www.elsevier.com/locate/jneuroim

Journal of Neuroimmunology 148 (2004) 162–171

Treatment with an acetylcholinesterase inhibitor in Alzheimer patients

modulates the expression and production of the pro-inflammatory

and anti-inflammatory cytokines

Marcella Realea, Carla Iarlorib, Francesco Gambic, Claudio Felicianid, Anatolia Salonec,Lucia Tomab, Giovanna DeLucab, Mirella Salvatoree, Pio Contia, Domenico Gambib,*

a Immunology Unit, Department of Oncology and Neuroscience, University UD’A, Via dei Vestini, 66013 Chieti, ItalybNeurology Unit, Department of Oncology and Neuroscience, University UD’A, Via dei Vestini, 66013 Chieti, ItalycPsychiatry Unit, Department of Oncology and Neuroscience, University UD’A, Via dei Vestini, 66013 Chieti, Italy

dDermatology Unit, Department of Oncology and Neuroscience, University UD’A, Via dei Vestini, 66013 Chieti, ItalyeDepartment MQIE-Faculty of Economics, Italy

Received 20 May 2003; received in revised form 27 October 2003; accepted 3 November 2003

Abstract

Elevated levels of cytokines have been detected in brains of Alzheimer’s disease (AD) patients, and altered peripheral levels of IL-1h,TNFa and IL-6 have been reported in these patients. We studied the ability of PBMC from patients with AD, matched with a control group,

to release pro- and anti-inflammatory cytokines, and the effect of AChEI treatment on cytokine release. Our data indicates that AChEI

treatment down-regulates IL-1, IL-6 and TNF, and up-regulates the expression and production of IL-4 in PBMC in AD patients, and that

AChEI leads to the remodelling of the cytokine network, probably acting on the lymphocytic cholinergic system.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Alzheimer’s disease; IL-1h; IL-6; TNFa; IL-4; PBMC

1. Introduction

Inflammatory mechanisms and immune activation have

been hypothesized to play a role in the pathogenesis of age-

associate disease (O’Mahony et al., 1998; Gerli et al., 2000)

and neurodegenerative processes such as dementia and

atherosclerosis (Benveniste, 1992; De Luca et al., 1998;

Porrini et al., 1998; Bruunsgaard et al., 1999; Iarlori et al.,

2000, 2002; Bruunsgaard and Pedersen, 2003).

Alzheimer’s disease (AD) is a chronic neurodegenerative

disorder causing progressive impairment of memory and

cognitive function. The amyloid cascade hypothesis sug-

gests that unregulated metabolism of the beta-amyloid (Ah)precursor protein (APP) followed by subsequent formation

of non-fibrillar and fibrillar Ah deposits leads to glial

activation and eventually to neurotoxicity, causing cognitive

impairment (Maat-Schieman et al., 1997). Ah up-regulates

0165-5728/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.jneuroim.2003.11.003

* Corresponding author. Tel./fax: +39-871-562019.

E-mail address: gambi@unich.it (D. Gambi).

and activates astrocytes, microglia and monocytes to act as

pro-inflammatory cells. These cells release a myriad of pro-

inflammatory cytokines, including TNF, IL-1 and IL-6

(Meda et al., 1999). In AD, immune and inflammatory-

related proteins have been implicated as mediators in

response to brain injury. Elevated levels of cytokines have

been detected in brains of AD patients. Altered peripheral

levels of IL-1h, TNFa and IL-6 have been reported in

patients with Alzheimer’s disease (Licastro et al., 2000).

Since the most recent successful therapeutic approach to AD

involve acetylcholinesterase inhibitors (AChEI) (Giacobini,

2001; Winblad et al., 2001), we investigated the ability of

peripheral blood mononuclear cells from a group of patients

with AD, matched with healthy controls (HC), to release

pro- and anti-inflammatory cytokines, and studied if the

stabilizing effect of AChEI treatment in vivo may relate to

the cytokine release or inhibition.

Biologically, IL-1 and TNF are closely related, because

of their multiple pro-inflammatory properties, these cyto-

kines contribute to many diseases and have strategic impor-

tance to the initiation and progression of inflammation

Table.1

Characteristics of AD patients and healthy controls at blood withdrawal

AD patients Age Gender Medication

1 77 M �2 75 F + (antibiotic)

3 63 F �4 80 F �5 81 M + (anxiolytic)

6 61 F �7 67 M + (analgesic)

8 74 M �9 76 F + (anxiolytic)

10 88 M + (vitamins)

11 77 F �12 70 F + (analgesic)

M. Reale et al. / Journal of Neuroimmunology 148 (2004) 162–171 163

(Dinarello, 1997). IL-1, a key molecule in systemic immune

responses in health and disease, has analogous roles in the

brain where it may contribute to neuronal degeneration

(Sheng et al., 1996) by triggering production of other

cytokines and nitric oxide (Rossi and Bianchini, 1996);

IL-1 promote Ah deposition through increased expression

and processing of amyloid precursor protein (APP) (Roth-

well and Hopkins, 1995) and may facilitate dystrophic

neuritic formation in diffuse non-neuritic plaques (Griffin

et al., 1995). In Alzheimer’s disease, TNF is up-regulated

and increases the production of Ah and inhibits the secre-

tion of neuroprotective, soluble amyloid precursor protein

(sAPPs). The up-regulation of Ah and ApoE by TNF would

then lead to increased neuritic plaque formation.(Perry et al.,

2001). High levels of protease inhibitors induced by IL-1

and IL-6 were responsible for an abnormal processing of

APP leading to high levels of insoluble Ah (Del Bo et al.,

1995). Over-expression of IL-6 in Alzheimer’s microglia are

associated with Ah plaque (Huell et al., 1995), and plasma

IL-6 concentrations in AD subjects was also significantly

higher than that of the control cases (Shibata et al., 2002).

The role of IL-4 in neurodegenerative disorders is still

unclear; however, IL-4, such as other anti-inflammatory

cytokines, regulates microglial responses to Ah in primary

murine microglia and in human monocyte cell line. IL-4 has

been reported to regulate Ah-induced production of the

inflammatory cytokines, IL-1 and IL-6 (Szcepanik et al.,

2001a). IL-4 is able to block or suppress IL-1, TNFa, IL-6

and IL-8 (Conti and Dempsey, 1990; Lee et al., 1995). IL-4

activity may also be associated with the pathophysiology of

AD (Abbas et al., 2002).

Our results demonstrate that, in AD patients the pro- and

anti-inflammatory peripheral cytokine system is affected

when compared with age/sex-matched HC, and that AChEI

treatment modulates cytokine production. Thus, at this stage,

we believe that AChEI treatment, besides restoring the

cholinergic system, reverses the cytokine imbalances ob-

served in AD.

13 68 F + (antibiotics plus vitamins)

14 84 M �15 77 M �16 80 M + (vitamins)

17 75 F �18 80 F + (vitamins plus anxiolytic)

19 67 M �20 72 F �21 73 F �

HC subject

1 77 M �2 69 M �3 81 F + (antibiotic)

4 72 M �5 76 F �6 75 F + (analgesic)

7 73 M �8 82 F �9 77 F �10 74 M + (analgesic)

2. Materials and methods

2.1. Subjects

Twenty-one AD patients, mean age 74.5F 7 (9 men,

42.8%; 12 women, 57.1%) who attended the Neurological

Clinic, Dept. of Oncology and Neuroscience, Chieti Univer-

sity, Chieti, Italy, were matched for age and habits with 10

HC (5 men, 50%; 5 women, 50%; mean age 75.5F 4). All

patients underwent clinical neuro-geriatric assessment and

routine laboratory analysis to exclude any possible influence

of other diseases which could be responsible for immune

activation. All patients were diagnosed with probable AD

according to the National Institute of Neurological and

Communicative Disorders and Stroke-Alzheimer’s Disease

and related Disorders Association (NINCDS-ADRDA) crite-

ria (McKhann et al., 1984). All patients were submitted to

the same research protocol and assessment of mental perfor-

mance and cognitive function was carried out using Fol-

stein’s Mini-Mental State Examination (MMSE) (Folstein et

al., 1975). Both patients and controls were not under med-

ication (especially analgesics and antibiotics) during the 2

weeks prior to testing and were asked not to use alcohol and

nicotine for at least 48 h before testing (Table 1). All patients

underwent early morning blood withdrawal before and after

1 month of AChEI treatment (Donepezil 10 mg/day, Pfeizer)

in the same hospital facility and by the same investigator. All

assays were run in parallel.

2.2. Cell separation and culture conditions

Blood samples for cytokine measurements were col-

lected into 4-ml peripheral blood mononuclear cells

(PBMC) endotoxin-free EDTA tubes (Vacutainer, Becton

Dickinson, NJ, USA). PBMC were isolated by Ficoll-

Hypaque density gradient centrifugation and resuspended

(5� 106/ml) in complete culture media consisting of

RPMI 1640 medium supplemented with 10% fetal calf

serum, 4 mM L-glutamine, 25 mM Hepes buffer, 50 U/ml

Fig. 1. Basal cytokine production (pg/ml/106 cells) in peripheral blood mononuclear cells cultures (incubated at 37 jC overnight) from AD patients (n= 21) and

age-matched HC (10). The cell-free supernatants were stored at � 80 jC until analysis. The ELISA values (in duplicates for each sample) have an error range

within 10%.

Fig. 2. Cytokine production (pg/ml/106 cells) after overnight stimulation with PHA mitogen in peripheral blood mononuclear cells cultures from AD patients

(n= 21) and age-matched healthy individuals (HC= 10). The ELISA values have an error range of within 10%.

M. Reale et al. / Journal of Neuroimmunology 148 (2004) 162–171164

M. Reale et al. / Journal of Neuroim

penicillin and 50 mg/ml streptomycin (all media and

components were purchased from Sigma, Italy). PBMC

were placed in polypropylene culture tubes (Bibby Sterilin

Italia, Italy) in a volume of 1 ml and cultured overnight,

at 37 jC in 95% humidified 5% CO2 cell culture in-

cubator, in complete culture medium, alone or stimulated

with PHA at 20 Ag/ml (Pharmacia, Sweden). Supernatants

were collected at the end of incubation and frozen at � 80

jC until assay. Pellet cells were also kept at � 80 jCuntil analysis. None of the reagents used contained endo-

toxin, as judged by Limulus amebocyte assay (minimum

detection level 0.1 Ag/ml) (E-Toxate).

Fig. 3. (A) RT-PCR shows IL-1h mRNA expression in PBMC isolated from AD pa

(T0) and from HC. Cells were stimulated with PHA (20 Ag/ml) overnight. The fi

expressed IL-1h mRNA levels relative to G3PDH mRNA. Date are meanF S.E.M

plots, where we have the data of IL-1h production (ELISA method) (basal and PH

distributed under the diagonal, that means after the treatment we have a reductio

2.3. Quantitation of chemokine and cytokine levels

Cytokine concentration in culture supernatants was

determined by a solid phase sandwich ELISA kit

(Endogen, Woburn, MA, USA) with monoclonal anti-

human IL-1h, IL-6, IL-4 and TNFa, according to the

manufacturers’ instructions. Samples from culture super-

natants were diluted in complete medium 1:10 for IL-1

h and IL-6, 1:2 for TNFa and undiluted for IL-4. All

steps were performed at room temperature. The color

reaction was stopped at 30 min and the OD was read at

450 nm within 30 min. Cytokine levels were then

munology 148 (2004) 162–171 165

tients after 1-month treatment with AChEI (T1), from untreated AD patients

gure shows one RT-PCR representative experiment. Densitometric analysis

. values for 21 (T0 and T1) to 10 (HC) individual samples. (B) In the scatter

A-stimulated) for untreated AD patients vs. treated AD patients, the data are

n in the quantities of these cytokines for all 21 AD patients.

M. Reale et al. / Journal of Neuroim166

calculated plotting the OD of each sample against the

standard curve. The assay sensitivity was < 1 pg/ml for

IL-6 and < 2 pg/ml for IL-1h, TNFa and IL-4, with an

assay range of 15.6–1000 pg/ml for TNFa and 10–400

pg/ml for IL-1h, IL-6 and IL-4. The intra- and inter-

assay reproducibility was >90%. Optical density values

obtained for duplicates was within 10% of the mean.

Duplicate values that differed from the mean by greater

than 10% were considered suspect and were repeated.

For convenience all results are expressed in pg/ml/106

cells.

Fig. 4. (A) RT-PCR shows IL-6 mRNA expression in PBMC isolated from AD

patients (T0) and from HC. Cells were stimulated with PHA (20 Ag/ml) overnigh

analysis expressed IL-6 mRNA levels relative to G3PDH mRNA. Date are mea

In the scatter plots, where we have the data of IL-6 production (ELISA metho

patients, the data are distributed under the diagonal, that means after the treatm

patients.

2.4. mRNA extraction and cDNA synthesis

Poly-A mRNA was extracted using a purification

system kit (Pharmacia Biotech, Milan, Italy). In brief,

about 10 million cells were dissolved in a solution

containing guanidinium thiocyanate 4 M and N-lauroyl-

sarcosine in order to preserve the RNA. The solution

was placed in oligo(dt)-cellulose at 25 Ag/ml suspended

in a storage buffer containing 0.15% Kathon CG (Phar-

macia LKB, Cologno Monzese (MI), Italy). After sev-

eral washes in salt buffers containing 10 mM Tris–HCl

munology 148 (2004) 162–171

patients after 1-month treatment with AChEI (T1), from untreated AD

t. The figure shows one RT-PCR representative experiment. Densitometric

nF S.E.M. values for 21 (T0 and T1) to 10 (HC) individual samples. (B)

d) (basal and PHA-stimulated) for untreated AD patients vs. treated AD

ent we have a reduction in the quantities of this cytokine for all 21 AD

M. Reale et al. / Journal of Neuroim

(pH 7.4), 1 mM EDTA, 0.5 M NaCl or 0.1 M NaCl in

the last two washes, the oligo(dt)-cellulose containing

the mRNA was placed in filter columns and the mRNA

was eluted in warm Tris–HCl 10 mM and precipitated

in chilled 95% ethanol overnight. After centrifugation,

the pellet was dissolved in 14 Al of DEPC-treated

sterile water and quantitated by spectrophotometric anal-

ysis. A total of 0.5 Ag of mRNA was transcribed into

cDNA using 200 U of superscript reverse transcriptase

(GIBCO BRL, Milan, Italy) and 50 ng of Random

Examers.

Fig. 5. (A) RT-PCR shows TNFa mRNA expression in PBMC isolated from A

patients (T0) and from HC. Cells were stimulated with PHA (20 Ag/ml)

Densitometric analysis expressed TNFa mRNA levels relative to G3PDH mR

individual samples. (B) In the scatter plots, where we have the data of TNFa p

patients vs. treated AD patients, the data are distributed under the diagonal, tha

cytokine for all 21 AD patients.

2.5. Reverse transcriptase PCR amplification (RT-PCR)

cDNAwas amplified with 2.5 U Taq polymerase (Perkin

Elmer Cetus, Milan, Italy) using 1.5 pM of the primers

specific for IL-1h, IL-6, IL-4 and TNFa and G3PDH. IL-1,

IL-6, IL-4, TNFa and G3PDH primers were purchased from

Clonetech Laboratory (Palo Alto, CA, USA).

Each sample was divided in half, one part was used for

the cytokine under investigation and the other half for the

G3PDH for semi-quantitative analysis. RT-PCR was con-

ducted with the following protocol: (1) predenaturation at

munology 148 (2004) 162–171 167

D patients after 1-month treatment with AChEI (T1), from untreated AD

overnight. The figure shows one RT-PCR representative experiment.

NA. Date are meanF S.E.M. values for 21 (T0 and T1) to 10 (HC)

roduction (ELISA method) (basal and PHA-stimulated) for untreated AD

t means after the treatment we have a reduction in the quantities of this

M. Reale et al. / Journal of Neuroimmunology 148 (2004) 162–171168

94 jC for 5 min, (2) denaturation at 94 jC for 2 min, (3)

annealing at 55 jC for 2 min, (4) extension at 72 jC for 2

min and (5) denaturation at 94 jC for 2 min.

The linear range of signal strength for each cytokine was

determined by performing titration for cDNA and cycle

numbers to obtain non saturated PCR reactions.

Five microliters of amplified products were electropho-

retically separated in a 2% agarose gel containing ethidium

bromide and finally analyzed for molecular size.

The following controls were used: cDNA without pri-

mers and cDNA from peripheral blood mononuclear cells

from HC.

Signals were analyzed using the Bio-profile software

(Vilber Lourmat, Nice, France) and semi-quantitative anal-

ysis was performed comparing the amplified product signals

with the G3PDH signal.

2.6. Statistical analysis

Treated and untreated AD patients were analyzed by two-

sample paired Student’s t-test to determine whether sample’s

means are distinct. We also compared the mean of the

Fig. 6. (A) RT-PCR shows IL-4 mRNA expression in PBMC isolated from ACh

patients (T0 = no treatment) and from age-matched HC. Cells were incubated with

experiment. Densitometric analysis expressed IL-4 mRNA levels relative to G3PD

individual samples. (B) Illustrates the production of interleukin-4 (ELISA method)

of IL-4. The data are all distributed above the diagonal, meaning that after the trea

patients.

untreated AD patients and the HC, and the AD patients

treated and the HC.

The t-test is significant, either comparing the untreated

AD patients and the HC in basal and the AD patients treated

or the HC in PHA-stimulated PBMC cultures.

3. Results

3.1. Cytokine production in unstimulated PBMC

Cytokine production was examined directly in periph-

eral blood mononuclear cells cultures of HC subjects and

AD patients. Immunoreactive IL-1h, IL-6, TNFa and IL-

4 were measured in an attempt to determine more closely

any possible correlation between the in vitro productions

of cytokines in the HC vs. the AD group of subjects. In

the unstimulated PBMC of the AD group, there was a

significant increase in IL-1h, IL-6 and TNFa production

when compared with the HC group. On the contrary,

levels of IL-4 released by unstimulated PBMC did not

differ between AD patients and HC ( p = 0.427) (Fig. 1).

EI-treated AD patients after 1 month (T1 = 1 month), from untreated AD

or without PHA (20 Ag/ml) overnight. The figure shows one representative

H mRNA. Date are meanF S.E.M. values for 21 (T0 and T1) to 10 (HC)

in basal and PHA stimulation from PBMC. The scatterplot shows the results

tment we have an increase in the quantities of this cytokines for all 21 AD

uroimmunology 148 (2004) 162–171 169

3.2. Cytokine production by PBMC after PHA stimulation

The ability of PBMC to produce IL-1h, IL-6, TNFa and

IL-4 when exposed to PHA was evaluated. The PHA

mitogen was chosen for its ability to aspecifically stimulate

the release of these cytokines. The results show that cyto-

kine production in AD patients after stimulation with PHA

was higher than in their matched HC. The difference

between the levels was significant ( p < 0.001) for each

cytokine studied (Fig. 2).

3.3. IL-1� , IL-6 and TNF� expression and production by

PBMC in AChEI-treated, untreated AD patients and HC

To determine the effect of the in vivo AChEI-treat-

ment, cells from treated and untreated patients were

cultured overnight with or without 20 Ag/ml PHA and

than extracted for mRNA. As shown in the representative

experiments (Figs. 3A, 4A and 5A) RT-PCR amplification

demonstrates that the three cytokines are detectable in

small quantities in HC subjects, but are expressed in

greater quantities in AD untreated patients. Treatment

with AChEI reduces the expression of pro-inflammatory

cytokines in AD patients. The densitometric analysis

confirmed the decreases in TNFa, IL-1h and IL-6 mRNA

expression relative to G3PDH mRNA expression in

AChEI-treated AD patients.

The treatment with AChEI reduces the production of

pro-inflammatory cytokines in AD patients: levels of IL-

1h, IL-6 and TNFa were reduced in AD patients after 30

days of therapy with AChEI (Figs. 3B, 4B and 5B). In all

21 patients, the following percent decreases of the cyto-

kines were seen: IL-1h in basal conditions decreased

57%, in PHA-treated cells 54%, IL-6 decreased 62% in

basal conditions and 34% in PHA-treated cells, and TNFa

decreased 32% in basal conditions, 54% in PHA-treated

cells.

3.4. IL-4 expression and production by PBMC in Alzheim-

er’s patients receiving AChEI or not

The expression of IL-4 in AD patients, HC and AChEI-

treated AD patients is shown in Fig. 6. RT-PCR shows that

IL-4 mRNA expression is increased in AChEI-treated AD

patients respect to untreated AD patients. Determination of

IL-4 in cell-free supernatants harvested from cultures of

PBMC from AD patients treated with AChEI revealed

increased levels of biologically active IL-4, compared with

cultures of PBMC from untreated AD patients. In basal

conditions, IL-4 increase 197% after treatment; after PHA

stimulation, IL-4 increased 200%; and in both cases, the

values were higher than the HC. When we compared HC

subject with AChEI-treated AD patients, the spontaneous

release of IL-4 was higher in AChEI-treated group ( p <

0.001), while PHA-induced IL-4 production was not signif-

icantly different ( p= 0.601) (data not shown).

M. Reale et al. / Journal of Ne

4. Discussion

There is an emerging concept that the net biological

response of pro and anti-inflammatory cytokines affects

the outcome of certain diseases such as neurodegenerative

disorders (O’Shea and Lipsky, 2002; Sredni-Kenigsbuch,

2002). Modified production of cytokines, with conflicting

data on circulating serum levels of IL-1h, TNFa and IL-

6 in AD patients, have been reported (Fillit et al., 1991;

Kalman et al., 1997; Lanzrein et al., 1998; Engelborghs

et al., 1999; Licastro et al., 2000). Several studies have

addressed the issue of peripheral inflammatory molecules

as possible markers for AD and other dementia condi-

tions (Paganelli et al., 2002), but it is still an open issue.

Cytokines found in plasma and/or serum may be pro-

duced by blood cells, endothelium or may originate from

the brain. We hypothesize that the inflammatory condition

in AD patients, such as Ah accumulation, may influence

the PBMC and drive these cells to an ‘‘active state’’,

producing high levels of cytokines, these activated T cells

can cross the blood brain barrier (BBB) and contribute to

neurodegeneration (Yates et al., 2000; Szczepanik et al.,

2001b).

Evaluation of the ability of PBMC of AD patients to

produce cytokines when exposed to mitogenic stimuli

may clarify the role of these cells in active immune

response occurring in AD and if changes in the respon-

siveness of these cells are related to changes in circulating

cytokines.

Experiments designed to determine the constitutive and

PHA-inducible production, of IL-1h, IL-6, TNFa and IL-4

by PBMC from AD patients were performed both at the

mRNA and protein levels and provide evidence that PBMC

from AD release more IL-1h, IL-6, TNFa and less IL-4 than

PBMC from HC, and that such production is regulated at the

mRNA level. Our study supports the hypothesis of a

systemic production of cytokines, changes in circulating

cytokines may contribute to the pathophysiology of AD and

pro-inflammatory cytokines may affect neuronal function.

Cholinesterase inhibitors (ChEI) are used in AD patients

to maintain cognitive function at a constant level. Recent

studies have postulated the effect of ChEI on Ah metabo-

lism to explain the stabilising effect of the drugs. Cells

exposed to pro-inflammatory cytokines have elevated levels

of APP, which may result in increased production of Ah,further exacerbating plaque deposition, which is a crucial

event in pathophysiology of AD (Goldgaber et al., 1989;

Chao et al., 1995).

In order to gain insight in the potential benefits of oral

administration of AChEI in AD patients, we have examined

its role in immunological responses. The interesting results

that emerge from this study is that transcription and pro-

duction of pro-inflammatory cytokines in PBMC of AD

patients are markedly enhanced, with respect to HC sub-

jects, and IL-1h, IL-6 and TNFa over-expression and

production, are in accordance with other studies (i.e. Lom-

M. Reale et al. / Journal of Neuroimmunology 148 (2004) 162–171170

bardi et al., 1999) that reported an increase of different pro-

inflammatory cytokines, including TNFa, IL-6 and IL-1h,in AD patients. We hypothesize that increased production of

multiple cytokines from AD patients could produce a pre-

activation state in circulating PBMC.

Interestingly, we found that oral AChEI-treatment in AD

patients reduces expression and secretion of the pro-inflam-

matory cytokines IL-1h, IL-6 and TNFa and up-regulates

the expression and production of IL-4 under basal and

stimulated conditions in PBMC.

These data indicates that AChEI plays a role in the

regulation of immune response by reducing the production

of functionally important inflammatory cytokines that may

contribute to the immunopathology associated with AD, and

promoting the Th2 response, leading to the remodelling of

the cytokine network, probably acting on the lymphocytic

cholinergic system. Recently, it has been demonstrated that

in vitro IL-4 inhibit almost 100% the secretion of IL-1h and

TNF, while the secretion of IL-6 was found to be reduced

from 70% to 85%. Pre-treatment of co-culture cells with IL-

4, an immunosuppressive cytokine, prevented neuronal cell

injury induced by activated microglia, in a dose-dependent

manner (Ledeboer et al., 2000). Moreover, co-treatment of

stimulated cells with IL-4 blocked the increase levels of

TNFa and IL-1h, the inhibition was manifest also at the

mRNA level and hypothesized that IL-4 could be involved

and/or responsible for an aberrant regulation of cytokines

which are crucial in regulating brain and endocrine function

under normal physiological conditions (Hart et al., 1989; te

Velde et al., 1990). Indeed, recent investigations have

provided evidence that PBMC possess most of the essential

components needed to constitute (Chao et al., 1993) a

cholinergic system and that ACh synthesized and released

from PBMC acts as an immunomodulator (Kawashima and

Fujii, 2003, Tayebati et al., 2001).

In the light of these data, we suggest that the mecha-

nism(s) whereby AChEI reduces the level of inflammatory

cytokines probably is represented by the increased levels of

IL-4. The mechanism by which IL-4 exerts its neuropro-

tective effect was found to involve the inhibition of IFNa

priming of microglia with a subsequent decrease in the

production of TNFa and nitric oxide (Chao et al., 1995).

These findings may have implications for the control of the

pathogenic mechanisms in neurodegeneration of Alzheim-

er’s disease and provide a basis for future studies related to

systemic cytokines and AD.

Acknowledgements

Thanks are due to Renato Barbacane for his technical

skills. This work was supported by MURST grants, prot.

CE00538159 ‘‘Biological and immunological markers in

neurodegenerative disorders’’ from the Ministry of Univer-

sity, Scientific and Technological Research, Italy for the

year 2000.

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