Journal of Neurochemistry, 2001, 76, 1326±1335

Central b-amyloid peptide-induced peripheral interleukin-6

responses in mice

Dong-Keun Song, Yeong-Bin Im, Jun-Sub Jung, Jaeyoung Cho, Hong-Won Suh and Yung-Hi Kim

Department of Pharmacology, College of Medicine, Institute of Natural Medicine, Hallym University, Chunchon, South Korea

Abstract

b-Amyloid peptides (Abs) share with lipopolysaccharide, a

potent pro-in¯ammatory agent, the property of stimulating glial

cells or macrophages to induce various in¯ammatory media-

tors. We recently reported that central administration of

lipopolysaccharide induces peripheral interleukin-6 responses

via both the central and peripheral norepinephrine system. In

this study, the effect of intracerebroventricular injection of

various synthetic Abs on plasma interleukin-6 levels was

examined in mice. Ab1242 dose-dependently increased plasma

interleukin-6 levels: `aged' Ab1242 was more effective than

fresh, whereas Ab4221 had no effect. `Aged' Ab1242

(205 pmol/mouse i.c.v.)-induced plasma interleukin-6 peaked

at 2 h post injection, which is earlier than the peak time of the

Ab1242-induced brain interleukin-6, tumor necrosis factor-a

and interleukin-1b levels, which was 4, 4 and 24 h, respec-

tively. Among various peripheral organs, Ab1242 (205 pmol/

mouse i.c.v.) signi®cantly increased interleukin-6 mRNA

expression in lymph nodes and liver. Ab1242 (205 pmol/

mouse i.c.v.) signi®cantly increased norepinephrine turnover

in both hypothalamus and spleen. Either central or peripheral

norepinephrine depletion effectively inhibited the Ab1242-

induced peripheral interleukin-6 response. Pretreatment with

prazosin (a1-adrenergic antagonist), yohimbine (a2-adrener-

gic antagonist), and ICI-118,551 (b2-adrenergic antagonist),

but not with betaxolol (b1-adrenergic antagonist), inhibited

Ab1242-induced plasma interleukin-6 levels. These results

demonstrate that centrally administered Ab1242 effectively

induces the systemic interleukin-6 response which is

mediated, in part, by central Ab1242-induced activation of

the central and the peripheral norepinephrine systems.

Keywords: Alzheimer's disease, 6-hydroxydopamine, intra-

cerebroventricular injection, norepinephrine, sympathetic

nervous system.

J. Neurochem. (2001) 76, 1326±1335.

Accumulating evidence demonstrates that the in¯ammatory

process plays an important role in Alzheimer's disease (AD)

(Aisen 1997; Hull et al. 1999; McGeer and McGeer 1999;

Combs et al. 2000). b-Amyloid peptide (Ab), a major

component of senile plaque, is postulated to play a pivotal

role in inducing the in¯ammatory process in the AD brain;

Ab can activate glial cells (astrocytes and microglia) in

culture to induce or potentiate the production of a variety of

in¯ammatory products, including interleukin (IL)-1b, tumor

necrosis factor (TNF)-a, IL-6, IL-8, nitric oxide (NO) and

reactive oxygen species (Araujo and Cotman 1992; Gitter

et al. 1995; Meda et al. 1995; Walker et al. 1995; Ii et al.

1996; McDonald et al. 1997). Furthermore, the glial/

macrophage-stimulating properties of Abs and some of

their signal transduction pathways are shared with lipo-

polysaccharide (LPS), a potent mediator of in¯ammation;

both stimuli similarly activate tyrosine kinases of src family,

such as Syk, and Lyn (Boulet et al. 1992; Crowley et al.

1996; McDonald et al. 1997; Combs et al. 1999), and

mitogen-activated protein kinases (MAPK) (McDonald et al.

1998; Pyo et al. 1998; Combs et al. 1999) in glia/

macrophages.

Interleukin-6 is a multifunctional cytokine involved in the

regulation of immune responses, hematopoiesis, and acute-

phase reactions (Hirano 1998). Head injury (Kossmann et al.

1995), and various neurological diseases, including ischemic

stroke (Fassbender et al. 1994), multiple sclerosis (Frei et al.

1326 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1326±1335

Received May 19, 2000; revised manuscript received October 2, 2000;

accepted October 6, 2000.

Address correspondence and reprints requests to D.-K. Song,

Department of Pharmacology and Institute of Natural Medicine,

College of Medicine, Hallym University, Chunchon, Kangwon-Do,

200-702, South Korea. E-mail: dksong@sun.hallym.ac.kr

Abbreviations used: Ab, b-amyloid peptide; AD, Alzheimer's

disease; IL, interleukin; i.c.v., intracerebroventricular; i.p., intra-

peritoneal; LPS, lipopolysaccharide; MAPK, mitogen-activated protein

kinases; NE, norepinephrine; NO, nitric oxide; 6-OHDA, 6-hydroxy-

dopamine; TNF-a, tumor necrosis factor-a.

1991), and AD (Kalman et al. 1997; Singh and Guthikonda

1997; Bonaccorso et al. 1998), have been associated with

increased plasma IL-6 levels. Recently, it has been shown

that the central administration of several potent mediators of

in¯ammation, such as IL-1b (De Simoni et al. 1990, 1993;

Romero et al. 1996; Kitamura et al. 1998), TNF (Benigni

et al. 1996) or LPS (Gottschall et al. 1992; Muramami et al.

1993; De Simoni et al. 1995), increases plasma levels of IL-

6 and IL-6 mRNA expression in peripheral organs,

demonstrating that the in¯ammatory process in the CNS

induces systemic IL-6 responses (Terreni and De Simoni

1998).

We hypothesized that central administration of Abs may

induce systemic IL-6 responses. In this study we examined

the effect of intracerebroventricular (i.c.v.) injection of Abs

on the plasma IL-6 levels and IL-6 mRNA expression in

various peripheral tissues in mice.

Materials and methods

Animals and i.c.v. injection

Male ICR mice weighing 25±30 g (Myung-Jin, Inc., Seoul, South

Korea) were used in all experiments. The animals were housed ®ve

per cage in a room maintained at 22 ^ 18C with an alternating 12-h

light/dark cycle. Food and water were available ad libitum. All

animal procedures were carried out as approved by the Animal

Care and Use Committee at Hallym University College of

Medicine. The i.c.v. administration was performed following the

procedure established by Laursen and Belknap (1986), which was

modi®ed from the method of Haley and McCormick (1957). The

animal was injected at bregma with a 50-mL Hamilton syringe

®tted with a 26-gauge needle, which was adjusted to be inserted

2.4 mm deep. The i.c.v. injection volume was 5 mL, and injection

sites were veri®ed by injecting the same volume of 1% methylene

blue into the site and then observing the distribution of the injected

dye in the ventricular space.

Reagents

Ab1242, Ab1240, Ab25235, Ab1228 (RBI, Natick, MA, USA) and

Ab4221 (Bachem, Bubendorf, Switzerland) were prepared as stock

solutions in sterile deionized water, and aliquots were stored at

2208C. Ab1240 and Ab1242 were used either fresh or after the

`aging' process, i.e. incubation of Abs in saline at ®nal concen-

tration at 378C for 3 days (Pike et al. 1993). Ab25235 and Ab1228

were used only in the `aged' state. Phentolamine mesylate, pro-

pranolol HCl, prazosin HCl, and ICI-118,551 HCl were purchased

from RBI, yohimbine HCl, and methyl ester of a-methyl-p-tyrosine

were from Sigma Chemical Co. (St Louis, MO, USA), and

betaxolol HCl was from Tocris Cookson Ltd. (Bristol, UK).

Prazosin HCl and yohimbine HCl were dissolved in saline con-

taining 10 and 20% dimethylsulfoxide, respectively. Other drugs

were dissolved in sterile saline.

Plasma and brain cytokine assay

Plasma was separated by centrifugation of the freshly drawn blood

from the retro-orbital venous plexus and stored at 2708C until

assayed. The plasma levels of IL-6 as well as IL-1b and TNF-a were

determined 1.5 h after an i.c.v. injection of Ab1242 with an ELISA

kit (Genzyme, Cambridge, MA, USA). Whole brain at various time

points after `aged' Ab1242 injection was homogenized with 10 vol.

of phosphate-buffered saline (pH 7.2) and centrifuged at 10 000 g

for 20 min twice, and the supernatant was used for cytokine assay.

Assays were performed exactly as described by the manufacturers.

Reverse transcription (RT) PCR

In the experiment on the tissue distribution of central Ab1242-

induced IL-6 mRNA expression various peripheral tissues [pituit-

ary, adrenals, kidney, heart, liver, spleen, and lymph nodes (iliac)]

were collected at 1 h after Ab1242 administration. The number of

animals used for each experiment was seven for pituitary, ®ve for

adrenals, and three for all other tissues. Total cellular RNAs were

extracted from tissues using a rapid guanidinium thiocyanate±

water saturated phenol±chloroform extraction and subsequent

precipitation with acidic sodium acetate (Chomczynski and Sacchi

1987). Total cellular RNAs in the aqueous phase were precipitated

using cold isopropyl alcohol. Isolated RNA samples were subjected

to spectrophotometric analysis at 260 and 280 nm, and samples

were stored at 2708C until use. RNA was denatured by incubating

at 708C for 5 min and then chilled quickly to 48C. cDNA synthesis

was conducted on 1 mg total RNA. The reaction mixture for the

synthesis of cDNA by RT reaction included the following: 15 mm

MgCl2; 5� reaction buffer containing 375 mm KCl and 250 mm

Tris±HCl (pH 8.3); 100 mm each dATP, dCTP, dGTP, and dTTP

(Pharmacia); ribonuclease inhibitor (RNasin, 40 U/mL, Promega);

and Moloney murine leukemia virus reverse transcriptase (200 U/mL,

GibcoBRL). One microgram of sample RNA was added to 20 mm

oligo(dT)16 primer, RT master mix, which contained 10 mm of

each dNTP, and 1 U of RNasin. The RT reaction mixture was

incubated in a Techine-PHCZ thermal cycler at 258C for 10 min,

378C for 60 min, 998C for 5 min, 48C for 5 min, and stored at

2208C. The PCR mixture contained the following: 15 mm MgCl2;

10� reaction buffer containing 500 mm KCl, 100 mm Tris±

HCl (pH 8.3), and 0.01% (w/v) gelatin; and Taq DNA polymerase

(5 U/mL; Perkin-Elmer). Primers for IL-6 and b-actin were

synthesized at Bohan Biomedical Inc. (Seoul, South Korea). The

sequences of these primers were as described previously (Faulkner

et al. 1995); b-actin, 5 0-TGGAATCCTGTGGCATCCATGAAAC-

3 0, 5 0-TAAAACGCAGCTCAGTAACAGTCCG-3 0 (348 bp); IL-6,

5 0-TGGAGTCACAGAAGGAGTGGCTAAG-3 0, 5 0-TCTGACCA-

CAGTGAGGAATGTCCAC-3 0 (155 bp). For each reaction, 30 mL

of master mix containing 10� reaction buffer, 0.5 U of Taq DNA

polymerase, and 20 mm of each primer was added to a tube

containing 3 mL of the cDNA synthesized in the RT reaction. The

tubes were incubated in a thermal cycler at 958C for 2 min (once),

948C for 45 s, 678C for 2 min, and 728C for 3 min (28±31 cycles),

and 728C for 10 min, and then held at 48C. PCR products were

visualized by ethidium bromide staining after agarose (1.2%) gel

electrophoresis. The density of each spot was quanti®ed using a

BIO-1D image analyzer. The unregulated mRNA encoding b-actin

was used as an internal loading control.

Depletion of either central or peripheral norepinephrine by

either central or peripheral injection of 6-hydroxydopamine

6-Hydroxydopamine (6-OHDA) hydrobromide (Sigma), dissolved

in sterile 0.1% ascorbic acid, was injected either i.c.v. or i.p. to

b-Amyloid peptide and interleukin-6 1327

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1326±1335

deplete either central or peripheral stores of norepinephrine (NE).

Three days after the 6-OHDA injection, NE contents were

measured in hypothalamus and spleen, as a representative tissue

for evaluating central and peripheral NE depletion, respectively.

Hypothalamus and spleen were homogenized in 0.1 m perchloric

acid containing 0.1 m sodium metabisul®te. After these samples

were centrifuged using a refrigerating microcentrifuge for 10 min,

the supernatant was ®ltered through a 0.45-mm Millipore HV-4

®lter. Ten microliters of sample was injected onto a C18 m-

Bondapak column (Waters, Milford, MA, USA). As a mobile

phase, 0.1 m KH2PO4 (adjusted to pH 3.8) containing sodium

octanesulfonic acid (0.25 mm), disodium EDTA (0.1 mm) and

acetonitrile (9%, v/v) was used. The ¯ow rate was 1 mL/min, and

the oxidation potential was 1 V. Previously, an i.c.v. injection of

6-OHDA at the dose of 50 mg/mouse was shown to cause a 73.1%

decrease in hypothalamic NE content without affecting dopamine

(DA) and serotonin content when measured 3 days after injection

(Suaudeau et al. 1995). We showed that the same dose of 6-OHDA

induced an 85.6% decrease in hypothalamic NE content without

affecting DA content 3 days after injection (Song et al. 1999a). In

contrast, an intraperitoneal injection of 6-OHDA (100 mg/kg)

induced an 86.6% decrease in NE content without changes in DA

content in spleen, when measured 3 days after injection (Song et al.

1999a). This treatment did not affect the hypothalamic NE and DA

content (Song et al. 1999a).

NE turnover

NE turnover was assessed by a decrease in tissue NE concentration

after the inhibition of NE biosynthesis with a-methyl-p-tyrosine.

Mice were injected i.c.v. with saline, Ab4221 (205 pmol/mouse), or

Ab1242 (205 pmol/mouse) and then i.p. with methyl ester of

a-methyl-p-tyrosine (300 mg/kg). Before (time 0) and 4 h after

a-methyl-p-tyrosine injection, NE levels in the hypothalamus and

spleen were determined.

Endotoxin assay

Levels of endotoxin in Abs solution were measured using an

E-Toxate kit (Sigma) and were not detected.

Statistical analysis

Statistical analysis was carried out by one-way (Fig. 1a) or two-

way (Figs 1b, 3, 4, 5b and c) analysis of variance (anova).

Bennett's test was used for post hoc comparisons. Student's t-test

was used for Table 1 and Figs 1(c) and 5(a). p-Values , 0.05 were

considered to indicate statistical signi®cance.

Results

Effect of i.c.v. Abs on plasma IL-6 levels

Various synthetic Abs, including Ab1242, Ab1240, Ab25235,

and Ab1228 were administered i.c.v., and plasma IL-6 levels

were measured at 1.5 h after injection. The native full-

length fresh Ab1242 induced a dose-dependent increase in

plasma IL-6 levels (F � 9.97, p , 0.001; Fig. 1a). Ab1242-

induced plasma IL-6 levels returned to a basal level by 6 h

after injection (Fig. 1b). Ab4221 (205 pmol) and boiled

Ab1242 (205 pmol) had no effect. `Aged' Ab1242 was more

effective than fresh (Fig. 1a); plasma IL-6 levels induced

by `aged' Ab1242 peaked at 2 h, remained increased at

6 h (Fig. 1b), and returned to basal levels 24 h after injec-

tion (data not shown). Because `fresh' Ab1242 effectively

induced an increase in plasma IL-6 in this study, we used

`fresh' Ab1242 in subsequent experiments. `Aged', but not

fresh, Ab1240 increased plasma IL-6 levels slightly at a

dose of 410 pmol/mouse i.c.v. (13 ^ 2 and 30 ^ 4 pg/mL

for saline-treated and Ab1240-treated animals, respectively;

p , 0.05). Intracerebroventricular Ab1228 and Ab25235 had

no effect at a dose of 205 pmol/mouse (data not shown).

IL-6, IL-1b and TNF-a are major pro-in¯ammatory

cytokines. Thus, it was of interest to study the effects of

i.c.v. administered Ab1242 on the plasma levels of IL-1b

and TNF-a. As shown in Fig. 1(c), Ab1242 (205 pmol/

mouse) induced a slight, but signi®cant, increase in plasma

IL-1b but not TNF-a levels. `Aged' Ab1240 (410 pmol/

mouse) also induced a slight but signi®cant increase in

plasma IL-1b (9 ^ 2 and 22 ^ 4 pg/mL for saline-,

Ab1240-treated animals, respectively; p , 0.05) but not

TNF-a levels.

Effect of i.c.v. Ab1242 on brain IL-6, IL-1b and

TNF-a levels

Intracerebroventricular injection of Ab1242 may increase

IL-6 in the brain tissue, which may leak into the systemic

circulation. Thus we examined the time-course of the

Ab1242-induced increase in IL-6 in the brain. As shown in

Fig. 2(a), Ab1242 (205 pmol/mouse) induced a marked

Table 1. Effects of intracerebroventricular injection of Ab1242 on norepinephrine turnover in the hypothalamus and spleen

4 h after a-methyl-p-tyrosine

Time 0 Saline Ab4221 Ab1242

Hypothalamus 2185� ^ 100 1123� ^ 98 1167� ^ 71 823� ^ 47*

Spleen 274� ^ 24 154� ^ 8 155� ^ 10 128� ^ 6*

Mice were injected intracerebroventricularly with Ab4221 (205 pmol/mouse) or Ab1242 (205 pmol/mouse) and then intraperitoneally with the methyl

ester of a-methyl-p-tyrosine (300 mg/kg) 4 h before norepinephrine assay. The data are means ^SE in ng/g tissue weight of six animals.

*p , 0.05, signi®cantly different from the saline-or Ab4221-treated controls.

1328 D.-K. Song et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1326±1335

increase in brain IL-6 levels, but the time course of the

increase peak at 4 h post injection was delayed compared

with that of plasma IL-6 levels. Therefore, it seems a remote

possibility that the Ab1242-induced plasma IL-6 levels were

derived mainly from the brain tissue, although it cannot be

excluded that a small portion of the plasma IL-6 may result

from brain tissue.

Plasma IL-6 levels are increased after central administra-

tion of IL-1b (De Simoni et al. 1990, 1993; Romero et al.

1996) and TNF-a (Benigni et al. 1996). In addition to IL-6,

i.c.v. injection of Ab1242 may increase IL-1b and TNF-a in

the brain tissue, which may mediate the Ab1242-induced

plasma IL-6 increase. Thus, we examined the time-course of

i.c.v. Ab1242-induced IL-1b and TNF-a in brain tissue. As

shown in Fig. 2(b,c), IL-1b and TNF-a levels in brain tissue

peaked at 24 h and 4 h after the i.c.v. Ab1242 injection,

respectively. Thus, it also seems a remote possibility that

central Ab1242-induced IL-1b and TNF-a in brain tissue

mediate the Ab1242-induced increase in plasma IL-6 levels.

Effect of i.c.v. Ab1242 on NE turnover

Intracerebroventricular administration of LPS induces an

increase in the NE turnover rate in various brain areas (Dunn

1992) and in peripheral tissues (Akiyoshi et al. 1990). Thus,

we examined the effect of i.c.v. administered Ab1242 on NE

turnover both in hypothalamus and spleen. As shown in

Table 1, i.c.v. administered Ab1242 induced a signi®cant

increase in NE turnover both in the hypothalamus and in the

spleen compared with Ab4221-administered animals. There

was no difference in central and peripheral NE turnover

between saline and Ab4221 administered groups.

Effect of 6-OHDA on Ab1242-induced plasma IL-6 levels

Both the central NE and the peripheral NE systems were

implicated in the central LPS-induced increase in plasma

IL-6 levels (Song et al. 1999b). Thus, we evaluated their

involvement in the central Ab1242-induced increase in

plasma IL-6 levels. 6-OHDA, a toxin speci®c for catechol-

amine neurons, can induce a selective depletion of NE in

brain tissue when i.c.v. administered at a certain dosage

(Suaudeau et al. 1995). In contrast, when i.p. administered,

it causes chemical sympathectomy in the periphery without

affecting central NE neurons, because 6-OHDA cannot cross

the blood±brain barrier (Kostrzewa and Jacobwitz 1977).

Thus, 6-OHDA is a useful experimental tool for the

selective depletion of either central or peripheral stores

of NE, depending on the routes of administration. The

effect of central or peripheral NE depletion with i.c.v. or

i.p. pretreatment with 6-OHDA on the effects of Ab1242

Fig. 1 Effects of Ab1242 injected intracerebroventricularly (i.c.v.) on

plasma IL-6 levels in mice (a). Either saline or various doses of fresh

and `aged' Ab1242 (7±205 pmol/mouse), boiled Ab1242 (205 pmol/

mouse), and Ab4221 (205 pmol/mouse) were administered i.c.v. and

plasma IL-6 levels were evaluated 1.5 h after injection. Time-course

of an increase in plasma IL-6 levels induced by an i.c.v. injection of

fresh and `aged' Ab1242 (b). Either saline, fresh or `aged' Ab1242

(205 pmol/mouse) was administered i.c.v. and blood was collected

at various times after the injection. `Aged' Ab1242 was used after

incubation in saline at the ®nal concentration at 378C for 3 days.

Effects of Ab1242 on plasma IL-1b and TNF-a levels in mice (c).

Plasma levels of cytokines were measured 1.5 h after i.c.v. injection

of either saline or Ab1242 (205 pmol). Values are given as means

^SEM of 8±10 animals. *p , 0.05, **p , 0.01, ***p , 0.001 signi®-

cantly different from saline-treated control animals. ²²p , 0.01,

signi®cantly different from fresh Ab1242 (205 pmol/mouse)-treated

animals.

b-Amyloid peptide and interleukin-6 1329

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1326±1335

(205 pmol/mouse)-induced increases in plasma IL-6 levels

was examined. anova revealed signi®cant effects for i.c.v.

6-OHDA (F � 6.01, p , 0.05) and Ab1242 (F � 93.21,

p , 0.001), and for i.p. 6-OHDA (F � 4.94, p , 0.05) and

Ab1242 (F � 62.77, p , 0.001). In mice pretreated with

i.c.v. 6-OHDA (50 mg/mouse) 3 days before the Ab1242

injection, the Ab1242-induced plasma IL-6 levels were

decreased by 34% (Fig. 3a). However, in mice pretreated

with i.p. 6-OHDA (100 mg/kg) 3 days before the Ab1242

injection, the Ab1242-induced plasma IL-6 levels were

decreased by 38% (Fig. 3b).

Effects of adrenoceptor antagonists on Ab1242-induced

plasma IL-6 levels

Next, to determine the adrenoceptor types involved in the

Ab1242-induced plasma IL-6 levels, either phentolamine,

an a-adrenoceptor antagonist, or propranolol, a b-adreno-

ceptor antagonist, was injected i.p. 15 min prior to Ab1242

injection. anova revealed signi®cant effects for antago-

nists (F � 16.77, p , 0.001) and Ab1242 (F � 56.14,

p , 0.001). As shown in Fig. 4(a), pretreatment with

either phentolamine mesylate (2 mg/kg) or propranolol HCl

(10 mg/kg) signi®cantly inhibited Ab1242 (205 pmol/

mouse, i.c.v.)-induced plasma IL-6 levels. To determine

the involvement of the subtypes of a- and b-adrenoceptors

in Ab1242-induced plasma IL-6 levels, prazosin HCl (an

a1-adrenoceptor antagonist; 0.5 mg/kg), yohimbine HCl (an

a2-adrenoceptor antagonist; 1 mg/kg), betaxolol HCl (a b1-

adrenoceptor antagonist; 2 mg/kg, i.p.), or ICI-118,551 HCl

(a b2-adrenoceptor antagonist; 2 mg/kg, i.p.) was injected

Fig. 2 Effects of Ab1242 injected intracerebroventricularly (i.c.v.) on

brain IL-6 (a), IL-1b (b) and TNF-a (c) levels in mice. At various time

points after injection of `aged' Ab1242 (205 pmol/mouse i.c.v.), whole

brain was homogenized and centrifuged, and the supernatant was

used for cytokine assay. Values are means ^SEM of six animals.

Fig. 3 Effects of pretreatment with (a) intracerebroventricular (i.c.v.)

or (b) intraperitoneal (i.p.) 6-hydroxydopamine (6-OHDA) on Ab1242-

induced increase in the plasma IL-6 levels. Mice were injected with

either vehicle (i.c.v. or i.p.) or 6-OHDA (50 mg/mouse i.c.v. or

100 mg/kg i.p.). Three days later, the effect of Ab1242 (205 pmol/

mouse i.c.v.) on the plasma IL-6 levels was evaluated 1.5 h after

injection. The vehicle was saline containing 0.1% ascorbic acid.

Values are means ^SEM of 14±20 animals. **p , 0.01, signi®-

cantly different from the respective controls without Ab1242.

²p , 0.05.

1330 D.-K. Song et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1326±1335

i.p. 15 min prior to the Ab1242 injection. anova revealed

signi®cant effects for antagonists (F � 7.95, p , 0.001) and

Ab1242 (F � 84.79, p , 0.001). As shown in Fig. 4(b and

c), pretreatment with prazosin, yohimbine or ICI-118,551,

but not betaxolol, signi®cantly inhibited the Ab1242

(205 ng/mouse, i.c.v.)-induced plasma IL-6 levels.

Effect of i.c.v. Ab1242 on IL-6 mRNA expression

Among the various organs, including pituitary, adrenals,

kidney, heart, liver, spleen, and lymph nodes, an i.c.v.

injection of Ab1242 (205 pmol/mouse) signi®cantly

increased IL-6 mRNA expression only in lymph nodes and

liver, as revealed by RT-PCR (Fig. 5a). Lymph nodes

displayed relatively high levels of basal IL-6 mRNA

expression (Fig. 5a). Next, the effect of central or peripheral

NE depletion on the i.c.v. Ab1242-induced IL-6 mRNA

levels in lymph nodes was examined. Both central and

peripheral NE depletion induced by an i.c.v. 6-OHDA

(50 mg/mouse) and an i.p. 6-OHDA (100 mg/kg), respec-

tively, blocked Ab1242-induced IL-6 mRNA expression in

lymph nodes (Fig. 5b,c). anova revealed signi®cant effects

for i.c.v. 6-OHDA (F � 133.9, p , 0.001) and Ab1242

(F � 116.3, p , 0.001), and for i.p. 6-OHDA (F � 222.2,

p , 0.001) and Ab1242 (F � 136.3, p , 0.001). In animals

pretreated with either i.c.v. or i.p. 6-OHDA, central Ab1242

injection did not signi®cantly increase hepatic IL-6 mRNA

expression (data not shown).

Discussion

Ab1242, which is the most insoluble and prone to aggregate,

and thus the most toxic of the various Abs (Giulian et al.

1996; Klein et al. 1999), is believed to be critically

important in AD (Iwatsubo et al. 1994). In this study,

we found a novel biological activity of Ab1242, i.e. the

induction of systemic IL-6 responses by centrally adminis-

tered Ab1242. The Ab1242-induced increase in plasma

cytokine levels extended to plasma IL-1b, but not to TNF-a.

Furthermore, we found for the ®rst time that i.c.v.

administered Ab1242 increases both central and peripheral

NE turnover. Because depletion of either central or

peripheral NE effectively inhibited the central Ab1242-

induced systemic IL-6 responses, it is suggested that the

Ab1242-induced activation of both central and peripheral

NE system mediates, at least in part, the central Ab1242-

induced systemic IL-6 responses. These novel in vivo

biological activities of Ab1242 are quite similar to those of

LPS; central injection of LPS is known to increase both the

central and peripheral NE systems (Akiyoshi et al. 1990;

Dunn 1992), and to induce systemic IL-6 responses

(Gottschall et al. 1992; Muramami et al. 1993; De Simoni

et al. 1995). We recently reported that central administration

of LPS induces systemic IL-6 responses, in part, via both the

central and the peripheral NE systems (Song et al. 1999b),

further illustrating the similarity between the actions of

Ab1242 and LPS. However, the intensity of centrally adminis-

tered Ab1242 in inducing systemic immune responses is

much smaller than that of LPS; the magnitude of the

Fig. 4 Effects of adrenoceptor antagonists injected intraperitoneally

(i.p.) on Ab1242-induced plasma IL-6 levels in mice. Either vehicle

(a) phentolamine mesylate (2 mg/kg) or propranolol HCl (10 mg/kg)

(b) prazosin HCl (0.5 mg/kg) or yohimbine HCl (1 mg/kg) (c) betaxo-

lol HCl (2 mg/kg) or ICI-118,551 HCl (2 mg/kg) was given as

pretreatment i.p. 15 min before the Ab1242 (205 pmol/mouse i.c.v.)

injection. Plasma IL-6 levels were evaluated 1.5 h after Ab1242 injec-

tion. Vehicle was saline for (a) and (c), and saline containing 20%

dimethylsulfoxide for (b). Values are means ^SEM of 8±14 animals.

*p , 0.05, **p , 0.01, ***p , 0.001 signi®cantly different from the

respective saline-treated controls. ²²p , 0.01, ²²²p , 0.001.

b-Amyloid peptide and interleukin-6 1331

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1326±1335

increase in plasma IL-6 induced by the maximal dose

of Ab1242 and LPS was < 100±200 pmol/mL, and

< 1500 pmol/mL (Song et al. 1999b), respectively. The

spectrum of peripheral organs that display increased IL-6

mRNA in response to central LPS (pituitary, adrenals, heart,

liver, spleen, and lymph nodes) (Song et al. 1999b) was

wider than that in response to central Ab1242 (liver and

lymph nodes) in this study. Furthermore, plasma TNF-a

levels were not increased by Ab1242, whereas central

injection of LPS effectively increased plasma TNF-a levels

(De Luigi et al. 1998).

In this study, we found for the ®rst time that central

injection of Ab1242 effectively induces an increase in brain

levels of various pro-in¯ammatory cytokines. However, a

time-course study showed that central Ab1242-induced

increases in brain IL-6, TNF-a, and IL-1b levels were

slower than that of plasma IL-6. Therefore, it seems that

central Ab1242-induced plasma IL-6 levels do not result

mainly from the central Ab1242-induced increase in brain

pro-in¯ammatory cytokine levels. In a similar vein, delayed

induction of brain IL-6 mRNA compared with peripheral

IL-6 mRNA was also reported in the central LPS-induced

IL-6 responses (Song et al. 1999b). However, the precise

intercellular mechanisms, including the involvement of

microglia, of the central Ab1242-induced plasma IL-6

response and activation of the NE neuronal system remain

to be further investigated.

Some investigators have reported elevated mean levels of

circulating IL-6 in AD patients (Kalman et al. 1997; Singh

and Guthikonda 1997; Bonaccorso et al. 1998; Licastro et al.

2000), whereas others have reported no signi®cant differ-

ences (van Duijn et al. 1990; Licastro et al. 1995a; Angelis

et al. 1998). However, even when the mean levels of

circulating IL-6 were not signi®cantly different, the positive

rate for detecting circulating IL-6 was higher in AD patients

than in controls [11% vs. 5% (van Duijn et al. 1990); 32%

vs. 15% (Licastro et al. 1995a); 27% vs. 19% (Angelis et al.

1998)]. Our ®nding that central administration of Ab1242

can induce systemic IL-6 responses may serve as an experi-

mental basis for the possibility that Ab1242±induced

in¯ammatory process in the AD brain may induce the

various derangement in systemic IL-6 systems reported in

some AD patients.

We found that i.c.v. Ab1242 injection selectively induced

IL-6 mRNA expression in lymph nodes and liver among

the various peripheral organs we examined. This suggests

that the Ab1242-induced IL-6 may particularly in¯uence

immunological and hepatic functions. Although the response

in liver was less marked than that in lymph nodes,

considering the much greater organ weight of the former

Fig. 5 Effects of Ab1242 on the IL-6 mRNA

levels in various peripheral organs (a).

Ab1242 (205 pmol/mouse) was administered

intracerebroventricularly (i.c.v.), and IL-6

mRNA expression was evaluated 1 h after

injection. Effects of pretreatment with (b)

i.c.v. or (c) intraperitoneal (i.p.) 6-hydroxy-

dopamine (6-OHDA) on the Ab1242-induced

increase in IL-6 mRNA levels in lymph

nodes. Mice were injected with either vehi-

cle (i.c.v. or i.p.) or 6-OHDA (50 mg/mouse

i.c.v. or 100 mg/kg i.p.). Three days later,

the effect of Ab1242 (205 pmol/mouse

i.c.v.) on the IL-6 mRNA expression was

evaluated at 1 h after injection. **p , 0.01,

***p , 0.001, signi®cantly different from

the respective saline-treated controls.

²²p , 0.01, ²²²p , 0.001. Values are

means ^SEM of three experiments, and

the gel data shown are from the representa-

tive experiments. The number of animals

used for each experiment was seven for

pituitary, ®ve for adrenals, and three for the

rest of the tissues.

1332 D.-K. Song et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1326±1335

compared with the latter, the contribution of liver to central

Ab1242-induced plasma IL-6 levels may not be less, or may

even be more, than that of lymph nodes. Because IL-6 is a

major cytokine involved in hepatic acute phase protein

synthesis, the Ab1242-induced increase in hepatic IL-6

mRNA in this study may be related to previous reports of

increased serum levels of a-antichymotrypsin, an acute-

phase protein, in some AD patients (Matsubara et al. 1990;

Brugge et al. 1992; Hinds et al. 1994; Altstiel et al. 1995;

Licastro et al. 1995a, b, 1997, 2000; Lieberman et al. 1995).

Among the various Abs with differing peptide lengths,

Ab1242 was the most effective; it increased plasma IL-6

levels even in the fresh state, whereas Ab1240 was margin-

ally effective only after the `aging' process, i.e. formation of

®brillar peptide, and other synthetic Abs were without

effect. Interestingly, this pattern of activity is almost iden-

tical to the neurotoxicity pro®le of various Abs via

stimulation of microglia (Giulian et al. 1996); in that report

Ab1242 is more potent than Ab1240 in activating microglia

to kill neurons, whereas other Abs, including Ab1228 and

Ab25235, have no effect. Similarly, non®brillar Ab1242 and

®brillar, but not non®brillar, Ab1240 have been shown to

increase LPS-induced IL-1b release from monocytes

(Lorton et al. 1996). The result of this study, which is in

line with the well-known phenomenon that the `aging'

process, i.e. the formation of ®brillar peptide, induces or

increases neurotoxicity (Pike et al. 1993) and the glial/

monocyte stimulating activity (Gitter et al. 1995; Lorton

et al. 1996; McDonald et al. 1997) of various Abs, suggests

that the activity of centrally administered Abs in inducing a

peripheral IL-6 response is also dependent on the degree of

aggregation or ®brillar formation. Furthermore, the lack of

effect of Ab4221 on NE turnover and plasma IL-6 levels

indicates the necessity for the speci®c conformation of

Ab1242 in activating NE system and in inducing peripheral

IL-6 responses.

In conclusion, this study demonstrates that centrally

administered Ab1242 effectively induces systemic IL-6

responses, which are mediated, at least in part, by central

Ab1242-induced activation of the central and the peripheral

NE systems. These activities of Ab1242, together with the

induction of pro-in¯ammatory cytokine levels in the brain

tissue, further illustrate the immune stimulating activity of

Ab1242 in vivo.

Acknowledgements

This work was supported by the Korea Ministry of Education

(Basic Medicine Research Fund 1997 (997-021-F0026) and

Brain Korea 21 Program), KOSEF through the Brain Disease

Research Center at Ajou University and the Korea Research

Foundation (1998-019-F00047). We thank Drs Yoo-Hun Suh

and Sung-Oh Huh for encouragement and kind advise,

Dr Choon-Gon Jang for statistical analysis and Mr Gordon

Slysz for revision of the English text.

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