Immunobiology 216 (2011) 8085

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Immunobiology
j our nal homepage: www. el sevi er . de/ i mbi o
Expression of inammatory 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
a r t i c l e i n f o
Article history:
Received 3 February 2010
Received in revised form 2 March 2010
Accepted 4 March 2010
Keywords:
Autism
Apoptosis
Inammation
Cytokines
Bcl2
Cathepsin D
a b s t r a c t
To determine whether inammation and apoptosis are involved in the pathogenesis of autism, we exam-
ined cytokines, Bcl2 expression and cathepsin Dprotease activity in the lymphoblasts of autistic subjects
and age-matched controls. We found increased expression levels of pro-inammatory 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 signicantly increased in autistic lymphoblasts. Conclusion: Our
ndings suggest that inammationandapoptosis mayplayasignicant roleinthepathogenesis of autism,
and cathepsin D may participate in the regulation of cytokine-induced inammation 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, decits 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 maybe associated
with disturbances of visuospatial-integration, impaired language,
apraxia, and agnosia. However, there is a paucity of neurochemical
data paralleling these neurohistologic ndings 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 inammatory
and apoptotic mechanisms being responsible for certain neuropsy-
chiatric disorders (Jarskog et al. 2000). A number of studies have
shown that inammatory 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 Wakeeld 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).
transforming growth factor (TGF)-1, macrophage chemoattrac-
tant protein (MCP)-1, IL-6, IL-8, IL-10, GM-CSF, TNF and IFN are
increasedinthefrontal 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 signicantly increased in the cerebrospinal
uid of autistic children (Vargas et al. 2005; Chez et al. 2007).
All this evidence suggests that inammation 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 specicity (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
0171-2985/$ see front matter. Published by Elsevier GmbH.
doi:10.1016/j.imbio.2010.03.001
M. Malik et al. / Immunobiology 216 (2011) 8085 81
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
thecytosol uponstimulationof apoptosis andcleavetheBcl2family
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 Dinitiates the secretion of cytokines, including IL-4, IL-8,
IL-10 and IL-13 (Fusek et al. 2007). On the other hand, inamma-
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), suggestingthat CathepsinDproteasemediates theapoptosis
induced by cytokines TNF- and IFN-.
Based on the above ndings, we hypothesize that in autism
there is an abnormal apoptotic activation induced by inammatory
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 showthat cytokines TNF- and IL-6
were signicantly elevated in the lymphoblasts of autistic subjects
compared to controls, while anti-apoptotic Bcl2 protein expres-
sion was signicantly reduced. In addition, our study shows that
cathepsin D was signicantly increased in lymphoblasts of autistic
subjects compared to controls. These ndings strongly support our
hypothesis and suggest that there is increased inammation 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: 8.40.27 years)
and 6 age-matched normal controls (mean age: 7.00.91 years)
were included in this study. Subjects t the diagnostic criteria for
autism of the Diagnostic and Statistical Manual-IV, as conrmed
by the Autism Diagnostic Interview-Revised. Participants were
excluded from the study if they had a diagnosis of fragile X syn-
drome, epileptic seizures, obsessivecompulsive 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
werethencollectedandresuspendedin10ml RPMI (noserum), and
centrifuged at 300g for 10min. The cell pellets were further sus-
pended in a mixture of 1ml RPMI containing 2g CSA/ml and 1ml
EBV stock and incubated in a round-bottom tube (Falcon 3033) at
37

C, 5% CO
2
. RPMI (complete) medium containing 1g 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 inlymphoblast. Eachcapturedanti-
body was coupled to a different bead set (Invitrogens Multiplex
Bead Immunoassays). The systemused a liquid suspension array of
10 sets of beads (Invitrogen, Human Cytokine 10-plex) internally
dyed with different ratios of two spectrally distinct uorochromes
to assigna unique spectral address. Eachset of beads was combined
withamonoclonal antibodyraisedagainst GM-CSF, IL-1, IL-2, IL-4,
IL-5, IL-6, IL-8, IL-10, IFN-, and TNF-. Beads were incubated rst
(2h, at room temperature) with diluted standards (serial dilutions
from 1.95 to 32,000pg/ml) or the lymphoblast lysates sample, and
then with biotinylated detector antibodies (30min, at room tem-
perature). They were washed twice in phosphate-buffered saline,
andincubatedfor 30minat roomtemperature withphycoerythrin-
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 uorescent 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,
100mMTris, pH6.8, 0.05%(w/v) Bromophenol blue, 2.5%SDS(w/v),
250mM 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 2h
at 120V at RT. The proteins were then electroblotted onto nitro-
cellulose membrane for 1h at 100V 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 (2min), the protein blots were further incubated with a
secondary antibody for 1h at RT (Sigma, goat ant-mouse IgG, HRP
conjugated, 1:5000) and followed by three washes in PBST (each
time for 10min). At the end, the blots were visualized using the
ECL detection system(AmershamPharmacia Biotech) and exposed
toHyper lmECL (AmershamPharmacia 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 quantied with background subtrac-
tion. Statistical analyses were conducted using paired t-tests with
signicance established at P<0.05.
Isolation of RNA
Total RNA was isolated from human lymphoblast cell cultures,
fromautistic and controls, using Tri-Reagent
TM
(SigmaAldrich, St.
Louis, MO) according to the manufacturers protocol. The nal pel-
lets were dissolved in DEPC-treated water in tubes treated with
RNase ZAP (SigmaAldrich) 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
23g 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 conrmation of sequence and
purication, 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 werethencollectedbyprecipitationwiththeadditionof
82 M. Malik et al. / Immunobiology 216 (2011) 8085
sodiumacetate/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 (3M NaCl, 0.3M Na
2
PO
4
, 25mM EDTA; pH
7.0). Pre-hybridization was performed in a heat-seal bag (2h, 68

C
water bath) using DIG Easy Hyb (10ml/cm
2
, supplemented with
100g/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 3ml/cm
2
of supplemented DIGEasy Hyb with the labeled anti-sense cathep-
sin D transcript at a concentration of 50ng/ml. Hybridization was
continuedovernight at 68

C. Themembranewas removedfromthe
heat-seal bag and washed twice at low-stringency (15min) at RT
with 2 SSPE containing 1% SDS, followed by two high-stringency
washes at 68

C in 0.5 SSPE containing 1% SDS (15min) at RT.

Detection was carried out with the DIG Luminescent Detection Kit.
This kit uses an AP-conjugated anti-digoxigenin antibody along
with a uorescent AP substrate (CSPD) for detection on X-ray lm.
Quantication 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 0.1M phosphate buffer (PB) for
1.5h at RT centrifuged at 2000rpm in the microfuge for 5min.
Supernatants were discarded. Pellets sere mixed with 2% agar,
cut to obtain a thin layer about 2mm thick slices. These slices
containing lymphocytes were xed in 1% paraformaldehyde/1%
glutaraldehyde in 0.1M PB for 1.5h washed in same PB for 20min.
The slices were then dehydrated with graded (50%, 70, 85, 95, 100)
ethanols (15min each step), mixture of 1 part of 100% ethanol and
1 part of 100% propylene oxide (PL) (30min), pure propylene oxide
(60min), mixture of 1 part of PL and 1 part of Spurr embedding
medium (60min), inltrated with 100% Spurr embedding medium
(overnight), embedded inpure Spurr medium, and nally polymer-
ized at 70

C oven overnight. EM sections were cut with diamond

knife, placed on Nickel grids, immersed in supernatant (10min)
of the saturated sodium metaperiodate (14,000rpm microfuge
10min). After blocking in 20% normal goat serum (NGS) with
FBS/PBS/NaN3 (1 drop of 100% NGS +1ml of FBS/PBS/NaN3) for
30min, 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 60min at RT and then jet washed
with FBS/PBS/NaN3 once, followed by immersing in the same solu-
tion for 3 5min. 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-inammatory 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 prole in the lymphoblasts of autistic subjects and the controls.
Control subjects (meanSE)
(pg/mg protein)
Autistic subjects (meanSE) (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
Invitrogens Multiplex Bead Immunoassays were used to detect the cytokine levels.
n=6.
**
P<0.01.
that pro-inammatory cytokines TNF-and IL-6 were signicantly
increased (P<0.01) in the autistic lymphoblasts as compared with
matched controls. The other cytokines showed no signicant dif-
ferences between the two groups (Table 1 and Fig. 1).
Bcl2 protein expression is decreased in the lymphoblast of autistic
subjects
Therewerenocorrelations betweenBcl2proteinconcentrations
and age in either group. In the autistic group, the bands represent-
ing the 26kDa 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 58) in the autis-
tic lymphoblasts as compared with the controls (Fig. 3A, lanes
14). Quantitative analysis using the image J programshowed that
the control lymphoblast RNA samples averaged 168.863.7ng of
cathepsin D mRNA (3.38% of the total RNA) whereas the autistic
lymphoblast RNA samples averaged 434.820.3ng (8.70% of the
total RNA). The analysis revealedanapproximately2.5foldincrease
in the level of cathepsin D mRNA in the autistic samples versus the
controls (Fig. 3B).
Fig. 1. Cytokine proles in lymphoblasts of autistic subjects.
Invitrogens 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) 8085 83
Fig. 2. Bcl2 expression in lymphoblasts of autistic subjects.
(A) Western blots of lymphoblast lysates using Bcl2 antibody (dilution 1:500). Lanes 14 represent autistic subjects and lanes 58 represent controls. The upper panel shows
actin bands (MW: 48kDa), the lower panel shows Bcl2 bands (MW: 26kDa).
(B) The blots shown in (A) were quantitated after normalization by actin. Data are shown as meanSE.
*
P<0.05, n=4 (ASD vs. control group).
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 14 represent the control subjects and lanes 58 represent the autistic subjects.
(B) Quantitative analysis of Northern blot studies using image J program. Data are shown as meanSE.
**
P<0.01, n=4 (ASD vs. control group).
Cathepsin D protein expression is increased in lymphoblasts of
autistic subjects
To further detect cathepsin Dexpression 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-
teinexpression(shownas white dots, Fig. 4A) inboththe 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
identied as characteristic of autism. Recent reports suggest that
a combination of environmental or perhaps in utero risk factors,
autoimmune risk factors and localized inammation 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-inammatory
cytokines TNF- and IL-6 were signicantly increased in autistic
lymphoblasts as compared with matched controls. TNF- and IL-6
are cytokines involvedincell-mediatedimmune response andtheir
production has been shown to be associated with tissue inamma-
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 inammation 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 anincreased 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 signicantly 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 includingfactor-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 inautistic
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) 8085
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 meanSE.
**
P<0.01, n=4 (ASD vs. control group).
levels of Bcl2 are low, may increase the risk of developing neuro-
developmental disorders suchas autismor schizophrenia. Recently
studies have shownthat Bcl2proteinlevel is decreasedinthe 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 autismand our nding 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, procathepsinD, is multifunctional withinandoutside the cell.
Elevated levels of procathepsin D occur in malignant tumors and
in organs experiencing chronic inammation. 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,
inammatory cytokines, including TNF- and IFN-, increases
extracellular procathepsin D in primary endothelial cell cultures
(Erdmann et al. 2008). It has been shown that acidication of
cytokine-treated media converts procathepsin D into cathepsin D
(Erdmannet al. 2008). Inaddition, cathepsinDhas beenreportedto
initiateapoptosis throughcaspase-8andhas beensuggestedtoplay
animportant roleintheregulationof cellular susceptibilitytoapop-
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
signicantly 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-inammatory 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-
inammatory cytokines TNF- and IL-6 in lymphoblasts of autistic
subjects. We also found that the Bcl2 expression is signicantly
decreased in autistic lymphoblasts, suggesting increased apoptosis
susceptibility in autism. In addition, we demonstrated that cathep-
sin D mRNA and protein expression are signicantly increased in
the autistic lymphoblasts as compared with the controls. These
ndings provide an evidence that inammation and apoptotic
change may be an important part of the pathogenesis of autism.
These ndings also suggest that cathepsin D may be signicantly
involved in the regulation of cytokine-induced inammation and
apoptosis in autism.
Acknowledgement
This work was supported by NYS ofce of Mental Retardation
and Developmental Disabilities.
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