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Immunobiology 216 (2011) 80–85

Immunobiology 216 (2011) 80–85 Contents lists available at ScienceDirect Immunobiology journal homepage: www.elsevier.de/imbio Expression of inflammatorynt studies have also de monstrated that Corresponding author. Tel.: +1 718 494 5265; fax: +1 718 494 2768. E-mail addresses: xiaohong.li@omr.state.ny.us , xiaohong.li@mssm.edu (X. Li). 0171-2985/$ – see front matter. Published by Elsevier GmbH. doi: 10.1016/j.imbio.2010.03.001 transforming growth factor (TGF)- 1, macrophage chemoattrac- tant protein (MCP)-1, IL-6, IL-8, IL-10, GM-CSF, TNF and IFN are increased in the frontal cortices of autistic brains ( Vargas et al. 2005; Li et al. 2009a, 2009b ). Furthermore, MCP-1, IL-6, IL-8, IFN and TNF were found to be significantly increased in the cerebrospinal fluid of autistic children ( Vargas et al. 2005; Chez et al. 2007 ). All this evidence suggests that inflammation and cytokines may play an important role in the pathogenesis of autism. In addition, a number of neuropathologic and biochemical studies implicate the apoptosis-related protein Bcl2 as being involved in several neuropsychiatric disorders, including schizophrenia ( Fatemi et al. 2000; Fatemi and Halt 2001; Guidotti et al. 2000; Jarskog et al. 2000 ), bipolar disorder ( Fatemi et al. 2000; Guidotti et al. 2000 ), major depression ( Fatemi et al. 2000 ), and lissencephaly ( Hong et al. 2000 ). Araghi-Niknam and Fatemi (2003) reported decreased levels of Bcl2 and increased levels of p53 proteins in the cortex of autistic subjects. Importantly, several studies demonstrated that apoptosis can be initiated by activation of a group of cytokines, including TNF- , IFN- and TGF- ( Laster et al. 1988; Trauth et al. 1989; Itoh et al. 1991; Lin and Chou 1992; Novelli et al. 1994; Suda and Nagata 1994; Deiss et al. 1995 ), and the apoptosis induced by cytokines TNF- and IFN- may be mediated by cathepsin D ( Deiss et al. 1996 ). Cathepsin D is the predominant lysosomal aspartic acid pro- tease, is abundantly expressed in the brain, and hydrolyzes select peptide bonds of target proteins with high specificity ( Whitaker and Rhodes 1983; Reid et al. 1986; Saftig et al. 1995 ). Cathepsin D was reported to initiate apoptosis through caspase-8 and to play an important role in the regulation of cellular apoptosis ( Deiss et " id="pdf-obj-0-5" src="pdf-obj-0-5.jpg">

Contents lists available at ScienceDirect

Immunobiology

journal homepage: www.elsevier.de/imbio

Immunobiology 216 (2011) 80–85 Contents lists available at ScienceDirect Immunobiology journal homepage: www.elsevier.de/imbio Expression of inflammatorynt studies have also de monstrated that Corresponding author. Tel.: +1 718 494 5265; fax: +1 718 494 2768. E-mail addresses: xiaohong.li@omr.state.ny.us , xiaohong.li@mssm.edu (X. Li). 0171-2985/$ – see front matter. Published by Elsevier GmbH. doi: 10.1016/j.imbio.2010.03.001 transforming growth factor (TGF)- 1, macrophage chemoattrac- tant protein (MCP)-1, IL-6, IL-8, IL-10, GM-CSF, TNF and IFN are increased in the frontal cortices of autistic brains ( Vargas et al. 2005; Li et al. 2009a, 2009b ). Furthermore, MCP-1, IL-6, IL-8, IFN and TNF were found to be significantly increased in the cerebrospinal fluid of autistic children ( Vargas et al. 2005; Chez et al. 2007 ). All this evidence suggests that inflammation and cytokines may play an important role in the pathogenesis of autism. In addition, a number of neuropathologic and biochemical studies implicate the apoptosis-related protein Bcl2 as being involved in several neuropsychiatric disorders, including schizophrenia ( Fatemi et al. 2000; Fatemi and Halt 2001; Guidotti et al. 2000; Jarskog et al. 2000 ), bipolar disorder ( Fatemi et al. 2000; Guidotti et al. 2000 ), major depression ( Fatemi et al. 2000 ), and lissencephaly ( Hong et al. 2000 ). Araghi-Niknam and Fatemi (2003) reported decreased levels of Bcl2 and increased levels of p53 proteins in the cortex of autistic subjects. Importantly, several studies demonstrated that apoptosis can be initiated by activation of a group of cytokines, including TNF- , IFN- and TGF- ( Laster et al. 1988; Trauth et al. 1989; Itoh et al. 1991; Lin and Chou 1992; Novelli et al. 1994; Suda and Nagata 1994; Deiss et al. 1995 ), and the apoptosis induced by cytokines TNF- and IFN- may be mediated by cathepsin D ( Deiss et al. 1996 ). Cathepsin D is the predominant lysosomal aspartic acid pro- tease, is abundantly expressed in the brain, and hydrolyzes select peptide bonds of target proteins with high specificity ( Whitaker and Rhodes 1983; Reid et al. 1986; Saftig et al. 1995 ). Cathepsin D was reported to initiate apoptosis through caspase-8 and to play an important role in the regulation of cellular apoptosis ( Deiss et " id="pdf-obj-0-16" src="pdf-obj-0-16.jpg">

Expression of inflammatory cytokines, Bcl2 and cathepsin D are altered in lymphoblasts of autistic subjects

Mazhar Malik, Ashfaq M. Sheikh, Guang Wen, Warren Spivack, William T. Brown, Xiaohong Li

Department of Neurochemistry, NY State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York, NY 10314, United States

article

info

Article history:

Received 3 February 2010 Received in revised form 2 March 2010 Accepted 4 March 2010

Keywords:

Autism

Apoptosis

Inflammation

Cytokines

Bcl2

Cathepsin D

abstract

To determine whether inflammation and apoptosis are involved in the pathogenesis of autism, we exam- ined cytokines, Bcl2 expression and cathepsin D protease activity in the lymphoblasts of autistic subjects and age-matched controls. We found increased expression levels of pro-inflammatory cytokines TNF- and IL-6, but decreased Bcl2 expression in lymphoblasts of autistic subjects. We also found that cathepsin

D mRNA and protein expression were significantly increased in autistic lymphoblasts. Conclusion: Our findings suggest that inflammation and apoptosismay play a significant role in the pathogenesis of autism, and cathepsin D may participate in the regulation of cytokine-induced inflammation and apoptosis in autistic lymphoblasts.

Published by Elsevier GmbH.

Introduction

Autism is a severe neuro-developmental disorder of childhood characterized by impairments in social interaction, deficits in ver- bal and non-verbal communication, and restricted repetitive and stereotyped patterns of behavior and interests (DSMIV criteria, American Psychiatric Association 1994). Many areas of brain in autism show abnormalities that include decreased Purkinje cell counts in cerebellar hemispheres and vermis (Ritvo et al. 1986), loss of granular cells (Bauman and Kemper 1994) and Purkinje cell atrophy (Fatemi et al. 2001). These abnormalities may be associated with disturbances of visuospatial-integration, impaired language, apraxia, and agnosia. However, there is a paucity of neurochemical data paralleling these neurohistologic findings in autism. Suscepti- bility to autism is clearly attributable to genetic factors (Folstein and Rosen-Sheidley 2001), but the etiology of this disorder is unknown, and no biomarkers have yet been proven to be char- acteristic of autism. Emerging evidence points to inflammatory and apoptotic mechanisms being responsible for certain neuropsy- chiatric disorders (Jarskog et al. 2000). A number of studies have shown that inflammatory cytokines including TNF- , IFN- , IL- 1 and IL-12 are elevated in the blood mononuclear cells, serum and plasma of autistic subjects (Jyonouchi et al. 2001, 2002; Singh 1996; Croonenberghs et al. 2002a; Molloy et al. 2006; Ashwood and Wakefield 2006). Recent studies have also demonstrated that

Corresponding author. Tel.: +1 718 494 5265; fax: +1 718 494 2768. E-mail addresses: xiaohong.li@omr.state.ny.us, xiaohong.li@mssm.edu (X. Li).

0171-2985/$ – see front matter. Published by Elsevier GmbH.

doi:10.1016/j.imbio.2010.03.001

transforming growth factor (TGF)- 1, macrophage chemoattrac- tant protein (MCP)-1, IL-6, IL-8, IL-10, GM-CSF, TNF and IFN are increased in the frontal cortices of autistic brains (Vargas et al. 2005; Li et al. 2009a, 2009b). Furthermore, MCP-1, IL-6, IL-8, IFN and TNF were found to be significantly increased in the cerebrospinal fluid of autistic children (Vargas et al. 2005; Chez et al. 2007). All this evidence suggests that inflammation and cytokines may play an important role in the pathogenesis of autism. In addition, a number of neuropathologic and biochemical studies implicate the apoptosis-related protein Bcl2 as being involved in several neuropsychiatric disorders, including schizophrenia (Fatemi et al. 2000; Fatemi and Halt 2001; Guidotti et al. 2000; Jarskog et al. 2000), bipolar disorder (Fatemi et al. 2000; Guidotti et al. 2000), major depression (Fatemi et al. 2000), and lissencephaly (Hong et al. 2000). Araghi-Niknam and Fatemi (2003) reported decreased levels of Bcl2 and increased levels of p53 proteins in the cortex of autistic subjects. Importantly, several studies demonstrated that apoptosis can be initiated by activation of a group of cytokines, including TNF- , IFN- and TGF- (Laster et al. 1988; Trauth et al. 1989; Itoh et al. 1991; Lin and Chou 1992; Novelli et al. 1994; Suda and Nagata 1994; Deiss et al. 1995), and the apoptosis induced by cytokines TNF- and IFN- may be mediated by cathepsin D (Deiss et al. 1996). Cathepsin D is the predominant lysosomal aspartic acid pro- tease, is abundantly expressed in the brain, and hydrolyzes select peptide bonds of target proteins with high specificity (Whitaker and Rhodes 1983; Reid et al. 1986; Saftig et al. 1995). Cathepsin D was reported to initiate apoptosis through caspase-8 and to play an important role in the regulation of cellular apoptosis (Deiss et

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al. 1996; Conus et al. 2008; Zheng et al. 2008; Benes et al. 2008). Recent studies also suggest that cathepsin D can be released into the cytosol upon stimulation of apoptosis and cleave the Bcl2 family member Bid to result in the subsequent release of cyt c from mito- chondria, as well as activation of caspase-9 and -3 (Heinrich and Junig 2004). In addition, a number of studies have shown that pro- cathepsin D initiates the secretion of cytokines, including IL-4, IL-8, IL-10 and IL-13 (Fusek et al. 2007). On the other hand, inflamma- tory cytokines, including TNF- and IFN- , were shown to increase extracellular procathepsin D in primary endothelial cell cultures (Erdmann et al. 2008). Deiss found that Pepstatin A, an inhibitor of cathepsin D, suppressed apoptosis in HeLa cells and interfered with the TNF- induced apoptosis in U937 cells as well (Deiss et al. 1996), suggesting that Cathepsin D protease mediates the apoptosis induced by cytokines TNF- and IFN- . Based on the above findings, we hypothesize that in autism there is an abnormal apoptotic activation induced by inflammatory cytokines and that cathepsin D may be involved in the regulation of this apoptosis. In the present study, we used lymphoblasts from autistic subjects and age-matched controls to test this hypothesis. Lymphoblast cell lines have been suggested to be a valuable tool for identifying genes associated with autism and provide an alterna- tive approach for understanding the biology and genetics of autism (Baron et al. 2006). Our results show that cytokines TNF- and IL-6 were significantly elevated in the lymphoblasts of autistic subjects compared to controls, while anti-apoptotic Bcl2 protein expres- sion was significantly reduced. In addition, our study shows that cathepsin D was significantly increased in lymphoblasts of autistic subjects compared to controls. These findings strongly support our hypothesis and suggest that there is increased inflammation and apoptosis present in autistic lymphoblasts. The elevated activation of cathepsin D in autistic lymphoblasts indicates that cathepsin D may be involved in the regulation of cytokine-induced apoptosis in ASD.

Material and methods

Study subjects

Lymphoblasts of 6 autistic subjects (mean age: 8.4 ± 0.27 years) and 6 age-matched normal controls (mean age: 7.0 ± 0.91 years) were included in this study. Subjects fit the diagnostic criteria for autism of the Diagnostic and Statistical Manual-IV, as confirmed by the Autism Diagnostic Interview-Revised. Participants were excluded from the study if they had a diagnosis of fragile X syn- drome, epileptic seizures, obsessive–compulsive disorder, affective disorders, or any additional psychiatric or neurological diagnoses.

Isolation and culture of lymphoblasts

Lymphoblasts were isolated from whole blood by Ficoll- diatrizoate density gradient centrifugation (Neitzel 1986). The cells were then collected and resuspended in 10 ml RPMI (no serum), and centrifuged at 300 × g for 10 min. The cell pellets were further sus- pended in a mixture of 1 ml RPMI containing 2 g CSA/ml and 1 ml EBV stock and incubated in a round-bottom tube (Falcon 3033) at 37 C, 5% CO 2 . RPMI (complete) medium containing 1 g CSA/ml was added once a week to maintain the culture.

Multiplexed analyses of cytokines in lymphoblast with the Bio-Plex system

A standard capture sandwich assay was used to determine the levels of the different cytokines in lymphoblast. Each captured anti- body was coupled to a different bead set (Invitrogen’s Multiplex Bead Immunoassays). The system used a liquid suspension array of

10 sets of beads (Invitrogen, Human Cytokine 10-plex) internally dyed with different ratios of two spectrally distinct fluorochromes to assign a unique spectral address. Each set of beads was combined with amonoclonal antibody raised against GM-CSF, IL-1 , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IFN- , and TNF- . Beads were incubated first (2 h, at room temperature) with diluted standards (serial dilutions from 1.95 to 32,000 pg/ml) or the lymphoblast lysates sample, and then with biotinylated detector antibodies (30 min, at room tem- perature). They were washed twice in phosphate-buffered saline, and incubated for 30 min at room temperature with phycoerythrin- conjugated streptavidin. Cytokine levels were measured with a Luminex 200 TM system (Bio-Rad Laboratories). Each measurement was taken in duplicate. Standard curves were generated by using the reference cytokine concentrations supplied by the manufac- turer. Raw data (mean fluorescent intensities) were analyzed by Bio-Plex Manager Software 4.1 version (Bio-Rad Laboratories) to obtain concentration values.

Western blot analysis

Lymphoblast cell lysates in SDS sample buffer (20% glycerol, 100 mM Tris, pH 6.8, 0.05% (w/v) Bromophenol blue, 2.5% SDS (w/v), 250 mM DTT) were denatured by heating at 100 C for three min- utes. Sixty micrograms of protein per lane per subject was loaded onto the 12% acryl-bisacrylamide gel and electrophoresed for 2 h at 120 V at RT. The proteins were then electroblotted onto nitro- cellulose membrane for 1 h at 100 V at 4 C. Afterwards, protein blots were blocked with 5% milk in PBS with 1% Tween (TBST) and then incubated with primary antibody for overnight at 4 C (anti- Bcl2, 1:1000, New England Biolabs, Ipswich, MA). After one wash in PBST (2 min), the protein blots were further incubated with a secondary antibody for 1 h at RT (Sigma, goat ant-mouse IgG, HRP conjugated, 1:5000) and followed by three washes in PBST (each time for 10 min). At the end, the blots were visualized using the ECL detection system (Amersham Pharmacia Biotech) and exposed to Hyper film ECL (Amersham Pharmacia Biotech). Sample densities were analyzed blind to the diagnosis, using a Bio-Rad densitome- ter and the Bio-Rad Multi Analyst software. The densities of 26-kDa Bcl2 and 46-kDa actin were quantified with background subtrac- tion. Statistical analyses were conducted using paired t-tests with significance established at P < 0.05.

Isolation of RNA

Total RNA was isolated from human lymphoblast cell cultures, from autistic and controls, using Tri-Reagent TM (Sigma–Aldrich, St. Louis, MO) according to the manufacturer’s protocol. The final pel- lets were dissolved in DEPC-treated water in tubes treated with RNase ZAP (Sigma–Aldrich) and stored at 80 C until needed. The RNA concentrations were determined spectrophotometri- cally.

Generation of sense and anti-sense cathepsin D RNA transcripts

With reverse transcription PCR approach, a 604 base- pair fragment of cathepsin D cDNA was generated using 2–3 g of human lymphoblast total RNA as the template (PCR primers are: 5 -ATCCCGCTGCACAA-GTTCACGTCCATCCGC- 3 and 5 -CCACCAGCTTCTGCTGCATCAGGTTGTCGAAGACG-3 ). The Cathepsin D cDNA was then subcloned into the pCR ® 2.1 TOPO vector using the TOPO TA Cloning ® Kit (Invitrogen/GIBCO-Life Technologies, New York). After confirmation of sequence and purification, the Cathepsin D cDNA were processed to generate sense and anti-sense RNA transcripts of the cathepsin D using the GeneScribe TM T7 In Vitro Transcription Kit (GeneScribe Kit). The transcripts were then collected by precipitation with the addition of

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M. Malik et al. / Immunobiology 216 (2011) 80–85

sodium acetate/ethanol and centrifugation, and the concentrations were determined spectrophotometrically.

Northern blot

Total RNA from autistic and control human lymphoblasts were fractionated on agarose/formaldehyde gels. The RNA was trans- ferred to a positively charged nylon membrane by capillary action using 20× SSPE buffer (3 M NaCl, 0.3 M Na 2 PO 4 , 25 mM EDTA; pH 7.0). Pre-hybridization was performed in a heat-seal bag (2 h, 68 C water bath) using DIG Easy Hyb (10 ml/cm 2 , supplemented with 100 g/ml salmon-sperm DNA, fragmented, heat-denatured and snap-chilled on ice) as the buffer. For the hybridization, the pre- hybridization solution was removed and replaced with 3 ml/cm 2 of supplemented DIG Easy Hyb with the labeled anti-sense cathep- sin D transcript at a concentration of 50 ng/ml. Hybridization was continued overnight at 68 C. The membrane was removed from the heat-seal bag and washed twice at low-stringency (15 min) at RT with 2× SSPE containing 1% SDS, followed by two high-stringency washes at 68 C in 0.5× SSPE containing 1% SDS (15 min) at RT. Detection was carried out with the DIG Luminescent Detection Kit. This kit uses an AP-conjugated anti-digoxigenin antibody along with a fluorescent AP substrate (CSPD) for detection on X-ray film. Quantification analysis is carried out by using Image J software (http://www.nih.gov). Band intensities in the lymphoblast samples were converted from arbitrary area units to nanograms using the measurements from the dilution series of the cathepsin D sense RNA transcript.

EM Immuno-gold labeling

Lymphoblasts were washed in 0.1 M phosphate buffer (PB) for 1.5 h at RT centrifuged at 2000 rpm in the microfuge for 5 min. Supernatants were discarded. Pellets sere mixed with 2% agar, cut to obtain a thin layer about 2 mm thick slices. These slices containing lymphocytes were fixed in 1% paraformaldehyde/1% glutaraldehyde in 0.1 M PB for 1.5 h washed in same PB for 20 min. The slices were then dehydrated with graded (50%, 70, 85, 95, 100) ethanols (15 min each step), mixture of 1 part of 100% ethanol and 1 part of 100% propylene oxide (PL) (30 min), pure propylene oxide (60 min), mixture of 1 part of PL and 1 part of Spurr embedding medium (60 min), infiltrated with 100% Spurr embedding medium (overnight), embedded in pure Spurr medium, and finally polymer- ized at 70 C oven overnight. EM sections were cut with diamond knife, placed on Nickel grids, immersed in supernatant (10 min) of the saturated sodium metaperiodate (14,000 rpm microfuge 10 min). After blocking in 20% normal goat serum (NGS) with FBS/PBS/NaN3 (1 drop of 100% NGS + 1 ml of FBS/PBS/NaN3) for 30 min, the EM sections of lymphoblasts were incubated with the primary antibody (Cathepsin D, 1:250, Sigma) overnight at 4 C. Afterwards, the lymphoblast sections were further incubated with secondary antibody (goat anti-mouse with gold particles, Amer- sham Life Science, Arlington) for 60 min at RT and then jet washed with FBS/PBS/NaN3 once, followed by immersing in the same solu- tion for 3× 5 min. Finally, the lymphoblast sections were washed with distilled water and mounted for EM examination.

Results

Elevated cytokines TNF-˛ and IL-6 in the lymphoblast of autistic subjects

Pro-inflammatory cytokines (IL-6, IL-1 , TNF- , GM-CSF), Th1 cytokines (IL-2 and IFN- ), Th2 cytokines (IL-4, IL-5 and IL-10) and chemokine IL-8 were analyzed in the lymphoblasts from both autistic subjects and the control subjects. Our results showed

Table 1

Cytokine profile in the lymphoblasts of autistic subjects and the controls.

Control subjects (mean ± Autistic SE) subjects (mean ± SE) (pg/mg protein)

(pg/mg protein)

IL-6

135.5 ± 24.8

255.3 ± 52.1 **

IL-1

14 ± 2.9

15.5 ± 2.98

GM-CSF

16.6 ± 3.41

12.9 ± 2.38

TNF

7.8 ± 3.1

38.1 ± 9.2 **

IFN

15.7 ± 3.2

12.9 ± 2.7

IL-2

15.9 ± 3.21

18.1 ± 4.0

IL-4

38.5 ± 8.2

52.7 ± 11.4

IL-5

8.1 ± 1.7

9.6 ± 1.9

IL-10

13.8 ± 2.81

13 ± 2.72

IL-8

269.3 ± 54.3

250.3 ± 54.6

Invitrogen’s Multiplex Bead Immunoassays were used to detect the cytokine levels.

n = 6.

**

P < 0.01.

that pro-inflammatory cytokines TNF- and IL-6 were significantly increased (P < 0.01) in the autistic lymphoblasts as compared with matched controls. The other cytokines showed no significant dif- ferences between the two groups (Table 1 and Fig. 1).

Bcl2 protein expression is decreased in the lymphoblast of autistic subjects

There were no correlations between Bcl2 protein concentrations and age in either group. In the autistic group, the bands represent- ing the 26 kDa Bcl2 protein expression from the lymphoblasts of autistic subjects were weaker than those from the control group (Fig. 2A). Quantitative analysis showed that the mean value of Bcl2 expression was decreased by 32% in autistic subjects as compared to control subjects (P < 0.05, Fig. 2B).

Cathepsin D mRNA expression is increased in the lymphoblast of autistic subjects

Using Northern blot assays, we detected stronger bands of cathepsin D mRNA expression (Fig. 3A, lanes 5–8) in the autis- tic lymphoblasts as compared with the controls (Fig. 3A, lanes 1–4). Quantitative analysis using the image J program showed that the control lymphoblast RNA samples averaged 168.8 ± 63.7 ng of cathepsin D mRNA (3.38% of the total RNA) whereas the autistic lymphoblast RNA samples averaged 434.8 ± 20.3 ng (8.70% of the total RNA). The analysis revealed an approximately 2.5 fold increase in the level of cathepsin D mRNA in the autistic samples versus the controls (Fig. 3B).

82 M. Malik et al. / Immunobiology 216 (2011) 80–85 sodium acetate/ethanol and centrifugation, and the68 C in 0.5 × SSPE c ontaining 1% SDS (15 min) at RT. Detection was carr i e d ou t w ith t he DIG Luminescent Detection Kit. This kit uses an AP-conjugated anti-digoxigenin antibody along with a fluorescent AP substrate (CSPD) for detection on X-ray film. Quantification analysis is carried out by using Image J software ( http://www.nih.gov ). Band intensities in the lymphoblast samples were converted from arbitrary area units to nanograms using the measurements from the dilution series of the cathepsin D sense RNA transcript. EM Immuno-gold labeling Lymphoblasts were washed in 0.1 M phosphate buffer (PB) for 1.5 h at RT centrifuged at 2000 rpm in the microfuge for 5 min. Supernatants were discarded. Pellets sere mixed with 2% agar, cut to obtain a thin layer about 2 mm thick slices. These slices containing lymphocytes were fixed in 1% paraformaldehyde/1% glutaraldehyde in 0.1 M PB for 1.5 h washed in same PB for 20 min. The slices were then dehydrated with graded (50%, 70, 85, 95, 100) ethanols (15 min each step), mixture of 1 part of 100% ethanol and 1 part of 100% propylene oxide (PL) (30 min), pure propylene oxide (60 min), mixture of 1 part of PL and 1 part of Spurr embedding medium (60 min), infiltrated with 100% Spurr embedding medium (overnight), embedded in pure Spurr medium, and finally polymer- ized at 70 C oven overnight. EM sections were cut with diamond knife, placed on Nickel grids, immersed in supernatant (10 min) of the saturated sodium metaperiodate (14,000 rpm microfuge 10 min). After blocking in 20% normal goat serum (NGS) with FBS/PBS/NaN3 (1 drop of 100% NGS + 1 ml of FBS/PBS/NaN3) for 30 min, the EM sections of lymphoblasts were incubated with the primary antibody (Cathepsin D, 1:250, Sigma) overnight at 4 C. Afterwards, the lymphoblast sections were further incubated with secondary antibody (goat anti-mouse with gold particles, Amer- sham Life Science, Arlington) for 60 min at RT and then jet washed with FBS/PBS/NaN3 once, followed by immersing in the same solu- tion for 3 × 5 min. Finally, the lymphoblast sections were washed with distilled water and mounted for EM examination. Results Elevated cytokines TNF- ˛ and IL-6 in the lymphoblast of autistic subjects Pro-inflammatory cytokines (IL-6, IL-1 , TNF- , GM-CSF), Th1 cytokines (IL-2 and IFN- ), Th2 cytokines (IL-4, IL-5 and IL-10) and chemokine IL-8 were analyzed in the lymphoblasts from both autistic subjects and the control subjects. Our results showed Table 1 Cytokine profile in the lymphoblasts of autistic subjects and the controls. Control subjects (mean ± Autistic SE) subjects (mean ± SE) (pg/mg protein) (pg/mg protein) IL-6 135.5 ± 24.8 255.3 ± 52.1 IL-1 14 ± 2.9 15.5 ± 2.98 GM-CSF 16.6 ± 3.41 12.9 ± 2.38 TNF 7.8 ± 3.1 38.1 ± 9.2 IFN 15.7 ± 3.2 12.9 ± 2.7 IL-2 15.9 ± 3.21 18.1 ± 4.0 IL-4 38.5 ± 8.2 52.7 ± 11.4 IL-5 8.1 ± 1.7 9.6 ± 1.9 IL-10 13.8 ± 2.81 13 ± 2.72 IL-8 269.3 ± 54.3 250.3 ± 54.6 Invitrogen’s Multiplex Bead Immunoassays were used to detect the cytokine levels. n = 6. P < 0.01. that pro-inflammatory cytokines TNF- and IL-6 were significantly increased ( P < 0.01) in the autistic lymphoblasts as compared with matched controls. The other cytokines showed no significant dif- ferences between the two groups ( Table 1 and Fig. 1 ). Bcl2 protein expression is decreased in the lymphoblast of autistic subjects There were no correlations between Bcl2 protein concentrations and age in either group. In the autistic group, the bands represent- ing the 26 kDa Bcl2 protein expression from the lymphoblasts of autistic subjects were weaker than those from the control group ( Fig. 2 A). Quantitative analysis showed that the mean value of Bcl2 expression was decreased by 32% in autistic subjects as compared to control subjects ( P < 0.05, Fig. 2 B). Cathepsin D mRNA expression is increased in the lymphoblast of autistic subjects Using Northern blot assays, we detected stronger bands of cathepsin D mRNA expression ( Fig. 3 A, lanes 5–8) in the autis- tic lymphoblasts as compared with the controls ( Fig. 3 A, lanes 1–4). Quantitative analysis using the image J program showed that the control lymphoblast RNA samples averaged 168.8 ± 63.7 ng of cathepsin D mRNA (3.38% of the total RNA) whereas the autistic lymphoblast RNA samples averaged 434.8 ± 20.3 ng (8.70% of the total RNA). The analysis revealed an approximately 2.5 fold increase in the level of cathepsin D mRNA in the autistic samples versus the controls ( Fig. 3 B). Fig. 1. Cytokine profiles in lymphoblasts of autistic subjects. Invitrogen’s Multiplex Bead Immunoassays were used to detect the cytokine con- centrations in lymphoblasts of autistic subjects and the control subjects P < 0.01, n = 6. " id="pdf-obj-2-265" src="pdf-obj-2-265.jpg">

Fig. 1. Cytokine profiles in lymphoblasts of autistic subjects.

Invitrogen’s Multiplex Bead Immunoassays were used to detect the cytokine con-

centrations in lymphoblasts of autistic subjects and the control subjects ** P < 0.01,

n = 6.

M. Malik et al. / Immunobiology 216 (2011) 80–85

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M. Malik et al. / Immunobiology 216 (2011) 80–85 83 Fig. 2. Bcl2 expression in lymphoblasts

Fig. 2. Bcl2 expression in lymphoblasts of autistic subjects.

  • (A) Western blots of lymphoblast lysates using Bcl2 antibody (dilution 1:500). Lanes 1–4 represent autistic subjects and lanes 5–8 represent controls. The upper panel shows

actin bands (MW: 48 kDa), the lower panel shows Bcl2 bands (MW: 26 kDa).

  • (B) The blots shown in (A) were quantitated after normalization by actin. Data are shown as mean ± SE. * P < 0.05, n = 4 (ASD vs. control group).

M. Malik et al. / Immunobiology 216 (2011) 80–85 83 Fig. 2. Bcl2 expression in lymphoblasts

Fig. 3. Cathepsin D mRNA expression in lymphoblasts of autistic subjects.

  • (A) Northern blot of lymphoblast RNA using an anti-sense cathepsin D probe. Lanes 1–4 represent the control subjects and lanes 5–8 represent the autistic subjects.

  • (B) Quantitative analysis of Northern blot studies using image J program. Data are shown as mean ± SE. ** P < 0.01, n = 4 (ASD vs. control group).

Cathepsin D protein expression is increased in lymphoblasts of autistic subjects

To further detect cathepsin D expression at the protein level, we conducted EM Immuno-gold labeling studies of the lymphoblasts from autistic and control subjects. We found that cathepsin D pro- tein expression (shown as white dots, Fig. 4A) in both the cytoplasm and nucleus of autistic lymphoblasts was increased as compared to the control lymphoblasts. Quantitative image J analysis after normalization by the background showed that cathepsin D pro- tein expression was increased approximately 3 times in autistic lymphoblasts as compared with controls (Fig. 4B).

Discussion

Although susceptibility to autism is clearly attributable to genetic factors (Folstein and Rosen-Sheidley 2001), the etiology of the disorder is unknown, and no biomarkers have yet been identified as characteristic of autism. Recent reports suggest that a combination of environmental or perhaps in utero risk factors, autoimmune risk factors and localized inflammation of the cen- tral nervous system may contribute to the pathogenesis of autism (Nelson et al. 2001; Vargas et al. 2005; Zimmerman et al. 2005; Chez et al. 2007). Emerging evidence also points to apoptotic mech- anisms being responsible for certain neuropsychiatric disorders including autism (Jarskog et al. 2000; Araghi-Niknam and Fatemi 2003). In the current study, we detected that the pro-inflammatory cytokines TNF- and IL-6 were significantly increased in autistic lymphoblasts as compared with matched controls. TNF- and IL-6 are cytokines involved in cell-mediated immune response and their production has been shown to be associated with tissue inflamma- tion and necrosis (Beutler and Cerami 1989). The role of TNF- as

a neuromodulating agent has also been related to brain develop- ment, and in modulating glutaminergic transmission (Pickering et al. 2005). Excessive glutamate excitotoxic effects acting via NMDA receptors may occur in the presence of excess TNF- (Pickering et al. 2005). This occurrence can lead to effects on microglial acti- vation, as well as on nuclear factor kappa-B (NF-kB). In addition, recent studies have shown that apoptosis can be initiated by acti-

vation of various cell surface receptors triggered by TNF- and IFN- (Laster et al. 1988; Trauth et al. 1989; Itoh et al. 1991; Lin and Chou 1992; Novelli et al. 1994; Suda and Nagata 1994; Deiss

et al. 1995). Thus elevations of TNF- and IL-6 in autistic lym- phoblasts suggest that autistic subjects may have a heightened immune response associated with brain inflammation and tissue necrosis. These results also imply the excessive TNF- and IL-6 may lead to increased apoptosis in autistic lymphoblasts. To determine whether there is an increased apoptotic process in autism, we examined Bcl2 protein expression in the lymphoblasts of autistic subjects and matched controls. Our results showed that Bcl2 protein expression was significantly decreased in autistic lymphoblasts as compared with the controls. Bcl2 is a membrane- bound protein, which strongly inhibits apoptosis and enhances cell survival, and has been shown to suppress apoptosis in a variety of cell systems including factor-dependent lymphohematopoietic and neural cells. Bcl2 protein regulates cell death by controlling mito- chondrial membrane permeability and inhibiting caspase activity either by preventing the release of cytochrome c from the mito- chondria and/or by binding to apoptosis-activating factor (APAF-1) (Harris and Thompson 2000). The decreased Bcl2 protein level therefore suggests that there may be increased apoptosis in autistic lymphoblasts. Previous studies showed that genetic vulnerability or prenatal stressors such as viral infections (Fatemi et al. 2000) or hypoxia (Margolis 1997) during periods when physiological

84

M. Malik et al. / Immunobiology 216 (2011) 80–85

84 M. Malik et al. / Immunobiology 216 (2011) 80–85 Fig. 4. Cathepsin D protein expression

Fig. 4. Cathepsin D protein expression in the lymphoblast of autistic subjects.

  • (A) EM Immuno-gold labeling studies on lymphoblasts of autistic and the control subjects. Cathepsin D protein expression is shown as white dots in both cytoplasm and

nucleus of lymphoblasts.

  • (B) Quantitative analysis using image J after normalization to the background measurement. Data are shown as mean ± SE. ** P < 0.01, n = 4 (ASD vs. control group).

levels of Bcl2 are low, may increase the risk of developing neuro- developmental disorders such as autism or schizophrenia. Recently studies have shown that Bcl2 protein level is decreased in the brains of autistic subjects (Fatemi et al. 2001; Araghi-Niknam and Fatemi 2003). In addition, our studies also found that the Bcl2 protein level is decreased and the BDNF-Akt-Bcl2 anti-apoptosis pathway is compromised in the frontal cortex of autistic subjects (Li et al. 2009a, 2009b). This evidence suggests that apoptosis may be par- tially responsible for the pathogenesis of autism and our finding of Bcl2 being decreased in autistic lymphoblasts further supports this hypothesis. In addition, we suggest that the alteration of apopto- sis in autistic lymphoblasts is likely to be induced by the elevated TNF- and IL-6 cytokines. The protease cathepsin D and its prototypically inactive pro- form, procathepsin D, is multifunctional within and outside the cell. Elevated levels of procathepsin D occur in malignant tumors and in organs experiencing chronic inflammation. Studies have shown that procathepsin D initiates secretion of cytokines, including IL- 4, IL-8, IL-10 and IL-13 (Fusek et al. 2007). On the other hand, inflammatory cytokines, including TNF- and IFN- , increases extracellular procathepsin D in primary endothelial cell cultures (Erdmann et al. 2008). It has been shown that acidification of cytokine-treated media converts procathepsin D into cathepsin D (Erdmann et al. 2008). In addition, cathepsin D has been reported to initiate apoptosis through caspase-8 and has been suggested to play an important role in the regulation of cellular susceptibility to apop- tosis and in cytokine-induced programmed cell death (Deiss et al. 1996; Conus et al. 2008; Zheng et al. 2008). To determine whether cathepsin D, like cytokines and apoptotic genes, is also abnor- mally regulated in autistic lymphoblasts, we examined cathepsin D mRNA and protein expression in autistic lymphoblasts. Our results showed that both cathepsin D mRNA and protein expression were significantly increased in autistic lymphoblasts as compared with the matched controls. Whether cathepsin D is involved in the reg- ulation of cytokines and apoptosis in autistic lymphoblasts will require further investigation. However the increase in cathepsin D, and the corresponding increase in pro-inflammatory cytokines (TNF- and IL-6) and apoptosis in autistic lymphoblasts, suggest that cathepsin D may play an important role in cytokine-induced apoptosis. These alterations may be important causes that lead to the pathogenesis of autism. In summary, we found increased expression levels of pro- inflammatory cytokines TNF- and IL-6 in lymphoblasts of autistic subjects. We also found that the Bcl2 expression is significantly decreased in autistic lymphoblasts, suggesting increased apoptosis susceptibility in autism. In addition, we demonstrated that cathep- sin D mRNA and protein expression are significantly increased in the autistic lymphoblasts as compared with the controls. These

findings provide an evidence that inflammation and apoptotic change may be an important part of the pathogenesis of autism. These findings also suggest that cathepsin D may be significantly involved in the regulation of cytokine-induced inflammation and apoptosis in autism.

Acknowledgement

This work was supported by NYS office of Mental Retardation and Developmental Disabilities.

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