control values, but it included two subjects only. The drug-treated spectrum patient group differed from normal controls relative to B lymphocyte increases, either as a percentage (P
TABLE
Bold typed values PC 0.05 versus
in the serum in the spectrum personality disorder group. No significant difference in immunoglobulin values could be found between patients and healthy subjects. We then looked into whether there were any differences, for sex, age, age of onset, duration of illness, family history, pathological findings on CT scans, BPRS and WAIS scores and obstetric complications at birth which could influence the immunological results. We did not find any significant differences among the groups. Finally we analyzed whether there was some association between immunological parameters and clinical or neuromorphological characteristics by comparing patients with immunological values above and below the mean of controls values plus 2 standard deviations: no difference could be detected. The clinical or neuromorphological charac-
4
Serum immunoglobulins in spectrum disorder subjects and healthy subjects Values expressed as mean k SD. Statistical analysis by Student’s t test, using Bonferroni’scorrection. Healthy subjects
IgA (mg/lOO ml) IgG (mg/lOO ml) IgM (mg/lOO ml)
231+87 1,348+336 209 & 80
Spectrum disorder subjects Drug naive
Drug-free
On drug
230+74 1,334 * 366 228 * 94
253 k 14 1,400+ 141 266+18
254 k 69 1,324&403 197+_66
213
teristics of the patients 5 and 6.
groups
are shown in Tables
DISCUSSION
Several immunological changes have been described in schizophrenic patients, though it is often quite hard to compare the results of different clinical populations. To the best of our knowledge, TABLE
5
Characteristics of schizophrenic patients Drug naive II=19
Age
Sex (M:F) Age of onset Illness length Family history (FH+:FH-) BPRS score WAIS IQ % IQ deterioration VBR Cortical atrophy (absent:present) Obstetric complications (absent:present)
TABLE
Dtugfree n=7
23.3k4.9 8:l 21.3+4.2 1.97& 1.7 217
22 k 2.9 7:o 18+3 3.7k2.6 5:2
57.8*20 101*18 8.4k8.5 3.2k 1.4 6:l
55.2*18 99* 14 10.5+ 18 4.2* 1.5 4:2
7:2
6:l
Dtugtreated n=15 23.7*4 9:6 19.4k3.7 4.2+3 4:ll 59.1*18 99+ 15 15*14 51+2.8 714 12:3
6
Chatacretistics of spectrum personality disorder patients
Age Sex (M:F) Age of onset Illness length Family history (FH+:FH-) BPRS score WAIS IQ % IQ deterioration VBR Cortical atrophy (absent:present) Obstetric complications (absent:present)
Drug naive n=6
Dtugfree n=2
Dtugtreated n=3
23.3 k4.6 3:3 19.6& 5.3 2.7* 1.8 1:5
22.5i2.1 0:2 19i2.8 3.8i4.5 1:l
23.li5.1 3:o 22+4 1.4* 1.5 1:2
45 k 2.7 102+7 21.7k8.5 7.8* 1.8 2:o 5:0
36 * 3.5 81 k4 0 l:o
66+5 98+8 1.3 f 7.5 2.2+0.1 2:o
1:l
3:o
our study presents the largest number of drug-free schizophrenic patients tested for lymphocyte subsets, with the exception of the work by Villemain (Villemain et al., 1989). 15 patients had never been treated with neuroleptic drugs and the other drugfree patients had a longer withdrawal period than those referred to in literature. The mean age of the patients and the duration of illness were lower than those of the patients referred to in previous research (DeLisi and Wyatt, 1982; Ganguli et al., 1987; Kaufman et al., 1987; Muller et al., 1987; Villemain et al., 1989). None of the patients had a history of long-term hospitalization and patients with schizophrenic spectrum disorders were also considered. We found an increase of T suppressor lymphocytes, either as a percentage or as an absolute number of cells/p1 of blood, in the drug-naive schizophrenic group as compared with healthy subjects. Schizophrenic patients on drugs differed from the healthy controls relative to higher T helper lymphocytes absolute number. Similar results were obtained by DeLisi (DeLisi and Wyatt, 1982) in a group of 38 chronic schizophrenic inpatients, seven of whom had been free from neuroleptics for at least 1 month. A difference between drug naive and drugtreated schizophrenic patients is present in the distribution of lymphocytes bearing the receptor for the OKT8 monoclonal antibody, which detects the T subpopulation with suppressor/cytotoxic function. With regard to the latter, our findings are similar to those of Kolyaskina et al. (1987), who reported a significant decrease of the T suppressor subpopulation in 53 chronic schizophrenic patients diagnosed as suffering from a malignant form of schizophrenia according to the ICD9 and free from drug medication for 3 weeks. In agreement with Kolyaskina’s hypothesis that psychotropic drugs could be responsible for immunological changes, we also found a trend towards lower IgM value in drug-treated schizophrenic patients versus drug-free ones. A reduction of serum IgA, IgM and IgG had also been reported by DeLisi et al. (1981) studying 3.5 chronic schizophrenic inpatients. A difference in the immunological assessment of drug naive, drug-free and drug-treated schizophrenic patients has not clearly emerged before. It is our guess that different immunological stages could be present during the longitudinal course of schizophrenia. We can sup-
274
pose that an initial T suppressor lymphocyte increase, perhaps caused by environmental (viruses) or genetic factors, is followed by an almost global activation of lymphocyte subsets, which is then replaced by T helper lymphocyte activation. This hypothesis may explain the contradictory reports of previous findings on lymphocyte subsets. Nevertheless neuroleptic drugs either directly, or by modulation of neurotransmitter functions, might be responsible for the immunological changes present in some drug-treated schizophrenic patients (Ferguson et al., 1978; Ganguli et al., 1987; Kereptic et al., 1987), and for masking the immunological picture preceding pharmacological treatment, such as, for example, an elevation of T suppressor subset. The meaning of the T suppressor increase might be linked with a previous or recent infection by some viruses, like cytomegalovirus, which may produce T suppressor elevation lasting for 10 or more months after the onset of clinical infection (Carney et al., 1981). On the other hand, a T suppressor increase, not confirmed by in vitro functionality tests, could mean a decreased activity of T suppressor/cytotoxic cells, as observed by Muller et al. (1987) in a population of previously drug-treated schizophrenic patients. Finally a T suppression increase could also express dopamine immunosuppressive activity in those schizophrenic patients with a dopaminergic hyperfunction. We can point out that specific immunogenetic characteristics, such as those found in several studies to be associated with schizophrenia (Smeraldi et al., 1976; Ivany et al., 1983) may influence the immunological reactivity of schizophrenic patients. Although the number of subjects is too small to draw some conclusions, we note the increase of B lymphocytes in the drug-treated spectrum patients. A subpopulation of B lymphocytes that express the CD5 (Leu-1) surface antigen has been reported to be elevated in 30% of drug-treated schizophrenic patients by McAllister et al. (1989). This subset of lymphocytes also is increased in patients affected by diseases of known autoimmune pathogenesis such as rheumatoid arthritis, Sjiigren’s syndrome and multiple sclerosis. In conclusion we think it could be useful to continue immunological studies in early stages of the disease in order to further determine the significance of these findings.
REFERENCES
Boyurn, A. (1968) Isolation of leucocytes from normal blood. Further observations. &and. J. Clin. Lab. Invest. 97 (suppl.), 31-50. Carney, W.P., Rubin, R.H., Hoffman, R.A., Hansen, W.P., Healey, K. and Hirsch, M.S. (1981) Analysis of T lymphocyte subsets in cytomegalovirus mononucleosis. J. Immunol. 126, 2114-2116. DeLisi, L.E. and Wyatt, R.J. (1982) Abnormal immune regulation in schizophrenic patients, Psychopharmacol. Bull. 18, 158-163. DeLisi, L.E., Weinberger, D.R., Potkin, S., Neckers, L.M., Shiling, D.J. and Wyatt, R.J. (1981) Quantitative determination of immunoglobulins of CSF and plasma of chronic schizophrenic patients. Br. J. Psychiatry 139, 5 13-5 18. DeLisi, L.E., Ortaldo, J.R., Maluish, A.E. and Wyatt, R.J. (1983) Deficient natural killer cell (NK) activity and macrophage functioning in schizophrenic patients. J. Neural Transm. 58, 966106. DeLisi, L.E., King, A.K. and Targum, S. (1984) Serum immunoglobulin concentrations in patients admitted to an acute psychiatric inpatient service. Br. J. Psychiatry 145, 661-664. Ferguson, R.M., Schmidtke, J.R. and Simmons, R.L. (1978) Effects of antipsychotic drugs on in-vitro lymphocyte activation. Birth Defects 14, 3799405. Ganguli, R., Rabin, B., Raghu, U. and Ulrich, R.S. (1987) T Lymphocytes in schizophrenics and normals and the effects of varying antipsychotic dosage. In: E.Kursak et al. (Eds.), Virus, Immunity and Mental Disorders. Plenum Press, New York, pp. 321-326. Hirata-Hibi, M., Higashi, S., Tachibana, T. and Watanabe, N. (1982) Stimulated lymphocytes in schizophrenia. Arch. Gen. Psychiatry 39, 82-87. Hofman, F.M., Billing, R.J., Parker, J.W. and Taylor, C.R. (1982) Cytoplasmatic as opposed to surface Ia antigens expressed on human peripheral blood lymphocytes and monocytes. Clin. Exp. Immunol. 49, 355363. Kaufmann, C.A., DeLisi, L.E., Torrey, F.E., Folstein, SE. and Smith, W.J. (1987) T. Lymphocyte subsets apd schizophrenia. In: E. Kursak et al. (Eds.), Virus, Immunity and Mental Disorders, Plenum Press, New York, pp. 3077320. Kerepcic, I., Bamburac, J. and Jurin, M. (1987) Is dopamine related to immunologic changes in schizophrenic patients. Ann. N.Y. Acad. Sci. 496, 737-739. Kolyaskina, G.I., Sekirina, T.P., Zozulya, A.A., Kushner, S.G., Zuzulkovskaya, M.Ya. and Abramova, L.I. (1987) Some aspects of immunologic studies in schizophrenia. In: E. Kursak et al. (Ed%), Virus, Immunity and Mental Disorders. Plenum Press, New York, pp. 285-294. Ivany, P., Does, J., Schreuder, G.M.Th., D’Amaro, J. and Van Rood, J.J. (1983) A search for association of HLA antigens with paranoid schizophrenia. A9 appears as a possible marker. Tissue Antigens 22, 1866193. Lehmann-Facius, H. (1937) iiber die Liquordiagnose der Schizophrenien. Klin. Wochenschr. 16, 1646-1648. McAllister, C.G., Rapaport, M.H., Pickar, D., Podruchny,
215
T.A., Chris&on, G., Alphs, L.D. and Paul, MS. (1989) Increased number of CDS+ B lymphocytes in schizophrenic patients. Arch. Gen. Psychiatry 46, 890-894. Muller, N., Ackenmeil, M., Eckstein, R., Hofschuster, E. and Mempel, W. (1987) Reduced suppressor cell function in psychiatric patients. Ann. N.Y. Acad. Sci. 486, 6866689. Overall, J.E. and Gorham, D.R. (1962) The brief Psychiatric Rating Scale. Psychol. Rep. 10, 7999810. Sacchetti, E., Vita, A., Calzeroni, A., Invernizzi, G. and Cazzullo, C.L. (1987) Neuromorphological correlates of schizophrenic disorders: Focus on cerebral ventricular enlargement. In: C.L. Cazzullo et al. (Eds.), Etiopathogenetic Hypotheses of Schizophrenia. MTP Press, Lancaster, pp. 67793. Smeraldi, E., Bellodi, L., Sacchetti, E. and Cazzullo, C.L. (1976) The HLA system and the clinical response to treatment with chlorpromazine. Br. J. Psychiatry 129, 4866489. Synek, V. and Reuben, J.R. (1976) The ventricular brain ratio, using planimetric measurement of EMI scans. Br. J. Radiol. 49, 233-237. Vartanian, M.E., Kolyaskina, G.L., Lozovsky, D.V., Burbaeva,
G.S. and Ignatov, S.A. (1978) Aspects of humoral and cellular immunity in schizophrenia. Birth Defects 18, 339-365. Vaughan, W.T., Sullivan, J.C. and Elmadjian, F. (1949) Immunity and Schizophrenia. Psychosom. Med. 11, 3277333. Villemain, F., Chatenoud, L., Guillbert, E., Pelicier, Y. and Bach, J.F. (1987) Decreased production of interleukin-2 in schizophrenia. Ann. N.Y. Acad. Sci. 496, 6699675. Villemain, F., Chatenoud, L., Galinowski, A., Homo-Delarche, F., Ginestet, D., Loo, H., Zarifian, E. and Bach, J.F. (1989) Aberrant T cell-mediated immunity in untreated schizophrenic patients: Deficient interleukin-2 production. Am. J. Psychiatry 146, 609-615. Vita, A., Sacchetti, E., Calzeroni, A. and Cazzullo, C.L. (1988) Cortical atrophy in schizophrenia: Prevalence and associated features. Schizophr. Res. 1, 3299337. Zarrabi, M.N., Zucker, S., Miller, F., Derman, R.M., Romano, G.S., Martinet& Y.A. and Varma, A.O. (1979) Immunologic and coagulation disorders in chlorpromazine treated patients, Ann. Intern. Med. 91, 194-199.
SCHIZO
Research,
00123
Lymphocyte Relationship Cinzia ‘Institute
269
3 (1990) 269-215
subsets in schizophrenic
with clinical, neuromorphological
Masserini’, Peruzzi’, of Psychiatry,
disorders
and treatment
variables
Antonio Vita i, Roberto Basile’, Raffaele Morselli’, Paolo Boato’, and Carlo Lorenzo Cazzullo’ Luigi Pugnetti 2, Pasquale Ferrante’
Cinzia
Milan University School of Medicine, Policlinico,Pad. Guardia II, via F. Sforza 35, 20122 Milan, Italy, and ‘Don Gnocchi Multiple Sclerosis Center, via Capecelatro 60. Milan, Italy. (Received
4 August
1989, accepted
6 March
1990)
Peripheral blood lymphocyte subsets and serum immunoglobulin levels were assessed in 42 patients and 37 healthy controls. 24 patients were free from neuroleptic medication and 15 had never been treated with neuroleptics. 31 patients had a diagnosis of schizophrenia (DSM-III criteria) and 11 a diagnosis of a disorder of the schizophrenic spectrum. As compared to healthy subjects, the drug naive schizophrenic patient group showed an increase of T suppressor lymphocytes, while the drug-treated schizophrenic group showed an increase of T helper lymphocytes. The drug-treated schizophrenic group differed from the drug naive one relative to a decrease ofT suppressor lymphocytes. As compared to healthy subjects, the drug naive spectrum disorder patients showed an increase of absolute number of lymphocytes, while the drug treated spectrum group showed an increase of B lymphocytes. These findings did not correlate with any clinical or neuromorphological variables taken into account. Key words: Schizophrenic
disorder;
Lymphocyte
subset;
Immunoglobulin;
INTRODUCTION
Immune system abnormalities in schizophrenic patients have been described previously. Evidence for the presence of antibrain antibodies in serum (Lehmann-Facius, 1937), morphological changes of lymphocytes (Hirata-Hibi et al., 1982), abnormal serum and CSF immunoglobulin distribution (Zarrabi et al., 1979; DeLisi et al., 1984), decreased cellular in vitro function of lymphocytes (Vartanian et al., 1978) and of natural killer cells (DeLisi et al., 1983) and abnormal distribution of lymphocyte subsets in peripheral blood have been reported. With regard to the last problem, there is evidence of T suppressor cell decrease (Kolyaskina et al., 1987; Muller et al., 1987), T helper increase (Ganguli et al., 1987), T total, T suppressor and B lymphocyte increase (DeLisi and Wyatt, 1982) Correspondence sitaria, via Besta
0920-9964/90/$03.50
to: C. Masserini, 1, 20161 Milan,
0
Clinica Psichiatrica Italy.
1990 Elsevier
Science
Univer-
Publishers
Neuroleptic
side effect
and/or reports of no changes at all (Kaufman et al., 1987; Villemain et al., 1987, 1989). These contradictory results have been found in chronic schizophrenic patients with a long history of disease and of neuroleptic treatment. Even when the patients were described as free from drugs, the withdrawal period did not exceed 4 weeks. To obviate this, we employed a population of patients affected by schizophrenia or schizophrenic spectrum disorders, a third of whom had never been treated with neuroleptic drugs, and studied the lymphocyte subsets and the immunoglobulin distribution in peripheral blood, dividing the patient group according to the presence or absence of neuroleptic medication. PATIENTS
AND METHODS
Patients 42 patients, referred to the Institute of Psychiatry
of the University
B.V. (Biomedical
Division)
of Milan both as in- or out-
270
patients over a period of 2 years, were included in the study. None of the patients had clinical evidence of a medical or neurological illness that could be responsible for psychiatric symptoms, nor any history of alcohol or drug abuse, head trauma with loss of consciousness, neurologic disorder or electroconvulsive therapy. No recent infections or diseases associated with immunological abnormalities were reported. 31 patients (24 males and seven females) fulfilled the DSM-III criteria for the diagnosis of schizophrenia or schizophreniform disorder and 11 patients (eight males and three females) had a diagnosis of schizophrenic spectrum personality disorder such as schizotypal (seven patients) or of schizoid (four patients) disorder. The study included 12 patients with disorganized, eight undifferentiated, seven paranoid subtypes of schizophrenia and four schizophreniform disorders. 15 subjects (nine schizophrenics and six personality disorders) had never been treated with neuroleptic medication (drug naives), and nine subjects (seven schizophrenics and two personality disorders) had been free of neuroleptic medication for at least 3 months (six patients for 3, one for 4, one for 6 months and one for 2 years). 18 subjects (15 schizophrenics and three personality disorders) had been on neuroleptic medication for a period lasting from 1 month to 3 years, with daily doses ranging from 2 to 13 mg of haloperidol. 37 healthy volunteer blood donors (22 males and 15 females, mean age+ SD, 30 + 10 years), screened for a history of psychiatric and immune disorders, served as a control group for the immunological parameters. There were no significant differences among controls and patients, as far as age was concerned. Methods The clinical evaluation of patients included a psychiatric interview and the Brief Psychiatric Rating Scale (Overall and Gorham, 1962). The immunological evaluation of subjects consisted in the assessment of peripheral blood lymphocyte subsets and serum immunoglobulins. Lymphocytes were separated from whole blood by the Bdyum method (Boyurn, 1968). Subsets of T and B lymphocytes and interleukin-2 receptor positive cells were determined by the immunoperoxidase staining method described by Hofman (Hofman et al., 1982), on cytocentrifugated smears previously coated with antibodies avaible monoclonal commercially
(OKT3, 0KT4, OKT8 from Ortho and anti-leu12 and anti-IL-2 receptor from Becton Dickinson). The percentage of lymphocytes positive to staining, i.e., having receptors for each monoclonal antibody, was determined by microscopical examination: 600 cells for each smear were counted. T total, helper, suppressor, B lymphocytes and IL-2 receptor bearing cells per microliter were calculated by multiplying the percentage of positive cells by the total number of lymphocytes/p1 obtained by a total and differential white cell blood count. The values of IgA, IgG and IgM serum immunoglobulins were assessed by the radial immunodiffusion technique on Nor-partigen plates (Behringwerke, Marburg). 28 patients underwent computerized tomographic (CT) scan examination of the brain. CT scans were performed with a model 1010 EM1 scanner to assess the presence of neuromorphological alterations. Ventricular size was measured on the tomographic slice which showed the greatest extension of the lateral ventricles by a manual planimetric method (Sacchetti et al., 1987) and expressed as the ventricular brain ratio (VBR) (Synek and Reuben, 1976). Cortical atrophy was measured using a fourpoint visual scale, assessing increasing degrees of atrophy (0 = absent, 1 = uncertain/mild, 2 = moderate, 3=severe) (Vita et al., 1988). The variables taken into account for correlation with immunological findings were: (a) age at the time of immunological evaluation; (b) sex; (c) age at disease onset, defined as the age of the patient’s first psychiatric referral; (d) duration of illness; (e) family history of schizophrenia in first degree relatives, assessed by means of interviews with the patient and at least one of his relatives; (f) intellectual functioning, expressed as the patient’s total IQ and percent of deterioration on the Wechsler Adult Intelligence Scale (WAIS); (g) ventricular brain ratio; (h) presence or absence of cortical atrophy. Statistical analysis of the results was performed by analysis of variance (ANOVA) followed by the Student’s t test, using Bonferroni’s correction, and the x2 test, using Yates’ correction when necessary.
RESULTS
Schizophrenic patients and those with a schizophrenic spectrum disorder did not differ signifi-
271
cantly on immunological results. Nevertheless the results of schizophrenic patients and of those with a schizophrenic spectrum disorder were analyzed and presented separately. The lymphocyte subset counts in the schizophrenic group are listed in Table 1. The drug naive schizophrenic patient group showed an increase of the percentage of T suppressor (PC 0.05) lymphocytes as compared with the control values. This difference also was evident by comparing the absolute number of T suppressor (PC 0.05) lymphocytes/p1 of blood. The percentage of T suppressor lymphocytes was significantly (P~0.05) higher than that present in the drugtreated patient group. The drug-free schizophrenic patients showed lymphocytes/pl of blood to be significantly higher than in healthy subjects (PcO.05) and an increase, as TABLE
absolute number, of T total, T suppressor and B lymphocytes (P-C 0.05). The drug-treated schizophrenic patient group differed from normal controls relative to a T helper cell absolute number increase (P < 0.006) but the T helper percentage did not reach statistical significance (P~0.10). No difference was detected in the distribution of IL-2 receptor positive cells among the four groups of subjects. Table 2 shows the immunoglobulin values found in the serum in the schizophrenic group. No significant difference of immunoglobulin values could be found between patients and healthy subjects. We observed a trend for a decrease of IgM values in drug-treated schizophrenic patients vs. drug-free ones (P~0.10). The lymphocyte subset counts in the spectrum personality disorder group are listed in Table 3.
1
Lymphocyte subsets in schizophrenic patients and healthy subjects Values expressed as mean f SD. Statistical analysis by Student’s t test, using Bonferroni’s healthy subjects. *P
Lymphocytes/p1 T total (%) (cells/PI) T helper (%) (cells/PI) T suppressor (%) (cells/PI) B (%) (cells/pi) IL2-r positive (X) (cells/pi)
TABLE
1,958+62 64k9 1,214+406 43&6 833 &274 31*6 619+208 14+3 268kll2 5&2 90+47
correction.
Bold typed values P < 0.05 versus
Schizophrenic subjects Drug naive (n= 7)
Drug-free (n=9)
On drug (n= 15)
2,251*717 69klO 1,587f696 44+9 1,200f432 38*8* 867 f 351 17*3 368kll6 7&3 145k66
2,789 + 714 73&6 1,987 f 605 41+5 1,068* 175 35&9 983k417 16+4 503 + 136 4+2 104+48
2,319*748 65+12 1,591+581 49&7 1,200*441 29+5 691+215 16+8 352+ 174 6k3 138+71
2
Serum immunoglobulins in schizophrenic palients and healthy subjects Values expressed as mean k SD. Statistical analysis by Student’s f test, using Bonferroni’s versus drug-free patients. Healthy subjects
IgA (mg/lOO ml) IgG (mg/lOO ml) IgM (mg/lOO ml)
231 f87 1,348 k 336 209 f 80
correction.
PC 0.10 IgM of patients
Schizophrenic subjects Drug naive
Drug-free
On drug
202 f 56 1,300&349 227 k 87
240&-111 1,344*252 283 k98
206 k 75 1,266 f 305 162k37
on drugs
212 TABLE
3
Lymphocyte subsets in spectrum disorders and healthy subjects Values expressed as mean + SD. Statistical analysis by Student’s t test using Bonferroni’s correction. healthy subjects. *PC 0.02 versus drug free patients and **PC 0.05 versus patients on drug. Healthy subjects (n=37)
Lymphocytes/p1 T total (%) (cells/pi) T helper (%) (cellslpl) T suppressor (X) (cells/pi) B (%) (cells/$) IL-2-r positive (%) (cells/fil)
Schizophrenic
1,958*62 64&9 1,214&406 43k6 833 + 274 31 k6 619+208 14_+3 268k112 5+2 90&47
spectrum personality
disorder subjects
Drug naive (n=6)
Drug-free (n=2)
On drug (n=4)
3,359 & 623 71&6 2,387*467*.** 46kl 1,524+211* 37+12 1,263 * 540 16&4 531+198 6+3 193+115
2,268*314 66k2 1,498*209 41+6 929k272 37&l 837&86 19*1 433 +93 7&4 165k118
2,838 + 1,255 63&6 1,687 + 565 43k2 1,258 + 593 29&9 865+ 147 22+9 801& 552 5*1 134*20
The drug naive spectrum patient group showed the number of lymphocytes/p1 of blood was higher than in healthy subjects (PC 0.005) while an increase of absolute number of T total, T helper and T suppressor lymphocytes was also present (PC 0.005). The drug naive patients also had an increase of the absolute number of T total and T helper lymphocytes vs. drug-free spectrum patients and of T total lymphocytes vs. drug-treated patients (PC 0.05). The drug-free spectrum patient group did not show any difference from normal control values, but it included two subjects only. The drug-treated spectrum patient group differed from normal controls relative to B lymphocyte increases, either as a percentage (P
TABLE
Bold typed values PC 0.05 versus
in the serum in the spectrum personality disorder group. No significant difference in immunoglobulin values could be found between patients and healthy subjects. We then looked into whether there were any differences, for sex, age, age of onset, duration of illness, family history, pathological findings on CT scans, BPRS and WAIS scores and obstetric complications at birth which could influence the immunological results. We did not find any significant differences among the groups. Finally we analyzed whether there was some association between immunological parameters and clinical or neuromorphological characteristics by comparing patients with immunological values above and below the mean of controls values plus 2 standard deviations: no difference could be detected. The clinical or neuromorphological charac-
4
Serum immunoglobulins in spectrum disorder subjects and healthy subjects Values expressed as mean k SD. Statistical analysis by Student’s t test, using Bonferroni’scorrection. Healthy subjects
IgA (mg/lOO ml) IgG (mg/lOO ml) IgM (mg/lOO ml)
231+87 1,348+336 209 & 80
Spectrum disorder subjects Drug naive
Drug-free
On drug
230+74 1,334 * 366 228 * 94
253 k 14 1,400+ 141 266+18
254 k 69 1,324&403 197+_66
213
teristics of the patients 5 and 6.
groups
are shown in Tables
DISCUSSION
Several immunological changes have been described in schizophrenic patients, though it is often quite hard to compare the results of different clinical populations. To the best of our knowledge, TABLE
5
Characteristics of schizophrenic patients Drug naive II=19
Age
Sex (M:F) Age of onset Illness length Family history (FH+:FH-) BPRS score WAIS IQ % IQ deterioration VBR Cortical atrophy (absent:present) Obstetric complications (absent:present)
TABLE
Dtugfree n=7
23.3k4.9 8:l 21.3+4.2 1.97& 1.7 217
22 k 2.9 7:o 18+3 3.7k2.6 5:2
57.8*20 101*18 8.4k8.5 3.2k 1.4 6:l
55.2*18 99* 14 10.5+ 18 4.2* 1.5 4:2
7:2
6:l
Dtugtreated n=15 23.7*4 9:6 19.4k3.7 4.2+3 4:ll 59.1*18 99+ 15 15*14 51+2.8 714 12:3
6
Chatacretistics of spectrum personality disorder patients
Age Sex (M:F) Age of onset Illness length Family history (FH+:FH-) BPRS score WAIS IQ % IQ deterioration VBR Cortical atrophy (absent:present) Obstetric complications (absent:present)
Drug naive n=6
Dtugfree n=2
Dtugtreated n=3
23.3 k4.6 3:3 19.6& 5.3 2.7* 1.8 1:5
22.5i2.1 0:2 19i2.8 3.8i4.5 1:l
23.li5.1 3:o 22+4 1.4* 1.5 1:2
45 k 2.7 102+7 21.7k8.5 7.8* 1.8 2:o 5:0
36 * 3.5 81 k4 0 l:o
66+5 98+8 1.3 f 7.5 2.2+0.1 2:o
1:l
3:o
our study presents the largest number of drug-free schizophrenic patients tested for lymphocyte subsets, with the exception of the work by Villemain (Villemain et al., 1989). 15 patients had never been treated with neuroleptic drugs and the other drugfree patients had a longer withdrawal period than those referred to in literature. The mean age of the patients and the duration of illness were lower than those of the patients referred to in previous research (DeLisi and Wyatt, 1982; Ganguli et al., 1987; Kaufman et al., 1987; Muller et al., 1987; Villemain et al., 1989). None of the patients had a history of long-term hospitalization and patients with schizophrenic spectrum disorders were also considered. We found an increase of T suppressor lymphocytes, either as a percentage or as an absolute number of cells/p1 of blood, in the drug-naive schizophrenic group as compared with healthy subjects. Schizophrenic patients on drugs differed from the healthy controls relative to higher T helper lymphocytes absolute number. Similar results were obtained by DeLisi (DeLisi and Wyatt, 1982) in a group of 38 chronic schizophrenic inpatients, seven of whom had been free from neuroleptics for at least 1 month. A difference between drug naive and drugtreated schizophrenic patients is present in the distribution of lymphocytes bearing the receptor for the OKT8 monoclonal antibody, which detects the T subpopulation with suppressor/cytotoxic function. With regard to the latter, our findings are similar to those of Kolyaskina et al. (1987), who reported a significant decrease of the T suppressor subpopulation in 53 chronic schizophrenic patients diagnosed as suffering from a malignant form of schizophrenia according to the ICD9 and free from drug medication for 3 weeks. In agreement with Kolyaskina’s hypothesis that psychotropic drugs could be responsible for immunological changes, we also found a trend towards lower IgM value in drug-treated schizophrenic patients versus drug-free ones. A reduction of serum IgA, IgM and IgG had also been reported by DeLisi et al. (1981) studying 3.5 chronic schizophrenic inpatients. A difference in the immunological assessment of drug naive, drug-free and drug-treated schizophrenic patients has not clearly emerged before. It is our guess that different immunological stages could be present during the longitudinal course of schizophrenia. We can sup-
274
pose that an initial T suppressor lymphocyte increase, perhaps caused by environmental (viruses) or genetic factors, is followed by an almost global activation of lymphocyte subsets, which is then replaced by T helper lymphocyte activation. This hypothesis may explain the contradictory reports of previous findings on lymphocyte subsets. Nevertheless neuroleptic drugs either directly, or by modulation of neurotransmitter functions, might be responsible for the immunological changes present in some drug-treated schizophrenic patients (Ferguson et al., 1978; Ganguli et al., 1987; Kereptic et al., 1987), and for masking the immunological picture preceding pharmacological treatment, such as, for example, an elevation of T suppressor subset. The meaning of the T suppressor increase might be linked with a previous or recent infection by some viruses, like cytomegalovirus, which may produce T suppressor elevation lasting for 10 or more months after the onset of clinical infection (Carney et al., 1981). On the other hand, a T suppressor increase, not confirmed by in vitro functionality tests, could mean a decreased activity of T suppressor/cytotoxic cells, as observed by Muller et al. (1987) in a population of previously drug-treated schizophrenic patients. Finally a T suppression increase could also express dopamine immunosuppressive activity in those schizophrenic patients with a dopaminergic hyperfunction. We can point out that specific immunogenetic characteristics, such as those found in several studies to be associated with schizophrenia (Smeraldi et al., 1976; Ivany et al., 1983) may influence the immunological reactivity of schizophrenic patients. Although the number of subjects is too small to draw some conclusions, we note the increase of B lymphocytes in the drug-treated spectrum patients. A subpopulation of B lymphocytes that express the CD5 (Leu-1) surface antigen has been reported to be elevated in 30% of drug-treated schizophrenic patients by McAllister et al. (1989). This subset of lymphocytes also is increased in patients affected by diseases of known autoimmune pathogenesis such as rheumatoid arthritis, Sjiigren’s syndrome and multiple sclerosis. In conclusion we think it could be useful to continue immunological studies in early stages of the disease in order to further determine the significance of these findings.
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