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
COMMUNICATIONS
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