Peripheral 5-HT7 receptors as a new target for prevention of lung injury and mortality in septic rats

Abstract

Sepsis is a complex pathophysiological event involving metabolic acidosis, systemic inflammatory response syndrome, tissue damage and multiple organ dysfunction syndrome. Although many new mechanisms are being investigated to enlighten the pathophysiology of sepsis, there is no effective treatment protocol yet. Presence of 5-HT7 receptors in immune tissues prompted us to hypothesize that these receptors have roles in inflammation and sepsis. We investigated the effects of 5-HT7 receptor agonists and antagonists on serum cytokine levels, lung oxidative stress, lung histopathology, nuclear factor κB (NF-κB) positivity and lung 5-HT7 receptor density in cecal ligation and puncture (CLP) induced sepsis model of rats. Agonist administration to septic rats increased survival time; decreased serum cytokine response against CLP; decreased oxidative stress and increased antioxidant system in lungs; decreased the tissue NF-κB immunopositivity, which is high in septic rats; and decreased the sepsis-induced lung injury. In septic rats, as a result of high inflammatory response, 5-HT7 receptor expression in lungs increased significantly and agonist administration, which decreased inflammatory response and related mortality, decreased the 5-HT7 receptor expression. In conclusion, all these data suggest that stimulation of 5-HT7 receptors may be a new therapeutic target for prevention of impaired inflammatory response related lung injury and mortality.

Full text

Immunobiology
218 (2013) 1271–
1283
Contents
lists
available
at
SciVerse
ScienceDirect
Immunobiology
jou
rn
al
hom
ep
age:
www.elsevier.com/locate/imbio
Peripheral
5-HT7
receptors
as
a
new
target
for
prevention
of
lung
injury
and
mortality
in
septic
rats夽
Elif
Cadircia,
Zekai
Halicib,∗,
Yasin
Bayirc,
Abdulmecit
Albayrakb,
Emre
Karakusd,
Beyzagul
Polata,
Deniz
Unale,
Sabri
S.
Atamanalpf,
Selina
Aksake,
Cemal
Gundogdug
aAtatürk
University,
Faculty
of
Pharmacy,
Department
of
Pharmacology,
Erzurum,
Turkey
bAtatürk
University,
Faculty
of
Medicine,
Department
of
Pharmacology,
Erzurum,
Turkey
cAtatürk
University,
Faculty
of
Pharmacy,
Department
of
Biochemistry,
Erzurum,
Turkey
dAtatürk
University,
Faculty
of
Veterinary
Medicine,
Department
of
Pharmacology
and
Toxicology,
Erzurum,
Turkey
eAtatürk
University,
Faculty
of
Medicine,
Department
of
Histology
and
Embryology,
Erzurum,
Turkey
fAtatürk
University,
Faculty
of
Medicine,
Department
of
General
Surgery,
Erzurum,
Turkey
gAtatürk
University,
Faculty
of
Medicine,
Department
of
Pathology,
Erzurum,
Turkey
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
3
December
2012
Accepted
21
April
2013
Available online 27 April 2013
Keywords:
Sepsis
5-HT7
receptor
CLP
Rat
NF-␬B
a
b
s
t
r
a
c
t
Sepsis
is
a
complex
pathophysiological
event
involving
metabolic
acidosis,
systemic
inflammatory
response
syndrome,
tissue
damage
and
multiple
organ
dysfunction
syndrome.
Although
many
new
mech-
anisms
are
being
investigated
to
enlighten
the
pathophysiology
of
sepsis,
there
is
no
effective
treatment
protocol
yet.
Presence
of
5-HT7
receptors
in
immune
tissues
prompted
us
to
hypothesize
that
these
receptors
have
roles
in
inflammation
and
sepsis.
We
investigated
the
effects
of
5-HT7
receptor
agonists
and
antagonists
on
serum
cytokine
levels,
lung
oxidative
stress,
lung
histopathology,
nuclear
factor
␬B
(NF-␬B)
positivity
and
lung
5-HT7
receptor
density
in
cecal
ligation
and
puncture
(CLP)
induced
sepsis
model
of
rats.
Agonist
administration
to
septic
rats
increased
survival
time;
decreased
serum
cytokine
response
against
CLP;
decreased
oxidative
stress
and
increased
antioxidant
system
in
lungs;
decreased
the
tissue
NF-␬B
immunopositivity,
which
is
high
in
septic
rats;
and
decreased
the
sepsis-induced
lung
injury.
In
septic
rats,
as
a
result
of
high
inflammatory
response,
5-HT7
receptor
expression
in
lungs
increased
significantly
and
agonist
administration,
which
decreased
inflammatory
response
and
related
mortality,
decreased
the
5-HT7
receptor
expression.
In
conclusion,
all
these
data
suggest
that
stimulation
of
5-HT7
receptors
may
be
a
new
therapeutic
target
for
prevention
of
impaired
inflammatory
response
related
lung
injury
and
mortality.
© 2013 Elsevier GmbH. All rights reserved.
Introduction
Sepsis
is
one
of
the
most
prevalent
diseases
and
one
of
the
main
causes
of
death
among
hospitalized
patients
(Alberti
et
al.
2002).
Sepsis/septic
shock
is
a
complex
pathophysiological
event
charac-
terized
by
profound
hypotension,
progressive
metabolic
acidosis,
systemic
inflammatory
response
syndrome,
tissue
damage
and
multiple
organ
dysfunction
syndrome,
acute
respiratory
distress
syndrome
(ARDS)
and/or
acute
lung
injury
(ALI)
and
even
death
(Ozdulger
et
al.
2003).
Although
its
pathophysiology
is
not
well
defined,
monocytes
orchestrate
the
innate
immunity
response
to
bacteria
by
expressing
a
variety
of
inflammatory
cytokines;
in
par-
ticular,
early
TNF-␣,
IL-1␤
and
IL-6
are
considered
to
participate
prominently
in
the
pathogenesis
of
sepsis
(Moine
and
Abraham
夽National
patent
application
has
been
made
for
this
study.
∗Corresponding
author.
Tel.:
+90
442
2346561;
fax:
+90
442
2360962.
E-mail
address:
hzekai@atauni.edu.tr
(Z.
Halici).
2004).
Nuclear
factor
␬B
(NF-␬B)
is
an
inducible
nuclear
transcrip-
tion
factor
that
plays
a
central
role
in
regulating
the
transcription
of
several
genes,
including
those
that
encode
the
proinflammatory
cytokines
(Shen
et
al.
2009;
Tergaonkar
2006)
such
as
TNF-␣,
IL-1␤,
IL-6,
adhesion
molecules
and
additional
proinflammatory
media-
tors
involved
in
severe
sepsis,
septic
shock,
ARDS
and
ALI
(Bone
1991;
Hotchkiss
and
Karl
2003).
Proinflammatory
cytokines
induce
release
of
secondary
cytokines,
lipid
mediators
and
reactive
oxygen
species
(ROS).
In
recent
studies,
oxidative
stress
has
been
shown
to
play
an
important
role
in
sepsis-related
mortality
and
organ
dys-
function
(Cadirci
et
al.
2010a;
Uyanik
et
al.
2010;
Albayrak
et
al.
2011).
Although
many
new
mechanisms
are
being
investigated
to
enlighten
the
pathophysiology
of
sepsis,
there
is
no
effective
treat-
ment
protocol
yet.
In
the
literature,
there
is
a
sizeable
body
of
clinical
and
experimental
studies
focusing
on
pathophysiology
and
treatment
of
sepsis,
showing
the
popularity
and
importance
of
this
issue.
After
a
detailed
literature
search,
we
decided
to
investigate
serotonin
receptors,
especially
5-HT7,
in
sepsis.
0171-2985/$
–
see
front
matter ©
2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.imbio.2013.04.012

1272 E.
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et
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/
Immunobiology
218 (2013) 1271–
1283
Serotonin
(5-hydroxytryptamine,
5-HT)
is
a
biogenic
monoamine
with
a
variety
of
functions
in
many
physiological
and
pathophysiological
processes
(Yang
et
al.
2006).
It
is
well
established
that
various
biological
effects
of
5-HT
are
mediated
through
different
serotonin
receptors
and
their
signal
transduction
pathways
(MaassenVanDenBrink
et
al.
2008).
Being
among
the
most
recently
discovered
receptors
for
serotonin,
the
5-HT7
recep-
tor
is
also
one
of
the
least
well
characterized
(Hedlund
et
al.
2004;
Hedlund
and
Sutcliffe
2004;
Hedlund
2009).
Subsequent
in
situ
hybridization
studies,
in
both
rats
and
guinea
pigs,
have
confirmed
that
5-HT7
receptor
mRNA
in
the
brain
is
found
predominantly
in
the
thalamus,
hypothalamus,
cerebral
cortex,
hippocampus
and
amygdala
(Neumaier
et
al.
2001;
To
et
al.
1995;
Tsou
et
al.
1994).
However,
studies
about
peripheral
effects
of
these
receptors
are
limited.
These
receptors
have
been
shown
in
arteries
such
as
pulmonary,
coronary
arteries
and
aorta
(Nilsson
et
al.
1999;
Ullmer
et
al.
1995).
The
presence
of
5-HT7
receptors
has
been
shown
in
T
cells:
5-HT7
receptor
stimulation
contributes
in
T-cell
functions
and
activations
(Leon-Ponte
et
al.
2007).
Soga
et
al.
showed
that
5-HT
prevents
monocytic
apoptosis
by
5-HT7
and/or
5-HT7
receptors
(Soga
et
al.
2007).
Stefuj
et
al.
showed
presence
of
5-HT7
receptor
mRNA
in
both
human
and
rat
immune
tissues
such
as
thymus,
spleen,
peripheral
lymphocytes
and
mitogen-activated
spleen
cells
(Stefulj
et
al.
2000).
Presence
of
5-HT7
receptors
in
both
human
(Heidmann
et
al.
1997)
and
rat
(Stefulj
et
al.
2000;
Shen
et
al.
1993)
immune
tissues
prompted
us
to
hypothesize
that
these
receptors
have
roles
in
immune
response
in
inflammation
and
sepsis.
However,
there
is
no
study
investigating
the
effects
of
5-HT7
receptors
in
sepsis
and
sepsis-induced
lung
damage.
In
light
of
the
above
data,
this
study
aimed
(1)
to
investigate
the
possible
role
of
5-HT7
receptors
in
cecal
ligation
and
puncture
(CLP)
induced
polymicrobial
sepsis
model,
(2)
to
determine
5-HT7
recep-
tor
presence
in
lung
tissue,
which
is
important
in
sepsis-related
mortality,
and
(3)
to
observe
the
effects
of
5-HT7
receptor
agonist
and
antagonist
administration
on
normal
progress
of
sepsis.
Materials
and
methods
Animals
Eighty
male
Wistar
rats
were
used
for
all
experiments,
each
weighing
200–220
g,
obtained
from
Ataturk
University’s
Experimental
Animal
Laboratory
of
Medicinal
and
Experimental
Application
and
Research
Center
(Erzurum,
Turkey).
Animal
exper-
iments
were
performed
in
accordance
with
the
national
guidelines
for
the
use
and
care
of
laboratory
animals
and
were
approved
by
Ataturk
University’s
local
animal
care
committee.
Rats
were
housed
in
standard
plastic
cages
on
sawdust
bedding
in
an
air-conditioned
room
at
22 ◦C
under
lighting
controls
(14-h-light/10-h-dark
cycle).
Chemicals
AS-19
(5-HT7
receptor
agonist)
(Cat.
No.
1968;
TOCRIS
Bio-
science)
and
SB
269970
hydrochloride
(5-HT7
receptor
antagonist)
(Cat.
No.
1612;
TOCRIS
Bioscience)
were
purchased
from
TOCRIS
(Ellisville,
MO,
USA).
All
other
chemicals
for
laboratory
exper-
imentation
were
purchased
from
Sigma
Chemical
Co.
(Munich,
Germany).
Experimental
groups
1.
Sham
(n
=
20)
2.
CLP
(n
=
20)
3.
CLP
+
agonist
AS-19
(n
=
20)
4.
CLP
+
antagonist
SB
269970
(n
=
20)
Drug
administration
A
10-mg/kg
dose
of
5-HT7
receptor
agonist
AS-19
(Hedlund
2009;
Bosker
et
al.
2009)
and
antagonist
SB
269970
(Forbes
et
al.
2002;
Thomas
et
al.
2003)
were
dissolved
in
100
mM
dimethyl
sulf-
oxide
(DMSO)
and
administered
intraperitoneally.
The
sham
and
CLP
groups
received
the
same
amount
of
DMSO
as
a
vehicle.
Drug
applications
were
performed
twice:
the
first
30
min
before
sepsis
induction,
and
the
second
8
h
after
sepsis
induction.
Polymicrobial
sepsis
model
Polymicrobial
sepsis
was
induced
by
cecal
ligation
and
two-
hole
puncture,
as
described
by
Wichterman
et
al.,
1980,
with
minor
modifications.
Anesthesia
was
induced
by
intraperitoneal
adminis-
tration
of
thiopental
25
mg/kg.
After
shaving
the
rats’
abdomen,
the
peritoneum
was
opened.
The
cecum
was
isolated
and
ligated
with
a
3–0
silk
ligature
just
distal
to
the
ileocecal
valve.
Two
punctures
were
made
with
a
16-gauge
needle
through
the
cecum
distal
to
the
point
of
ligation,
and
the
cecum
was
returned
to
the
peritoneal
cavity.
The
abdominal
incision
was
then
closed
with
a
4–0
sterile
synthetic
absorbable
suture.
The
wound
was
bathed
in
a
1%
lido-
caine
solution
to
provide
analgesia.
Laparotomy
was
performed
on
the
sham-operated
groups,
and
the
cecum
was
manipulated,
but
not
ligated
or
perforated.
All
animals
were
given
2
mL/100
g
body
weight
of
normal
saline
subcutaneously
at
the
time
of
surgery
and
also
6
h
postoperatively,
for
fluid
resuscitation.
Postoperatively,
the
rats
were
observed
for
the
next
16
h.
At
the
16th
hour,
six
rats
were
randomly
selected
from
each
group
for
biochemical,
histopatho-
logical
and
molecular
analyses.
The
selected
rats
were
killed
16
h
later
by
an
overdose
of
a
general
anesthetic
(thiopental
sodium,
50
mg/kg).
Cardiac
blood
samples
were
collected
immediately
and
transferred
to
the
laboratory
for
the
estimation
of
TNF-␣,
IL-1␤
and
IL-6
levels
in
serum.
The
lungs
were
rapidly
removed
from
all
rats
and
washed
in
ice-cold
saline.
Half
of
the
lung
tissues
were
transferred
to
a
biochemistry
laboratory
to
be
kept
at
80 ◦C
for
bio-
chemical
and
molecular
analyses;
the
other
half
of
the
lung
tissues
were
fixed
in
10%
formalin
solution
for
histopathological
analyses.
Surviving
rats
from
each
group
were
observed
for
survival
times
for
72
h.
Biochemical
analyses
Serum
was
obtained
from
blood
samples
and
TNF-␣
(Invitro-
gen;
KRC33012),
IL-1␤
(Invitrogen;
KRC0012)
and
IL-6
(RayBiotech;
Cat-ELR-IL6-001)
concentrations
were
analyzed
with
ELISA
accord-
ing
to
the
manufacturer’s
instructions.
Also,
all
rats’
lungs
were
analyzed
for
oxidative
stress.
For
this
purpose,
8-isoprostan
(Cell
Biolabs
Inc.;
STA-337),
superoxide
dismutase
(SOD)
(Cayman;
706002),
and
glutathione
(GSH)
(Cell
Biolabs;
STA-312)
were
eval-
uated
with
ELISA
kits
according
to
the
manufacturer’s
instructions.
All
measurements
were
performed
twice
and
their
means
were
evaluated
statistically.
Histopathologic
analyses
Histopathological
procedure:
Each
lung
sample
was
fixed
in
buffered
10%
formalin
solution
for
48–55
h,
dehydrated
in
a
graded
alcohol
series,
embedded
in
paraffin
wax
and
sectioned
using
a
microtome
(Leica
RM2125RT).
Five-␮m
sections
were
mounted
onto
glass
slides
for
analysis.
For
light
microscopic
histological
examination,
sections
were
stained
with
hematoxylin
and
eosin
(H&E).
The
slides
were
covered,
and
photographs
were
taken
using
a
light
microscope
with
a
camera
attachment
(Nikon
Eclipse
E600,
Japan).

E.
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Immunobiology
218 (2013) 1271–
1283 1273
Inflammation
scoring
in
lungs:
Sections
were
obtained
randomly
and
were
scored
depending
on
the
degree
of
inflammation
in
the
perivascular
area
as
follows:
0:
no
inflammatory
cells
(ICs)
1:
a
few
ICs
2:
many
ICs
in
the
peripheral
parts
of
the
perivascular
area
3:
numerous
ICs
in
the
perivascular
area
(Singh
et
al.
2005)
Immunohistochemistry
by
5HT-7
and
NF-B
in
paraffin
sections:
Sections
were
mounted
on
polylysine
slides
and
stained
with
5HT-
7
Ab
Leica
Bond
Polymer
Refine
Detection
Kit
and
NF-␬B
in
an
immunohistochemical
staining
machine
(Leica
Bond-Max).
Statistical
analyses
Results
were
expressed
as
mean
±
standard
deviation.
All
para-
metric
data
were
analyzed
with
one
way
analyses
of
variance,
Tukey’s
test.
p
<
0.05
was
accepted
as
significant
when
compared
to
CLP
group.
Results
Survival
time
In
the
experimental
groups,
there
was
no
death
in
the
sham
group
during
the
72-h
observation
period.
The
rats
in
the
CLP
group
survived
for
16.71
±
5.17
h;
however,
the
rats
in
the
CLP
+
agonist
group
survived
for
35.43
±
21.27
h.
Mean
survival
time
of
the
rat
groups
were
determined
as
CLP:
16.71;
CLP
+
agonist:
35.43
(p
<
0.05)
and
CLP
+
antagonist:
17.14
±
4.82
(p
>
0.05).
There
was
Fig.
1.
The
number
of
surviving
rats
during
72
h.
Fig.
2.
Serum
TNF-␣
levels
(*p
<
0.05
significant
when
compared
to
CLP
group).
Fig.
3.
Serum
IL-1␤
levels
(*p
<
0.05
significant
when
compared
to
CLP
group).
Fig.
4.
Serum
IL-6
levels
(*p
<
0.05
significant
when
compared
to
CLP
group).
Fig.
5.
Lung
8-izoprostan
concentration
(*p
<
0.05
significant
when
compared
to
CLP
group).
Fig.
6.
Lung
GSH
concentration
(*p
<
0.05
significant
when
compared
to
CLP
group).

1274 E.
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/
Immunobiology
218 (2013) 1271–
1283
Fig.
7.
Lung
SOD
activity
(*p
<
0.05
significant
when
compared
to
CLP
group).
no
significant
difference
between
survival
times
of
the
CLP
and
CLP
+
antagonist
groups
(Fig.
1).
Biochemical
results
Cytokines
TNF-␣,
IL-1␤
and
IL-6
levels
were
significantly
high
in
the
CLP
group
when
compared
to
the
sham
group.
Agonist
treatment
in
sep-
tic
rats
decreased
TNF-␣,
IL-1␤
and
IL-6
levels
significantly
when
compared
to
CLP
rats.
There
was
no
significant
difference
between
cytokine
levels
of
CLP
rats
and
CLP
+
antagonist
rats
(Figs.
2–4).
Fig.
8.
Scoring
results
of
conventional
light
microscopical
data
(*p
<
0.05
significant
when
compared
to
CLP
group).
Oxidative
stress
As
a
result
of
ELISA
analyses
performed
in
lung
tissues,
8-
isoporostan
levels,
a
sensitive
indicator
of
lipid
peroxidation,
were
found
to
be
significantly
increased
in
the
CLP
group
when
com-
pared
to
the
sham
group
(Fig.
5).
Agonist
administration
to
septic
rats
significantly
decreased
8-isoprostan
levels;
however,
antag-
onist
administration
was
not
effective.
The
levels
of
superoxide
dismutase
(SOD)
glutathione
and
(GSH),
indicators
of
tissue
antiox-
idative
system,
were
significantly
decreased
in
the
CLP
group
when
compared
to
the
sham
group.
Agonist
administration
to
septic
rats
Fig.
9.
SHAM
group
lung
sections.
Bv:
blood
vessel,
a:
alveolus,
TB:
terminal
bronchiole.
Dye:
H&E,
section
thickness:
5
␮m
(mean
score:
0.84).

E.
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Immunobiology
218 (2013) 1271–
1283 1275
Fig.
10.
CLP
group
lung
sections.
Bv:
blood
vessels,
a:
alveolus,
TB:
terminal
bronchiole.
Dye:
H&E,
section
thickness:
5
␮m
(mean
score:
2.71).
ameliorated
the
decrease
in
SOD
and
GSH
levels;
however,
antag-
onist
administration
had
no
effect
(Figs.
6
and
7).
Histopathological
results
Mean
scoring
of
histopathological
analyses
is
summarized
in
Fig.
8.
Detailed
histopathological
results
were
as
follows:
SHAM
group
When
terminal
bronchiole,
alveoli
and
vascular
structure
were
evaluated,
they
were
detected
as
normal
histological
appearance
(Fig.
9A–D).
Terminal
bronchioles
were
composed
with
simple
cuboidal
epithelium
and
Clara
cells
bulged
into
the
lumen
(Fig.
9D).
A
smooth
muscle
layer
was
observed
around
the
terminal
bronchi-
oles.
Interstitial
cells
were
seen
outside
of
the
terminal
bronchioles
(Fig.
11D).
A
vascular
structure
with
their
endothelial
cells
and
smooth
muscle
layer
were
detected
as
normal
(Fig.
9A
and
C).
Alveoli
had
thin
walls
and
abundant
capillaries
(Fig.
9A–D).
CLP
group
Inflammatory
cell
infiltrations
in
the
interstitial
area
were
remarkable
at
the
first
glance
(Fig.
10A).
Additionally
these
infiltra-
tions
were
observed
at
bronchial
walls
and
vascular
walls
(Fig.
10B
and
C).
When
alveoli
were
detected,
thickness
and
irregularities
in
septa
and
capillary
dilation
were
observed
(Fig.
10A,
B
and
D).
CLP
+
agonist
groups
Inflammatory
cell
infiltration
in
the
whole
area
decreased
com-
pared
to
the
CLP
group
(Fig.
11A–C);
thickening
and
irregularities
of
alveoli
and
capillary
dilations
decreased
compared
the
CLP
group
(Fig.
11B
and
D).
CLP
+
antagonist
groups
In
this
group’s
section,
inflammatory
cell
infiltration
and
eryth-
rocytes
extravagation
were
increased
when
compared
to
the
CLP
group
(Fig.
12A–D).
Additionally,
total
obstruction
in
vessel
and
necrotic
focuses
were
determined
in
this
group’s
section
(Fig.
12C).
Thickening
and
irregularities
of
alveoli
and
capillary
dilations
were
much
more
than
other
groups
(Fig.
12A–D).
Immunohistochemical
results
of
5HT-7
SHAM
group
In
this
group’s
section,
extremely
low
immunopositivity
was
observed.
This
positivity
was
restricted
in
interstitial
cells
(Fig.
13A
and
B).
CLP
Immunopositive
cell
numbers
were
much
greater
than
the
sham
group.
Positivity
in
interstitial
area
was
grouped
around
the
terminal
bronchioles.
Endothelial
cells
of
the
vessel
also

1276 E.
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Immunobiology
218 (2013) 1271–
1283
Fig.
11.
CLP
+
5HT7
agonist
given
group
lung
sections.
Bv:
blood
vessel,
a:
alveolus,
TB:
terminal
bronchiole.
Dye:
H&E,
section
thickness:
5
␮m
(mean
score:
1.86).
showed
immunopositivity
(Fig.
14A).
Immunopositivity
was
quite
detectable
in
interstitial
cells
and
type
I
pneumocytes
(Fig.
14B).
CLP
+
agonist
group
Immunopositive
cell
numbers
were
restricted
by
1–2
cells
around
the
periphery
of
terminal
bronchioles
(Fig.
15A).
Addition-
ally,
immunopositivity
in
interstitial
cells
and
alveolar
cells
were
not
as
low
as
in
the
CLP
group
(Fig.
15B).
CLP
+
antagonist
group
Although
immunopositivity
was
not
observed
in
the
epithelia
of
terminal
bronchioles,
strong
immunopositivity
was
detected
in
the
interstitial
cells
and
alveolar
cells
(Fig.
16A).
Also,
epithelia
of
small
bronchioles
showed
strong
immunopositivity
(Fig.
16B).
Immunohistochemical
results
of
NF-B
SHAM
group
In
this
group’s
section,
very
few
terminal
bronchioles
basal
cells
showed
nuclear
positivity
(Fig.
17A).
Additionally,
cytoplasmic
positivity
was
observed
in
cells
of
alveoli
and
vascular
structures
(Fig.
17B).
CLP
group
Epithelial
cells
of
terminal
bronchioles
had
nuclear
posi-
tivity.
There
were
also
inflammatory
cells
that
had
nuclear
immunopositivity
around
terminal
bronchioles
(Fig.
18A).
Alveolar
cells
and
endothelial
cells
of
the
vascular
structure
showed
nuclear
immunopositivity
(Fig.
18B).
CLP
+
agonist
group
There
were
some
nuclear
positive
cells
in
both
terminal
bron-
chioles’
epithelia
and
alveoli
(Fig.
19A
and
B),
but
there
were
fewer
nuclear
immunopositive
cells
than
in
the
CLP
group.
CLP
+
antagonist
group
In
this
group’s
section,
many
nuclear
immunopositive
cells
in
bronchioles
and
endothelia
of
vessel
were
observed
(Fig.
20A).
Whole
alveoli
and
interstitial
cells
showed
nuclear
immunoposi-
tivity
(Fig.
20B).
Discussion
This
study
demonstrated
the
role
of
5-HT7
receptors
in
CLP-
induced
polymicrobial
sepsis
of
rats.
Also,
we
investigated
the
presence
of
5-HT7
receptors
in
lung
tissue,
which
is
important
in
sepsis-related
mortality,
and
the
effects
of
5-HT7
receptor
agonist
and
antagonist
on
CLP-induced
acute
lung
injury.
Serum
cytokine
levels,
histopathological
changes
and
oxidative
stress
parameters
in
lungs
were
also
investigated.
In
immunochemical
staining,
lung
NF-␬B
and
5-HT7
concentration
were
evaluated.

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Immunobiology
218 (2013) 1271–
1283 1277
Fig.
12.
CLP
+
5HT7
antagonist
given
group
lung
sections.
Bv:
blood
vessel,
a:
alveolus,
TB:
terminal
bronchiole.
Dye:
H&E,
section
thickness:
5
␮m
(mean
score:
2.77).
Fig.
13.
SHAM
group
5HT-7
Ab
dyed
lung
sections.
TB:
terminal
bronchiole,
a:
alveolus,
arrow:
5HT7
immunopositive
cell.
Dye:
5HT-7
Ab,
section
thickness:
5
␮m,
bars:
10
␮m.

1278 E.
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/
Immunobiology
218 (2013) 1271–
1283
Fig.
14.
CLP
group
5HT-7
Ab
dyed
lung
sections.
TB:
terminal
bronchiole,
a:
alveolus,
arrow:
5HT7
immunopositive
cell.
Dye:
5HT-7
Ab,
section
thickness:
5
␮m,
bars:
10
␮m.
Sepsis
resulting
from
CLP
in
animals
is
an
accepted
animal
model
that
mimics
the
clinical
situation
of
bowel
perforation
and
bacterial
infection
in
humans
(Chen
et
al.
2001;
Hubbard
et
al.
2005; ˙
Iskit
2005).
A
period
of
16–20
h
is
the
late
phase
of
sepsis
in
the
CLP-
induced
intra-abdominal
sepsis
model
(Wang
et
al.
1997).
In
the
present
study,
the
late
phase
of
sepsis
was
accepted
to
be
equal
to
the
experimental
model
at
the
16th
hour.
Rats
were
observed
for
72
h
to
evaluate
mortality
rate.
There
was
no
death
in
the
sham
group
during
72
h.
In
the
CLP
group,
mean
survival
time
was
16.71
h.
Administration
of
5-HT7
receptor
agonist,
AS-19
(Hedlund
2009;
Bosker
et
al.
2009),
to
septic
rats
increased
the
mean
survival
time
to
35.43
h.
However,
there
was
no
significant
difference
in
antagonist
SB
269970
(Forbes
et
al.
2002;
Thomas
et
al.
2003)
administered
CLP
rats
when
compared
to
CLP
group.
Survival
times
of
rats
were
also
in
line
with
histopathologic
analyses.
Namely,
when
we
scored
inflammatory
cell
infiltration
in
the
lungs
of
septic
rats,
we
showed
that
inflammation
scores
in
septic
lungs
were
significantly
higher
than
those
of
the
sham
group.
Agonist
administration
to
septic
rats
significantly
reduced
the
inflammation
scores
while
antagonist
had
no
effect.
As
it
was
demonstrated
in
previous
studies
(Coskun
et
al.
2011;
Otero-Anton
et
al.
2001),
mortality
is
quite
high
in
septic
rats;
acute
lung
injury
has
been
suggested
to
be
the
most
important
cause
of
death
(Cadirci
et
al.
2010b;
Vincent
2011).
We
have
pre-
viously
shown
that
in
rats
with
lower
lung
injury,
mortality
rate
also
decreased
significantly
(Cadirci
et
al.
2010a,
2011).
We
also
demonstrated
that
stimulation
of
5-HT7
receptors
is
effective
for
both
increasing
survival
times
and
decreasing
sepsis-induced
lung
injury.
Presence
of
5-HT7
receptors
in
immune
response
cells
such
as
monocytes
(Soga
et
al.
2007),
T
cells
(Leon-Ponte
et
al.
2007),
Fig.
15.
CLP
+
agonist
given
group
5HT-7
Ab
dyed
lung
sections.
TB:
terminal
bronchiole,
a:
alveolus,
arrow:
5HT7
immunopositive
cell.
Dye:
5HT-7
Ab,
section
thickness:
5
␮m,
bars:
10
␮m.

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/
Immunobiology
218 (2013) 1271–
1283 1279
Fig.
16.
CLP
+
antagonist
given
group
5HT-7
Ab
dyed
lung
sections.
TB:
terminal
bronchiole,
a:
alveolus,
RB:
respiratory
bronchiole,
arrow:
5HT7
immunopositive
cell.
Dye:
5HT-7
Ab,
section
thickness:
5
␮m,
bars:
10
␮m.
thymus,
peripheral
lymphocytes,
spleen
and
mitogen-activated
spleen
cells
(Stefulj
et
al.
2000)
made
us
think
that
stimulation
of
these
receptors
mediates
their
protective
effects
on
septic
lungs.
In
previous
studies,
5-HT
has
been
shown
to
be
released
by
mast
cells,
basophiles
and
enterochromaffin
cells
in
peripheral
tissues,
thus
extracellular
5-HT
levels
increase
in
inflammatory
condi-
tions
(Matsuda
et
al.
1997).
Thus,
5-HT
can
be
accepted
as
an
immunomodulatory
mediator.
Soga
et
al.
reported
that
serotonin
activates
human
monocytes
and
prevents
apoptosis
via
5-HT1
and
5-HT7
receptors
(Soga
et
al.
2007),
and
they
suggested
that
this
effect
may
result
from
increased
intracellular
cyclic
adenosine
monophosphate
and
induced
production
of
cytokines,
such
as
IL-
1b
and
IL-6,
in
lipopolisaccaride
LPS-primed
monocytes
via
5-HT7
activation
(Durk
et
al.
2005).
In
experimental
sepsis
models
and
clinical
trials,
proinflam-
matory
and
potential
auto-toxic
mediators
were
released.
TNF-␣,
IL-1␤
and
IL-6,
which
are
macrophage-derived
proinflammatory
cytokines,
have
important
roles
in
sepsis
and
sepsis-related
multi-organ
failure
(Damas
et
al.
1992).
These
proinflammatory
mediators
are
also
involved
in
the
pathogenesis
of
sepsis-induced
acute
lung
injury
(Bhatia
and
Moochhala
2004).
In
this
study,
while
TNF-␣,
IL-1␤
and
IL-6
levels
were
significantly
increased
in
CLP
rats,
stimulation
of
5-HT7
receptors
with
AS-19
prevented
this
increase
significantly.
These
findings
are
also
in
line
with
pre-
vious
studies
in
which
increased
cytokine
levels
were
observed
(Cadirci
et
al.
2010b;
Li
et
al.
2009).
TNF-␣
contributes
to
many
important
processes
in
inflammatory
responses
(Kuwano
and
Hara
2000).
Fig.
17.
SHAM
group
Nf␬B
Ab
dyed
lung
sections.
TB:
terminal
bronchiole,
a:
alveolus,
Bv:
blood
vessel,
arrow:
nuclear
Nf␬B
immunopositivity.
Dye:
Nf␬B
Ab,
section
thickness:
5
␮m,
bars:
10
␮m.

1280 E.
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/
Immunobiology
218 (2013) 1271–
1283
Fig.
18.
CLP
group
Nf␬B
Ab
dyed
lung
sections.
TB:
terminal
bronchiole,
a:
alveolus,
Bv:
blood
vessel,
arrow:
nuclear
Nf␬B
immunopositivity.
Dye:
Nf␬B
Ab,
section
thickness:
5
␮m,
bars:
10
␮m.
IL-6
has
an
important
role
in
neutrophil
migration
and
con-
tributes
in
sepsis
pathogenesis.
Therefore
it
is
a
useful
marker
for
determination
of
sepsis
severity
(Li
et
al.
2009;
Remick
et
al.
2005).
Durk
et
al.
reported
that
5-HT7
receptors
contribute
in
the
release
of
cytokines
like
IL-1␤,
IL-6
and
TNF-␣
from
monocytes
(Durk
et
al.
2005).
Our
results
showed
that
5-HT7
agonist
adminis-
tration
to
septic
rats
decreased
the
serum
cytokine
levels,
and
CLP
administration
increased
NF-␬B
immunopositivity
in
lung
tissues.
Also,
stimulation
of
5-HT7
receptors
decreased
NF-␬B
immunopos-
itivity
in
septic
lungs.
NF-␬B
activation
and
subsequent
increase
in
expression
of
many
proinflammatory
molecules
contributes
to
inflammation
pathogenesis
(Sakurai
et
al.
1999)
and
plays
a
cen-
tral
role
in
sepsis-induced
cytokine
release
such
as
TNF-a,
IL-6,
etc.
(Shen
et
al.
2009).
In
addition,
our
findings
showed
that
in
septic
rats
with
increased
NF-␬B
immunopositivity,
serum
TNF-␣,
IL-6
and
IL-1␤
levels
were
also
increased.
Oxidative
stress
up-regulates
NF-␬B
transcription
and
antiox-
idants
down-regulate
the
increased
NF-␬B
transcription
(van
den
Berg
et
al.
2001).
This
study
suggests
that
the
potential
therapeu-
tic
effects
of
AS-19
in
the
beginning
and
continuation
of
various
inflammatory
diseases
can
be
attributed
to
NF-␬B
inhibition
and
subsequent
decrease
in
both
oxidative
stress
and
proinflammatory
cytokines.
The
increased
generation
of
ROS
from
neutrophils
has
been
proposed
as
an
important
mechanism
of
lung
injury
(Fink
2002).
It
has
been
reported
that
the
antioxidative
system
was
severely
compromised
and
plasma
lipid
peroxidation
(LPO)
end
products
were
significantly
increased
in
septic
cases,
suggesting
an
increased
oxidative
stress
in
patients
with
sepsis-induced
lung
Fig.
19.
CLP
+
agonist
given
group
Nf␬B
Ab
dyed
lung
sections.
TB:
terminal
bronchiole,
a:
alveolus,
Bv:
blood
vessel,
black
arrow:
nuclear
Nf␬B
immunopositivity,
white
arrow:
nuclear
Nf␬B
immunonegativity.
Dye:
Nf␬B
Ab,
section
thickness:
5
␮m,
bars:
10
␮m.

E.
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/
Immunobiology
218 (2013) 1271–
1283 1281
Fig.
20.
CLP
+
antagonist
given
group
Nf␬B
Ab
dyed
lung
sections.
TB:
terminal
bronchiole,
a:
alveolus,
Bv:
blood
vessel.
Dye:
Nf␬B
Ab,
section
thickness:
5
␮m,
bars:
10
␮m.
injury
(Metnitz
et
al.
1999).
Superoxide
dismutase
and
GSH
are
the
main
enzymes
involved
in
the
antioxidative
system.
All
of
these
antioxidants
limit
the
cytotoxic
effects
of
toxic
free
radi-
cals
(Fink
2002),
and
many
sepsis
studies
have
demonstrated
reduced
antioxidant
levels
in
lung
tissues
(Coskun
et
al.
2011;
Cadirci
et
al.
2010a,
2011;
Starr
et
al.
2011;
Qiao
et
al.
2011).
In
this
study,
AS-19
increased
antioxidant
enzyme
activity
in
septic
lung
tissues.
Both
GSH
and
SOD
levels
were
significantly
higher
in
agonist-administered
septic
rats’
lungs
when
compared
to
the
non-treated
CLP
group.
Agonist
administration
to
septic
rats
signif-
icantly
reduced
the
increased
levels
of
8-isoprostan
levels
which
is
an
end
product
of
tissue
lipid
peroxidation
in
the
CLP
group,
suggesting
AS-19
decreases
oxidative
stress
in
lung
tissue.
Lipid
peroxidation,
which
is
an
indicator
of
oxidative
stress,
affects
mem-
brane
permeability
thus
protein
degradation
and
lysis
in
cells
(Garcia
et
al.
1997).
Previously
it
was
shown
that
in
clinical
and
experimental
sepsis
tissue,
LPO
levels
are
increased
(Ortolani
et
al.
2000).
We
showed
that
8-isoprostan
level,
which
is
a
sensitive
indi-
cator
of
tissue
LPO
concentration
(Wood
et
al.
2003),
increased
in
lungs,
as
previously
shown
(Razavi
et
al.
2005).
Agonist
adminis-
tration
decreased
8-isoprostan
levels
in
lung
tissue,
prompting
us
to
think
that
stimulation
of
5-HT7
receptors
protected
lung
tis-
sue
by
reducing
oxidative
stress.
Therefore,
stimulation
of
5-HT7
receptors
might
be
a
potential
therapeutic
target
for
prevention
of
sepsis-induced
lung
injury.
Protective
effects
of
5-HT7
receptor
stimulation
in
lungs
caused
us
to
hypothesize
that
these
receptors,
whose
presence
in
the
brain
is
well
known
but
only
limited
information
exists
about
peripheral
tissues
(Segura
et
al.
2010),
are
also
expressed
in
lungs,
and
an
inflammatory
stimulus
such
as
sepsis
might
increase
their
expression.
Many
studies
demonstrated
the
presence
of
5-
HT2a
receptors
in
airway
smooth
muscles
(Cazzola
and
Matera
2000;
Rhoden
et
al.
2000;
Yang
et
al.
1994).
However,
there
is
limited
study
investigating
5-HT7
receptors’
pulmonary
localiza-
tion
(Segura
et
al.
2010).
In
our
study,
lung
sections
mounted
on
polylysine
slides
were
stained
with
5-HT7
Ab,
and
immunopositiv-
ity
was
shown
to
be
significantly
higher
in
the
CLP
group
than
the
sham
group.
Previously,
Segura
et
al.
(2010)
showed
that
SB269970
(5-HT7
antagonist)
decreased
the
antigen-induced
airway
hyper
responsiveness
in
guinea
pigs
and
LP44
(5-HT7
agonist)
induced
a
significant
increase
in
contractile
response
of
isolated
in
tra-
cheal
rings
(Segura
et
al.
2010).
Segura
et
al.
also
suggested
roles
of
5-HT2A
and
5-HT4
receptors
in
airway
hyper
responsiveness;
however,
RT-PCR
analyses
showed
that
neither
sensitization
nor
antigen
challenge
modified
the
5-HT2A
receptor
mRNA
levels.
As
in
our
study,
this
study
suggests
that
5-HT7
receptors
are
involved
in
abnormal
conditions.
However,
though
the
authors
suggested
that
this
involvement
is
mediated
by
pulmonary
neuro-endocrine
cells,
they
did
not
analyze
pulmonary
5-HT7
receptor
presence.
In
this
regard,
our
study
is
the
first
study
immunohistochemically
showing
the
presence
of
5-HT7
receptors
in
lung
tissue.
Immunopositivity
was
highest
in
the
CLP
group,
and
agonist
(AS-19)
administration
decreased
5-HT7
immunopositivity.
This
could
be
due
to
the
anti-
inflammatory
effects
of
5-HT7
receptor
stimulation
and
related
reduction
in
inflammatory
response
resulting
in
decreased
5-HT7
receptor
density
in
lungs.
However,
the
increase
of
immunopositiv-
ity
in
septic
lungs
can
also
be
related
to
accumulation
of
monocytes,
T
cells
and
peripheral
lymphocytes,
which
have
5-HT7
receptor
expression
(Leon-Ponte
et
al.
2007;
Soga
et
al.
2007;
Stefulj
et
al.
2000).
Conclusion
This
study
demonstrated
for
the
first
time
that
peripheral
5-HT7
receptors
have
very
important
roles
in
CLP-induced
sepsis.
Namely,
the
reason
why
5-HT-7
receptor
agonist
treatment
increased
sur-
vival
time
may
be
related
to
(1)
a
decrease
in
serum
cytokine
response
against
CLP,
(2)
a
decrease
in
oxidative
stress
in
lung
and
increase
in
antioxidant
system,
(3)
a
decrease
in
the
tissue
NF-␬B
immunopositivity,
which
is
high
in
septic
rats,
and
finally
(4)
a
decrease
in
the
sepsis-induced
lung
injury.
In
conclusion,
all
these
data
suggest
that
stimulation
of
5-HT7
receptors
may
be
a
new
therapeutic
target
for
prevention
of
impaired
inflammatory
response
related
lung
injury
and
mortality.
Financial
support
None.
Conflict
of
interest
Nothing
to
declare.

1282 E.
Cadirci
et
al.
/
Immunobiology
218 (2013) 1271–
1283
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