Expression of anti-cardiolipin antibodies and inflammatory associated factors in patients with schizophrenia

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Expression of anti-cardiolipin antibodies and in

ﬂammatory associated factors in

patients with schizophrenia

Sheng-Huang Chang

a,b

, Szu-Yi Chiang

c

, Chun-Ching Chiu

d,e

, Chun-Chou Tsai

e

, Huei-Huang Tsai

b

,

Chih-Yang Huang

f,g,h,1

, Tsai-Ching Hsu

e,i,j,1

, Bor-Show Tzang

a,j,k,1,

⁎

a

Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan

b

Tsao-Tun Psychiatric Center, Department of Health, Executive Yuan, Taiwan

c

Department of Health, Executive Yuan, Hua-Lien Hospital, Hua-Lien, Taiwan

d

Department of Neurology and Department of Medical Intensive Care Unit, Chunghua Christian Hospital, Chunghua, Taiwan

e_{Institute of Immunology, Chung Shan Medical University, Taichung, Taiwan} f

Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan

g

Institute of Basic Medical Science, China Medical University, Taichung, Taiwan

h

Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan

i

Division of Allergy, Immunology and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan

j

Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan

k

Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan

a b s t r a c t

a r t i c l e i n f o

Article history: Received 20 March 2009

Received in revised form 2 April 2010 Accepted 26 April 2010 Available online xxxx Keywords: Schizophrenic (SZ) Interleukin (IL) TLR (toll-like receptor) Autoimmune disorder

Numerous evidences have implicated a connection between schizophrenia and autoimmune disorders. However, the precise relationship and underlying mechanism are still obscure. To further identify the association between autoimmune disorders and schizophrenia, the mRNA expressions of various cytokines and Toll-like receptors (TLRs) in monocytes are examined by using RT-PCR. Additionally, ELISA and zymography were performed to determine the anti-cardiolipin antibody (aCL) and MMP9 activity in serum form schizophrenic patients. Notably, significantly increased interleukin (IL)-6 and IL-10 mRNA were observed in schizophrenic patients, whereas the significant reductions of TLR-3 and TLR-5 mRNA were detected. Moreover, significantly increased aCL antibody and higher frequency of positive-MMP9 activity were detected in serum from patients with schizophrenia. Meanwhile, no significant association was found between each of the medication and aCL activity. These findings demonstrated the autoimmune related phenomena in schizophrenic patients and further suggested a connection between schizophrenia and autoimmune disorders.

1. Introduction

Schizophrenia (SZ) is an exhausting sickness. Along with the

behavioral and mental deterioration observed in patients with

schizo-phrenia, many neuro-imaging and postmortem

ﬁndings reﬂect

signif-icant neuro-degenerative process (

Lieberman, 1999

). Recently, a study

indicated that history of any autoimmune disease was associated with a

45% increase in risk for schizophrenia (

Eaton et al., 2006

). Although the

mechanism underlying is still obscure, increasing evidences have been

associated autoimmune disorders with schizophrenia.

Previously, schizophrenia has been proposed to be an autoimmune

disorder in which antibodies are elevated against speci

ﬁc self-antigen in

brain (

Bergen et al., 1980; Heath et al., 1989; Henneberg et al., 1994

). In a

clinical study, anti-brain antibodies were detected in sera and

cerebro-spinal

ﬂuid from 26 out of 54 schizophrenic patients but in none of 27

healthy controls (

Pandey et al., 1981

). Another study indicated that

increased CD5 positive B-lymphocytes in schizophrenic patients were

observed and recognized to play a role in the pathogenesis of

schizophrenia (

McAllister et al., 1989

). Moreover, autoantibodies against

platelet or nicotinic acetylcholine receptors in patients with

schizophre-nia were also reported (

Shinitzky et al., 1991; Mukherjee et al., 1994

).

Signi

ﬁcant increases of various natural autoantibodies, including

antinuclear, anti-double-stranded DNA, anti-Sm, and

anti-single-strand-ed DNA autoantibodies, were signi

ﬁcantly more frequent in

schizo-phrenic patients than in normal subjects (

Sirota et al., 1993

). These

reports did suggest the association between schizophrenic and

autoim-mune disorders.

Evidences have indicated that IL-6 is a key mediator of various

autoimmune diseases such as SLE and RA (

Cronstein, 2007

) and targeting

IL-6 might be a promising therapy of autoimmune diseases (

Fujimoto

et al., 2008

). Apparently, similar phenomena have been reported in

schizophrenia patients. Various cytokines are also recognized to be

Psychiatry Research xxx (2010) xxx–xxx

⁎ Corresponding author. Department of Biochemistry, Institute of Biochemistry and Biotechnology, Chung Shan Medical University, No.110, Sec.1, Jianguo N. Rd., Taichung 402, Taiwan. Tel.: +886 4 23248168; fax: +886 4 23248175.

E-mail address:[email protected](B.-S. Tzang).

1_{These authors contributed equally to this work.}

PSY-06487; No of Pages 6

Contents lists available at

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Psychiatry Research

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p syc h r e s

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involved in the development of schizophrenia. Abnormal cytokine pro

ﬁle

and increasing activated CD4±and CD16±natural killer cells were

observed in schizophrenic patients (

Theodoropoulou et al., 2001

).

Another study indicated the elevated level of IL-6 in schizophrenia

(

Naudin et al., 1996

) as well as the increased levels of IL-2 and IL-8 in

serum from neuroleptic-free schizophrenia was reported in other studies

(

Ganguli et al., 1994; Zhang et al., 2002

). These evidences suggested that

IL-6 play a role between autoimmune diseases and schezophrenia.

Toll-like receptors (TLRs) have been known to play important roles in

innate immunity and associated with autoimmune diseases (

Abdollahi-Roodsaz et al., 2007; Barrat and Coffman, 2008

). Genetic variations in

TLR5 and TLR9 have been associated with the disease activity of

autoimmune disease such as SLE (

Hawn et al., 2005; De Jager et al.,

2006

). A recent study demonstrated that TLR-7 is speci

ﬁcally required

for the production of RNA-reactive autoantibodies and the development

of glomerulonephritis in pristane-induced murine lupus mice (

Savarese

et al., 2008

). Another study indicated that TLR9 knockout mice revealed

exacerbated autoimmune disorders including activated lymphocytes,

plasmacytoid dentric cells, increased serum IgG and IFN-alpha whereas

TLR7 knock out mice revealed ameliorated disease and failed to generate

antibody against Smith (Sm) Ag (

Christensen et al., 2006

). However, the

relationships between autoantibody and innate immunity such as

Toll-like receptors (TLRs) in patients with schizophrenia are still unknown. In

the current study, we

ﬁrstly reported the decreased mRNA expression of

TLR-3 and TLR5 in monocytes from schizophrenic patients, which is

associated with the increased anti-cardiolipin antibodies and mRNA

expression of IL-6 and IL-10, and suggested a connection between

schizophrenia and autoimmune disorders.

2. Materials and Methods 2.1. Patients and monocytes isolation

Twenty-two healthy individuals and forty-six volunteer in-patients from a psychiatric unit participated in this study, approved by Institutional Review Board (IRB), Tsao-Tun Psychiatric Center, Department of Health, Executive Yuan, Taiwan. Patients were recruited if they experienced psychosis (hallucinations or delusions) during or just prior to admission. All patients willing to volunteer were accepted without exclusion and diagnosis was made by a single board certiﬁed psychiatrist (Huei-Huang Tsai). The majority of patients were on neuroleptic medication. The monotyes were isolated from the peripheral blood monocyte cells (PBMC) of healthy and schizophrenic subjects by using Histopaque-1077 (Sigma Chemicals, Poole, Dorset, UK) according to the manufacture's instructions.

2.2. RT-PCR

All studies were carried out in a designated PCR-clean area. RNA was extracted from isolated cells using a Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Total RNA was isolated from the monocytes of healthy individuals and schizophrenic subjects. RNA samples were resuspended in diethyl pyrocarbonate (DEPC)-treated water, quantified, and then stored at−80 °C until used. RNA concentration and purity were determined by a spectrophotometer by calculating the ratio of optical density at wavelengths of 260 and 280 nm. Thefirst-strand of cDNA for RT– PCR was synthesized from the total RNA (2 µg) using the Promega RT–PCR system (Promega, Madison, Wisconsin, USA). Specific primer sets are listed inTable 1. The amplification was performed in a 50 µl reaction volume containing 1×reaction buffer (Promega), 1·5 µM of MgCl2, 200 µM of dNTPs, 1 µM of each primer and 2·5 units of Taq DNA polymerase (Promega, Madison, WI, USA) using a Perkin-Elmer Gene Amp PCR system 2400. Each cycle consisted of denaturation at 95 °C for 1 min, annealing at 60 °C for 45 s and amplification at 72 °C for 45 s. The RT–PCR-derived DNA fragments, obtained by 25 PCR cycles, were subjected to electrophoresis in a 4% acrylamide gel. Following staining with ethidium bromide, the gels were photographed. The specific RNA level of every sample was expressed as the product's intensity and the cDNA encoding glyceraldehyde-3-phosphate -dehydrogenase (GAPDH) was amplified as internal control.

2.3. ELISA

The serum samples were collected from healthy and schizophrenic subjects. The antibodies against cardiolipin were measured with commercial QUANTA Lite ACA IgG III enzyme-linked immunoabsorbent assay kits (INOVA Diagnostic Inc., San Diego, CA, USA). Anti-human IgG-HRP conjugate was used as the secondary antibody and the color developed with chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB). The enzymatic reaction is directly proportional to the amount of antibodies present in the sample.

2.4. Gel Zymography

MMP-9 and MMP-2 activities were analyzed by gelatin zymography. Ten microliters of diluted serum was separated on an 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel polymerized with 1 mg/ml gelatin. Gels were washed once for 30 mins in 2.5% Triton X-100 to remove the SDS and then soaked in the reaction buffer containing 50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl2, and 0.02% (w/v) Brij 35 (Sigma, St. Louis, MO; pH 7.5) for 30 mins. The reaction buffer was changed to a fresh one, and the gels were incubated at 37 °C for 24 hrs. Gelatinolytic activity was visualized by staining the gels with 0.5% Coomassie brillant blue and quantiﬁed by densitometry (Appraise; Beckman-Coulter, Brea, CA).

2.5. Statistical analysis

All the statistical analyses were performed using SPSS 10.0 software (SPSS Inc, Chicago, IL). Three independent experiments were repeated. Statistical analyses were performed using the one-way ANOVA or Chi-square. Pb0.05 was considered statistically signiﬁcant.

3. Results

3.1. Up-regulation of IL-6 and IL-10 mRNA in monocytes from

schizophrenic patients

Forty-six volunteer patients with schizophrena and 22 healthy

subjects agreed to donate peripheral blood and the analyses.

Schizo-phrenic subject age at time of enrollment ranged from 24 to 61 years

(average 40± 10.42). The gender of the participants was all male and

the racial background was 100% Taiwanese. The age of 22 healthy

subjects was range from 21 to 55 (average 33± 5.88). No signi

ﬁcant

differences of various biochemistry and blood-cell parameters between

schizophrenic and healthy subjects were observed (

Table 2

). To

investigate the mRNA expression of cytokines in monocytes from

healthy and schizophrenic subjects, RT-PCR was performed to

Table 1

Primer pairs used for RT-PCR.

Gene type Sequence Size IL-12 F 5’-CAGACCCAGGAATGTTCCCA-3’ 310 bp R 5’-TCTTGAACTCCACCTGGTAC-3’ IL-10 F 5’-GGACAACATACTGCTAACCGAC-3’ 256 bp R 5’-AAAATCACTCTTCACCTGCTCC-3’ IL-13 F 5’-ATGCATCCGCTCCTCAATCC-3’ 286 bp R 5’-TCTTCTCGATGGCACTGCAG-3’ IFN-γ F 5’-CTTGGCTGTTACTGCCAGGA-3’ 240 bp R 5’-TTCCTTGATGGTCTCCACAC-3’ IL-6 F 5’-ATGAACTCCTTCTCCACAAGCGC-3’ 454 bp R 5’-GGATCAGGACTTTTGTACTCATC-3’ IL-5 F 5’-GCTAGCTCTTGGAGCTGCCT-3’ 370 bp R 5’-CTATTATCCACTCGGTGTTC-3’ TGF-ß F 5’-CTGCTACCGCTGCTGTGGCT-3’ 211 bp R 5’-GGGTGCTGTTGTACAGGGCG-3’ TLR-4 F 5’-ATGATGTCTGCCTCGCGCCT-3’ 349 bp R 5’-CCCAGGGCTAAACTCTGGAT-3’ TLR-1 F 5’-ATGACTAGCATCTTCCATTTTGCC-3’ 408 bp R 5’-GCCAAACTCTTTGCATATAGGC-3’ TLR-3 F 5’-ATGAGACAGACTTTGCCTTG-3’ 279 bp R 5’-TCTGGCACAATTCTGGCTCC-3’ TLR-5 F 5’-ATGGGAGACCACCTGGACCTTC-3’ 319 bp R 5’-TCTTACTACTTCCCAGGTCCAAG-3’ TLR-2 F 5’-ATGCCACATACTTTGTGGATGG-3’ 221 bp R 5’-CCTCTGTAGGTCACTGTTGCTA-3’ TLR-9 F 5’-ATGGGTTTCTGCCGCAGCGC-3’ 200 bp R 5’-CTGGTGACATTGCCACGGGG-3’ CD14 F 5’-CGTCCTGCTTGTTGCTGCTG-3’ 309 bp R 5’-CCAGTAGCTGAGCAGGAACC-3’ TLR-6 F 5’-ATGTCTCAGAACTACATCGCTG-3’ 240 bp R 5’-GATGGGCAGGGCCTTGAAATCAT-3’ GAPDH F 5’-ATGGGGAAGGTGAAGGTCGG-3’ 310 bp R 5’-TGGTGAAGACGCCAGTGGAC-3’

IL indicates interleukin. IFN indicates interferon. TGF indicates tumor growth factor. TLR indicates toll-like receptor. CD indicates cluster of differentiation. GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase. F indicates forward primer. R indicates reverse primer.

2 S.-H. Chang et al. / Psychiatry Research xxx (2010) xxx–xxx

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determine the mRNA expression of IL-5, IL-6, IL-10, IL-12, IL-13, IFN-

γ,

and TGF-

β (

Fig. 1

A). Signi

ﬁcant difference in IL-6 and IL-10 mRNA

expression between healthy and schizophrenic subjects was observed

(

Table 3

). Five out of 22 healthy subjects (22.7%) express IL-6 mRNA

whereas 43 out of 46 (93.5%) schizophrenic subjects express IL-6 mRNA.

Ten out of 22 healthy subjects (45.5%) express IL-10 mRNA whereas 41

out of 46 (89.1%) schizophrenic subjects express IL-10 mRNA. No

signi

ﬁcant difference in IL-5, IL-12, IL-13, IFN-γ, and TGF-β mRNA

expression was detected.

3.2. Down-regulation of TLR-3 and TLR-5 mRNA in monocytes from

schizophrenic patients

To study the association between innate immunity and

schizo-phrenic, expression of various TLR mRNA were determined.

Fig. 1

B

revealed the RT-PCR results of TLR-1, TLR2, TLR-3, TLR4, TLR-5, TLR-6,

TLR-9, and CD14. Signi

ﬁcant difference in TLR-3 and TLR-5 mRNA

expression between healthy and schizophrenic subjects was observed

(

Table 4

). Sixteen out of 22 healthy subjects (72.7%) express TLR-3

mRNA whereas only 15 out of 46 (32.6%) schizophrenic subjects express

TLR-3 mRNA. All 22 healthy subjects (100%) express TLR-5 mRNA

whereas 3 out of 46 (6.5%) schizophrenic subjects express TLR-5 mRNA.

No signi

ﬁcant difference in TLR-1, TLR2, TLR4, TLR-6, TLR-9, and CD14

mRNA expression was detected.

3.3. Increased binding activity of anti-cardiolipin antibody and MMP9

activity in serum from schizophrenic patients

To further verify the connection between schizophrenia and

autoimmunity, aCL antibody was detected in serum from healthy and

schizophrenic patients. ELISA was performed to detect the binding

activity of antibody against cardiolipin and the result was shown in

Fig. 2

A. Notably, binding activity of aCL antibody was signi

ﬁcantly higher

in serum of schizophrenic patients as compared to those from healthy

subjects (

Fig. 2

A). Moreover, MMP9 activity was also detected, which is

known as an in

ﬂammatory indicator and has been associated with

autoimmune diseases.

Fig. 2

B shows the representative results of

zymography. Signi

ﬁcantly higher frequency of positive MMP9-activity

was detected in serum from patients with schizophrenia as compared to

those from healthy subjects (

Table 5

). Additionally, to verify whether

the medications have in

ﬂuences on cardiolipin autoantibody

develop-ment in patients with schizophrenia, statistic assay was performed. All

patients were taking at least one and some were taking several

anti-psychotic medications (

Table 6

). Notably, no signi

ﬁcant associations

between any of these neuroleptic medications and aCL antibody

development were found.

Table 2

Characteristics of healthy controls and schizophrenic (SZ) subjects.

SZ subjects (N = 46) Normal subjects (N = 22) Age 40 ± 10.42 33 ± 5.88 Gender Male 46 22 Female 0 0 Age ofﬁrst episode 30 ± 9.81 -Years of illness 10 ± 6.30 -CGI score 6 1 -5 4 -4 7 -3 27 -2 7 -Glucose 89.2 ± 15.70 88.6 ± 11.2 Bilirubin 10.3 ± 3.49 11.3 ± 2.7 Uric Acid 5.5 ± 1.75 6.4 ± 1.1 Creatinine 0.9 ± 0.17 0.9 ± 0.1 Aspartate aminotransferase 23.0 ± 7.94 21.0 ± 4.1 Alanine aminotransferase 24.0 ± 14.27 22.7 ± 7.4 Cholesterol 165.9 ± 34.18 176.6 ± 20.2 Triacylglycerol 112.3 ± 75.98 114.2 ± 43.2 Bili-D 0.1 ± 0.05 0.1 ± 0.1 Bili-T 0.7 ± 0.36 1.0 ± 0.2 r-Glutamyl Transpeptidase 24.1 ± 23.10 22.0 ± 11.1 Alkaline phosphatase 65.4 ± 22.38 58.6 ± 15.1 Red blood cell 465.4 ± 45.0 483.2 ± 30.2 Hemoglobulin 14.3 ± 1.2 14.8 ± 0.74 Ht 48.0 ± 9.2 42.8 ± 2.7 White blood cell 7287.3 ± 2230.7 5909 ± 1100 Platelet 230.1 ± 57.4 250.8 ± 44.3 Segmental cell 58.0 ± 11.9 55.9 ± 6.6 Lymphocyte 31.1 ± 11.0 33.2 ± 5.7

CGI indicates clinical global impression. Fig. 1. Expression of various cytokines and Toll-like receptors. The mRNA of monocytes from schizophrenic and normal subjects were isolated and extracted for analysis. The mRNA expressions of (A) cytokines and (B) TLRs were detected by RT-PCR. P, SZ, and Normal indicate positive, schizophrenia, and healthy controls, respectively. Three independent experiments were performed and similar results were observed.

Table 3

Presence of different cytokine mRNA in control individuals and schizophrenic patients. Gene type Control (n = 22) Patients (n = 46) χ2

df P-valuea IL-12 0/22 (0) 0/46 (0) 0 1 -IL-10 10/22 (45.5) 41/46 (89.1) 12.8992 1 b0.001⁎ INF-γ 21/22 (95.5) 45/46 (97.8) 0.05275 1 N0.05 IL-6 5/22 (22.7) 43/46 (93.5) 32.56 1 b0.001⁎ IL-5 0/22 (0) 0/46 (0) - 1 -IL-13 19/22 (86.4) 42/46 (91.3) 0.04 1 N0.05 TGF-β 21/22 (95.5) 45/46 (97.8) 0.05275 1 N0.05 Numbers in parentheses are percentages.

χ2_{indicates the Chi-Square value.}

df indicates the degree of freedom.

a

Chi-Square was performed to determine the P-value. ⁎ indicates statistically signiﬁcant deviation.

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4. Discussion

Schizophrenia has been associated with a variety of immune

abnormalities than heretofore suspected. Irregular levels or activity of

lymphocytes, cytokines, or cytokines receptors, or symptoms of

autoimmune disease were observed in patients with schizophrenia,

which may play a role in the etiology of schizophrenia (

Fudenberg et al.,

1983; Ganguli et al., 1989; Smith, 1991; Gilmore and Jarskog, 1997;

Eaton et al., 2006

). However, the underlying psychopathologic

mechanisms of schizophrenia and the underlying mechanism in

immune regulation are still obscure. In current study, we reported the

increased mRNA expression of IL-6 and IL-10, MMP-9 activity, and

reduced TLR-3 and TLR-5 mRNA in monocytes from schizophrenic

patients and suggested a connection with the increased activity of

anti-cardiolipin antibody.

Cytokines are known to play crucial roles in many physiological and

pathological processes including schizophrenia and autoimmune

dis-orders. Signi

ﬁcantly increased serum levels of IL-6 and IL-8 were also

founded in patients with schizophrenia (

Naudin et al., 1996; Ganguli

et al., 1994; Zhang et al., 2002

). Meanwhile, increased levels of IL-4, IL-6,

and IL-10 were reported in CSF of schizophrenic patients (

Zhang et al.,

2002; Muller and Schwarz, 2006

) that has been demonstrated to be

associated with the pathological development of autoimmune diseases

(

Cronstein, 2007

). In current study, similar results were observed that

elevated expression of IL-6 and IL-10 mRNA was observed in monocytes

from schizophrenic patients. Moreover, signi

ﬁcantly higher binding

activity of aCL antibody and frequency of MMP9-positive was founded in

schizophrenic patients, which have been known as important indicators

for autoimmune diseases such as SLE (

Sammaritano and Gharavi, 1992;

Ainiala et al., 2004; Robak et al., 2006; Hsu et al., 2008

). Indeed, these

ﬁndings implied an association between schizophrenia and

autoim-mune disorders and may provide a more credible detection in patients

with schizophrenia although the precise mechanism underlying still

needs further investigations.

Recently, toll-like receptors (TLRs) are known to play crucial roles in

innate immunity and the development of autoimmune disorders (

Hawn

et al., 2005; Abdollahi-Roodsaz et al., 2007; Barrat and Coffman, 2008

). A

recent study indicated that TLR-7 is important for the production of

Table 4

Presence of different TLR mRNA in control individuals and schizophrenic subjects. Gene type Control (n = 22) Patients (n = 46) χ2 _df _P-valuea

TLR4 21/22 (95.5) 46/46 (100) 0.1435 1 N0.05 TLR1 0/22 (0) 0/46 (0) - 1 -TLR3 16/22 (72.7) 15/46 (32.6) 8.1068 1 b0.001⁎ TLR5 22/22 (100) 3/46 (6.5) 51.98877 1 b0.001⁎ TLR2 22/22 (100) 44/46 (95.6) 0.050856 1 N0.05 TLR9 22/22 (100) 46/46 (100) - 1 -CD14 22/22 (100) 46/46 (100) - 1 -TLR6 22/22 (100) 46/46 (100) - 1 -Numbers in parentheses are percentages.

χ2_{indicates the Chi-Square value.}

df indicates the degree of freedom.

a

Chi-Square was performed to determine the P-value. ⁎ indicates statistically signiﬁcant deviation.

Fig. 2. Detection of anti-cardiolipin antibody and MMP9/2 activity. The serum from schizophrenic and normal subjects was collected and (A) the reactivity of anti-cardiolipin antibody was detected. (B) Theﬁgure shows representative results of MMP9/2 zymography. M, SZ, and Normal indicate standard MMP9/2 marker, schizophrenia, and healthy controls, respectively. Three repeated experiments were performed and similar results were obtained.

Table 5

Frequency of MMP9 activity in serum of control individuals and schizophrenic patients. Control (n = 22) Patients (n = 46) P-valuea

MMP9 7/22 (31.2) 39/46 (84.8) b0.001⁎ Numbers in parentheses are percentages.

a_{Chi-Square was performed to determine the P-value.}

(*) indicates statistically signiﬁcant deviation.

Table 6

Neuroleptic medications.

Medication Number of subject Serum aCL positive P Anxiedin 4 2 0.507 Apa-Risdol 14 4 0.966 Apo-Divalproex 6 0 0.164 Apo-Haloperidol 5 1 0.715 Ativan 8 2 0.831 Betamac 3 1 0.905 Biperiden 2 0 0.438 Cebotval 5 2 0.687 Clopine 22 4 0.363 Clozaril 8 5 0.123 Dogmatyl 3 0 0.338 Domilium 3 0 0.338 Eszo 16 4 0.737 Etumine 3 1 0.905 Euglucon 2 0 0.438 Eurodin 2 1 0.648 Fluanxol 2 0 0.438 Gendergin SR 2 1 0.348 Haldol 2 1 0.648 Halin 2 2 0.175 Inderal 17 4 0.586 Lodopin 5 0 0.208 Luvox 10 2 0.585 Mezapin 6 1 0.577 Rivotril 6 1 0.577 Seroquel 3 0 0.338 Silence 3 1 0.905 Solian 3 0 0.338 Surin 2 2 0.175 Switane 14 8 0.054 Tegretol 2 0 0.438 Uspen 3 1 0.905 All patients in the study received pharmaceutical administration at the time of serum collection. No signiﬁcant association was found between each of the medication and aCL activity.

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RNA-reactive autoantibodies and the development of

glomerulonephri-tis (

Savarese et al., 2008

). In a gene-knocked lupus-prone mice, the

de

_{ﬁciency of TLR9 gene led to exacerbated autoimmune disorders such}

as activated lymphocytes, plasmacytoid dentric cells, increased serum

IgG and IFN-alpha (

Christensen et al., 2006

). Although irreverent innate

immunity has been reported in patients with schizophrenic such as

increasing activated CD4+/CD16+ natural killer cells in schizophrenic,

no study reported the pro

ﬁles of TLRs in patients with schizophrenic

(

Theodoropoulou et al., 2001

). However, no study of TLRs expression in

schizophrenic patients was reported. In current study, we

ﬁrstly

reported the reduced expression of TLR-3 and TLR-5 mRNA in

monocytes from patients with schizophrenia as well as the signi

ﬁcantly

increased binding activity of anti-cardiolipin antibody that has been

known to play crucial roles in various autoimmune disorders. These

ﬁndings may provide a clue in understanding the association of

abnormal innate immunity in schizophrenic patients.

Several observational studies have suggested a link between the

use of antipsychotics drugs and the phenomena of autoimmune

disorders, such as increased thrombosis and aCL antibodies. Indeed,

clozapine have been reported to induce venous thromboembolism in

psychiatric patients (

Brenner and Metz, 1995; Hägg et al., 2000

).

Recently, the association between conventional antipsychotics and

venous thrombosis has been strengthened by an epidemiological

research (

Hägg and Spigset, 2002; Liperoti et al., 2005

). Additionally,

the correlation between the treatment of neuroleptic drugs and serum

level of aCL, especially clozapine, has also been reported (

Knudsen et

al., 2000; Shen et al., 2009

). A higher serum clozapine level is

associated with an increased level of aCL antibodies in schizophrenia

patients (

Shen et al., 2009

). Although these epidemiological data

support an association among the treatment of neuroleptic drugs and

increased thrombosis and aCL antibodies in psychiatric patients, the

biological mechanisms involved in the pathogenesis are still obscure.

In the current study, no signi

ﬁcant correlations between any of the

neuroleptic medications and increased aCL antibody level were found,

including clozapine. Relatively, predominant autoimmune

phenom-ena including increased MMP-9 activity and mRNA of IL-6 and IL-10

were reported in this study. Therefore, these

ﬁndings demonstrated

the autoimmune related phenomena in schizophrenic patients and

suggested further connections between schizophrenia and

autoim-mune disorders. Although the in

ﬂuence of medications in psychiatric

patients cannot be neglected and needed further investigations, this

study did provide an alternative understanding for schizophrenic

patients in developing the autoimmune disorders.

Contributors

Authors TCH conceived this study, drafted the manuscript, and

performed the performed statistical analyses. CCT performed the

RT-PCR and Zymography. YCT performed the Zymography. HHC provided

the data necessary for our analysis. SHK, SHC and CYH provided

material support and encouragement for this work. BST designed the

study and provided material support, and drafted signi

ﬁcant portions

of the manuscript. All authors contributed to and have approved the

ﬁnal manuscript.

Role of Funding Source

Funding for this study was provided by Department of Health

(DOH), Taiwan, ROC and had no further role in study design; in the

collection, analysis and interpretation of data; in the writing of the

report; and in the decision to submit the paper for publication.

Con

ﬂict of interest

There are no con

ﬂicts of interest.

Acknowledgments

The authors wish to thank all participating physician and patients.

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