Vol.

174,

February

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

14, 1991

Pages

GASTRIC CYTOPROTECTION INTERLEUKIN-16 Andre Adalsteinn

BY INTRACISTERNAL IN THE RAT

Robert*, Esteban Saperas#, Weirong i!hang*, 5. Olafsson*, Cleo Lancaster*, Daniel E. Tracey**, John G. Chosay**

*Safety

1117-1124

Pharmacology, The Upjohn

and Yvette Tachk#

**Hypersensitivity Diseases Company, Kalamazoo, MI

Research,

# CURE, VA Wadsworth Medical Center, Department of Medicine and Brain Institute, UCLA, Los Angeles, CA Received

December

5, 1990

SUMMARY:

The effects of intracisternal (ic) injection of recombinant interleukin-lB(IL-1) on absolute ethanol-induced gastric necrotic lesions were studied in conscious rats. IL-1 given ic inhibited ethanol-induced gastric lesions. The cytoprotective effect was dose dependent (ED175 rig/rat), long lasting with a maximal action when given l-3 h prior to ethanol, blocked by ic injection of a IL-l receptor antagonist protein (IRAP), and by intraperitoneal injection of indomethacin. IL-l, injected ic, was detected in the peripheral blood. However, IL-1 serum levels were lower after IL-l injection ic than after ip at a dose giving equal gastric protection. These data show that ic IL-l induces long lasting gastric protection mediated by interaction with IL-l receptors and prostaglandin pathways at central and/or peripheral sites that remain to be localized. 0 1991 Academicpress, Inc.

Interleukin-18 important

role

, a 153 amino in immune

neuroanatomical in the and

nervous

receptors

have

cerebrospinal

fluid

sympathetic

activity

(10).

centrally

of prostaglandin We recently acid secretion secretion induced

system been

processes

have

(S),

in the

pathways (13). been

Several shown

by stress or central

brain

(3-5).

sites

induces

peripheral

biological

actionsof

to play

(1, 2).

indicates

that

IL-l

immunoreactivity

IL-l

injected

into

a pyrogenic immune

IL-l require

acts the

response

secretion

cellular

an

Recent

IL-l

pituitary-adrenal

decreases

(6,8, that

brain

activates

mediated

reported

evidence

as a neuromodulator.

(CSF) or specific (9), and

is recognized

inflammatory

located

hyperinsulinemia Several

and

polypeptide,

and neuropharmacological

central

(6,7),

acid

and response

the integrity

11-13).

IL-1 injected peptides

acting

to prevent injection

ic acts in the brain in the brain the

to inhibit

development

of thytotropin

to inhibit

releasing

gastric

gastric

of gastric hormone

acid

lesions (14)

Vol.

174, No. 3, 1991

BIOCHEMICAL

AND BIOPHYSICAL

However, there is little evrdence that these centrally agents introduced

RESEARCH COMMUNICATIONS

actrng peptides offer

cytoprotection

against necrotizing

in the stomach (15-18).

Cytoprotection

has been linked to activation of prostaglandin

pathways (19),

and the antisecretory effect of IL-l ic (13) and ip (20,21) is mediated through prostaglandin mechanisms. In the present studies, we investigated ic injection of IL-l preventsethanol-induced gastric lesions.

whether

MATERIAL AND METHODS Animals: Experiments were performed in male and female Spra ue-Dawley -300 g) (Harlan SD Laboratories) fasted for 18 h in isolate 8 cages with free accessto water up to 2 h before the beginning of the experiments. Gastric necrotic lesions: Absolute ethanol (lml) was given through an oroesophageal tube to conscious rats at various times after either ic or ip (intraperitoneal) injection of vehicle [O.l% bovine serum albumin (BSA)] or IL1. Rats were killed by CO2 inhalation 1 h after absolute ethanol. The stomachs were removed, randomized, opened along the greater curvature and rinsed with running water. The mucosa was examined with a 5 x binocular magnifier by an investigator unaware of the treatment given, and the number of gastric lesions was counted. Some stomachs in control and ILl-treated groups were processed by standard techniques for histological examination. Druqs and treatments: IL-l 8 (human recombinant) used in Figures 1, 3 and 4 w-by Dr. Alan R. Shaw (Glaxo Institute for Molecular Biology, S.A, Geneva, Switzerland). It was diluted in 100 mM Tris-HCI and 2 mM sodium azide buffer pH 7.8, aliquoted to a concentration of 1 mg/lO ml, and stored at -70°C. In the other studies, human recombinant IL-l B produced in the Upjohn Laboratories, Kalamazoo, Ml, was used (22). The specific activity of the Glaxo lot was 2.5 x 107 LAF units/mg protein (lymphocyte activator factor), and that of the Upjohn lot was 2 x 107 units LAF/mg protein. IL-l or vehicle was injected either ic in 10 ~1 or ip in 0.5 ml under ether anesthesia at various doses and time intervals before ethanol administration. The IL-l 8 receptor antagonist protein, IRAP (Upjohn Laboratories, Kalamazoo, Ml), a specific IL-l receptor anta onist (23), was diluted in 0.1% BSA and injected ic under ether anesthesia 940 ug/rat in 5 ul) immediately before ic injection of IL-l (500 rig/rat in 5 ul). lndomethacin (5 mg/k ; Sigma Chemical Co., St. Louis, MO) was dissolved in 0.5 ml 1% NaHCO3 an c?injected ip 1 h prior to ic injection of IL-l. Measurements of blood levels of IL-l: 125l-IL-18 (specific activity 2890 Ci/mmol; Upjohn Laboratories, Kalamazoo MI) was administered either ic or ip (I .9 ng containing 106 cpm). Nonradioactive IL-l Bwas added to make total doses of 75, 150, 300 and 500 rig/rat. One hour after injection, aortic blood was collected. Radioactivity was counted in 100 ul serum in a gamma counter. The sera were also assayed for IL-l bioactivity in a sensitive comitogenegis assay employing the murine T-lymphoma cell lines LBRM-33lA5 and HT-2, as described previously (24). The sera were diluted to 1:500 to avert the inhibitory effects of serum on detection of IL-l Statistical analysis: The results are presented as means f SEM. Comparisons between two groups were calculated by the Student t test and the Fisher’s exact test. Multiple group comparisons were performed by ANOVA followed by post hoc multiple comparisons. Differences between means with a p < 0.05 were considered statistically significant. 1118

Vol.

174,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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RESULTS Dose-dependent (100-500

prevention

rig/rat)

injected

dose dependent

cytoprotection the

completely limited

for both was

massive

prevented to

routes

by ic or ip IL-l

the

monolayer

levels

of IL-l

at a 500

necrosis (500

surface

ng).

epithelium,

in a

lesions

(Fig.

(175 rig/rat). given

by 100%

The

IL-1

resulted

necrotic

ng dose

caused

ip IL-l.

ethanol

gastric

of administration

observed

mucosal

by ic and

to absolute

of ethanol-induced

1). The ED50 was similar complete

lesions

ic or ip 1 h prior

inhibition

Histologically,

of ethanol

only

which

Near

ic or ip.

ethanol

was

damage

seen was

showed

areas

of

exfoliation. Serum measured

In the serum

of unlabeled effect

were

ic and

1 h after

ic injection

IL-1 at various

of the transport

1251.IL-I

followinq

from

significantly

unlabeled

IL-1 was

difference

in the serum

of IL-1 measured

doses

with

after

sensitive

Co-administration polypeptide

However, after

was

the serum

had

no

levels

of

ip administration. of

150

rig/rat,

(Fig. 2 LEFT).

specific

bioassay

When the Serum

mean levels

rose. in a dose

lntracisternal

IT

75 IL-

150 I

Gastric lesions were mean -+ SEM of 5-12

200

DOSE

Fiaure 1. Dose dependent prevention IL-l injected either ic or ip. Absolute with vehicle-treated

labeled

(p < 0.01)

and

Radioactivity

lzsl-IL-l.

at a dose

levels was 36%

0

vehicle or IL-l. represents the

the

tc than

co-admrnistered

15

of

the CSF to blood. lower

by the

ip injection.

300

500

(rig/rat)

of ethanol-induced gastric lesions by ethanol (1 ml) was injected 1 h after

counted 1 h after rats. * p < 0.01,

group. 1119

ethanol. Each column ** p < 0.001 compared

Vol.

174,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

IL-l: Bioassay

‘251-IL-1 120

600-

1 7

500-

$

400

t 8

300-

;-: 1 T T

200-

40

v= i!

z

-

loo-

20

01

I

O’Y,,,,, 0

100

200

300

400

0

500

100

IL-l: rig/rat

200

IL-l:

300

400

500

rig/rat

Figure 2. Serum levels of IL-l following ~c or ip injection of IL-l. Serum obtained 1 h after treatment with IL-l. LEFT: Radioactivity after IIs-IL-1 given ic or ip together with various amounts of unlabeled IL-l. RIGHT: IL-l measured by bioassay after IL-1 given ic or ip. n = 6. dependently values 0.05)

(75-500

were

47%

ngkat),

lower

1 h after

for ic than

ic Injection

ip injection

of IL-l.

The

bioassay

at a dose of 300 rig/rat

(p

Gastric cytoprotection by intracisternal interleukin-1 beta in the rat.

The effects of intracisternal (ic) injection of recombinant interleukin-1 beta (IL-1) on absolute ethanol-induced gastric necrotic lesions were studie...
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