Apoptosis (2007) 12:1989–2001
DOI 10.1007/s10495-007-0125-1
ORIGINAL PAPER
Lung injury after ischemia-reperfusion of small intestine in rats
involves apoptosis of type II alveolar epithelial cells mediated
by TNF-a and activation of Bid pathway
Shucai An Æ Yoshitaka Hishikawa Æ Jie Liu Æ
Takehiko Koji
Published online: 5 September 2007

Springer Science+Business Media, LLC 2007
Abstract Although ischemia-reperfusion (I/R) of small
intestine is known to induce lung cell apoptosis, there is
little information on intracellular and extracellular molec-
ular mechanisms. Here, we investigated the mechanisms of
apoptosis including the expression of Fas, Fas ligand
(FasL), Bid, Bax, Bcl-2, cytochrome c, and activated cas-
pase-3 in the rat lung at various time-points (0–24 h)
of reperfusion after 1-h ischemia of small intestine. As
assessed by TUNEL, the number of apoptotic epithelial
cells, which were subsequently identified as type II alveolar
epithelial cells by electron microscopy and immunohisto-
chemical double-staining, increased at 3 h of reperfusion in
the lung. However, intravenous injections of anti-TNF-a
antibody decreased the number of TUNEL-positive cells,
indicating involvement of tumor necrosis factor-a (TNF-a)
in the induction of lung cell apoptosis. Western blotting
and/or immunohistochemistry revealed a marked up-regu-
lation of Fas, FasL, Bid, Bax, cytochrome c and activated
caspase-3 and down-regulation of Bcl-2 in lung epithelial
and stromal cells at 3 h of reperfusion. Our results indicate
that I/R of small intestine results in apoptosis of rat alveolar
type II cells through a series of events including systemic
TNF-a, activation of two apoptotic signaling pathways and
mitochondrial translocation of Bid.
Keywords Ischemia-reperfusion injury
TNF-a

Apoptosis
Apoptosis-related proteins
Rat lung
S. An
Y. Hishikawa
J. Liu
T. Koji (&)
Department of Histology and Cell Biology, Unit of Basic
Medical Science, Nagasaki University Graduate School of
Biomedical Sciences, 1-12-4, Sakamoto, Nagasaki 852-8523,
Japan
e-mail: tkoji@net.nagasaki-u.ac.jp
Introduction
Intestinal ischemia-reperfusion (I/R) appears to induce
remarkable inflammatory response, including both local
and remote organ injuries, such as lung, kidney and liver as
a secondary effect [1, 2]. In fact, lung injury is a common
cause of death and complications after intestinal I/R and
that was caused by systemic inflammatory response
syndrome [3, 4], which is characterized by pulmonary
neutrophil accumulation and increased vascular leakage [5,
6]. Importantly, the I/R intestine may be the source of pro-
inflammatory mediators [4] and release of cytokines such
as tumor necrosis factor-a (TNF-a) [7, 8] and interleukin-6
[9, 10]. In fact, TNF-a was reported as an essential cyto-
kine in the inflammatory response after intestinal I/R [8],
and as a pleiotropic cytokine that might trigger either cell
death or proliferation, acting through two receptors; TNF
receptor (TNFR) 1 and 2 [11].
Apoptosis is an active mode of cell death to maintain
tissue mass homeostasis under physiological and patho-
logical conditions including acute lung injury and intestinal
I/R [12, 13]. In mammals, two distinct apoptotic signaling
pathways have been identified, which can be induced
through the extrinsic (death receptor) or intrinsic mito-
chondrial pathways [14]. The binding of TNF-a to TNFR1
or TNFR1 is activated by other ligands, such as Apo2
ligand or TNF-related apoptosis-inducing ligand through
death receptor 4 (DR4) and DR5 leads to the formation of
Fas-associated death domain (FADD), and then FADD
further causes the activation of caspase-8 [15–17]. Fas
ligand (FasL) can also stimulate apoptotic cell death by
binding to Fas, with recruitment of FADD to the Fas-FasL
complex and subsequent activation of caspase-8 [18, 19],
leading to the cleavage of Bid protein [20]. On the other
hand, the mitochondria-dependent pathway is tightly
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regulated by Bcl-2 family proteins (e.g., Bid, Bax and
Bak). These proteins promote mitochondrial release of
cytochrome c, whereas Bcl-2 or Bcl-xL suppresses this
process [21]. Thus, Bid is supposed to allow the intrinsic
pathway to be significantly influenced by factors from
extrinsic pathway (such as caspase 8), and Bid is ‘‘partly’’
responsible for cytochrome c release and the downstream
caspase activities.
TNF-a is produced by many cells including macro-
phages, monocytes, lymphocytes and fibroblasts. TNF-a is
also shown to play important roles in regulating cell pro-
liferation and differentiation, inflammatory responses,
immune functions, and apoptosis as well as in a variety of
acute lung injury models [22, 23]. TNF-a increases lung
vascular permeability, and modulate the eventual recruit-
ment and activation of neutrophils as well as an important
trigger for up-regulation of pulmonary endothelial and
neutrophil adhesion molecules after intestinal I/R [1, 23].
The present study was designed to determine the
molecular mechanism of induction of apoptosis in the rat
lung after I/R of small intestine. We hypothesized that
TNF-a initiates induction of apoptosis of lung epithelial
cells through Bid activation during small intestinal I/R. To
test this hypothesis, we first analyzed the kinetics of
apoptotic cell death by terminal deoxynucleotidyl trans-
ferase (TdT)-mediated dUTP-biotin nick end labeling
(TUNEL) and electron microscopy in the rat lung after I/R
of small intestine. Second, to gain further insights into the
molecular pathway, we performed Western blotting anal-
ysis and/or immunohistochemistry for apoptosis-related
proteins, such as Fas, FasL, Bid, Bax, Bcl-2, cytochrome c,
and activated caspase-3. Finally, to examine whether TNF-
a is a trigger of induction of apoptosis, we investigated the
effect of anti-TNF-a antibody on the induction of
apoptosis.
Materials and methods
Chemicals and biochemicals
Nembutal was purchased from Dainippon Pharmaceutical
Co. (Osaka, Japan). Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) reagents were purchased
from Daiichi Pure Chemicals (Tokyo, Japan). Immobilon-
polyvinylidene difluoride membrane was purchased from
Millipore (Bedford, MA, USA). The protein assay kit was
purchased from Bio-Rad Laboratories (Hercules, CA,
USA). Anti-TNF-a antibody was obtained from R&D Sys-
tems Inc. (Minneapolis, MN, USA). Paraformaldehyde
(PFA) was purchased from Merck (Darmstadt, Germany)
and glutaraldehyde and Epon 812 resin were obtained from
Taab Laboratories (Aldermastton, Berkshire, UK).
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Apoptosis (2007) 12:1989–2001
Ethylenediaminetetraacetic acid disodium salt dihydrate
(EDTA) and 3, 30 -diamino-benzidine-4 HCl (DAB) was
purchased from Dojin Laboratories (Kumamoto, Japan). 4-
chloro-1-naphthol was purchased from Tokyo Kasei Kogyo
Co. (Tokyo, Japan). Tris [hydroxymethyl] aminomethane
(Tris), phenylmethylsulfonylfluoride (PMSF), 2-mercapto-
ethanol, Triton X-100, proteinase K, 3-aminopropyl-
triethoxysilane, bovine serum albumin (BSA; minimum
98%, c-globulin free) and Brij 35 were purchased from
Sigma Chemical Co. (St Louis, MO, USA). Biotin-16-
dUTP and terminal deoxynucleotidyl transferase (TdT)
were from Roche Diagnostics (Mannheim, Germany). All
other reagents used in this study were from Wako Pure
Chemicals (Osaka, Japan) and were of analytical grade.
Antibodies
Anti-Fas (P4; 1:800) serum [24, 25] was prepared by
immunization of rabbits against synthetic oligopeptides
corresponding to the intracellular domain (P4; amino acids
292–306) of mouse Fas [26]. Anti-FasL (P5; 1:100) serum
[27] was generated with a synthetic peptide corresponding
to the intracellular domain (P5; amino acids 41–55) of rat
FasL [28]. Rabbit polyclonal anti-Bid (FL-195), rabbit
polyclonal anti-Bax (P-19), rabbit polyclonal anti-Bcl-2
(N-19), rabbit polyclonal anti-cytochrome c (H-104) and
goat polyclonal anti-surfactant protein-A (SP-A) (N-19)
were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Anti-human/mouse caspase-3 active was
from Techne Corporation (Minneapolis, MN, USA).
Horseradish peroxidase (HRP)-conjugated goat anti-rabbit
IgG was from MBL (Nagoya, Japan). HRP-conjugated
rabbit anti goat IgG was from Wako Pure Chemicals. HRP-
conjugated goat anti-mouse IgG was from Chemicon
International (Temecula, CA, USA). HRP-anti-biotin was
from Vector Laboratories (Burlingame, CA, USA). Normal
rabbit IgG, normal goat IgG were purchased from Sigma.
Normal rabbit serum was purchased from DAKO (Glost-
rup, Denmark).
Animals
Male Wistar rats (7–8 weeks) weighing 200–300 g were
purchased from Ohtsubo Experimental Animal Co. (Isa-
haya, Japan) and each experimental group consisted of five
rats. The animals were fasted for 12 h before the experi-
ments but had free access to water. Animal care and
experimental procedures were performed in accordance
with the Guidelines for Animal Experimentation of Naga-
saki University with approval of the Institutional Animal
Care and Use Committee.
Apoptosis (2007) 12:1989–2001
Ischemia-reperfusion of the rat small intestine
Rats were anesthetized with an intraperitoneal injection of
Nembutal (40 mg/kg body weight) and the abdomen was
opened. The superior mesenteric artery (SMA) was
occluded with an atraumatic microvascular clamp for a
period of 60 min. Collateral blood supply was blocked
according to the method of Megison [29] by ligating
arcades between the descending branch of the right colic
artery and the ileocolic artery and between jejunal branches
just proximal and distal to the point of SMA. At the end of
the ischemic period, the clamp was removed (time 0) and
the intestine was allowed to reperfuse for 0, 3, 6, 12 and
24 h. At various time-points of reperfusion, segments of
lung were dissected out and removed. In the sham-operated
group, laparotomy was performed, and the SMA was
exposed but not occluded. Vascular occlusions and sham
operations were always performed at the same time of the
day (between 0900 and 1100).
Treatment with anti-TNF-a antibody
Anti-TNF-a antibody, which was dissolved in 0.5 ml of
0.9% NaCl at a dose of 37 lg per rat, and was administered
intravenously at the end of ischemia or 15 min after rep-
erfusion [1, 30]. Control animals were treated with an
equivalent volume of 0.9% NaCl instead of anti-TNF-a
antibody.
Assessment of arterial blood gas exchange
The blood samples for gas measurement were collected
from the arterial blood line at the end of 3 h of reperfusion
using the rats of intestinal I/R groups and intestinal I/R plus
anti-TNF-a antibody treatment groups. These samples were
analyzed immediately for pH, PO2, PCO2, and HCO–3 by
the i-STAT Portable Clinical Analyzer (i-STAT corpora-
tion, Princeton) with G3+ cartridges (Abbott Point of Care
Inc., NJ) in accordance with the manufacture’s recom-
mendations. Briefly, an approximately 70 ll of blood was
dispensed into the G3+ cartridge up to the full mark, and
then correct filling and absence of bubbles was performed.
The measurement was triggered by inserting the G3+ car-
tridge into the analyzer, upon which the results were
displayed on the screen.
Tissue preparation
Lung tissue samples were fixed in 4% PFA in phosphate-
buffered saline (PBS) at room temperature (RT) overnight
and embedded in paraffin using standard procedures. The
1991
tissues were cut into 5-lm-thick sections and mounted onto 3-
aminopropyltriethoxysilane-coated slides and adjacent sec-
tions were stained with hematoxylin and eosin for histological
analysis. Some pieces of the tissues were quickly frozen,
stored at –80C and later used for Western blotting. One piece
of each lung was fixed with 2.5% glutaraldehyde in 0.1 M
sodium phosphate buffer, pH 7.4, and then processed rou-
tinely and embedded in Epon 812 for electron microscopy.
Electron microscopy
Epon-embedded lung specimens were cut into semi-thin
and ultra-thin sections. The semi-thin sections were stained
with toluidine blue, and the ultra-thin sections were stained
with uranium acetate and lead nitrate. The ultra-thin sec-
tions were observed under a JEOL 1200 EX electron
microscope at accelerated voltage of 60 KV, as described
previously [13].
TUNEL staining
TUNEL assay was carried out according to the method of
Gavrieli [31] with a slight modification, as described pre-
viously [13]. Briefly, the paraffin-embedded sections were
deparaffinized with toluene and rehydrated in serially gra-
ded ethanol solutions. After washing with PBS, the sections
were treated with 0.5 lg/ml proteinase K in PBS for 15 min
at 37C. Then the sections were incubated with 1 · TdT
buffer (25 mM Tris-HCl buffer, pH 6.6, containing 0.2 M
potassium cacodylate and 0.25 mg/ml BSA) alone for
30 min at RT. After incubation, the slides were reacted with
100 U/ml TdT dissolved in TdT buffer supplemented with
1.5 mM CoCl2, 1 lM biotin-16-dUTP, 20 lM dATP and
0.1 mM dithiothreitol for 90 min at 37C. The reaction was
terminated by washing with 50 mM Tris-HCl buffer (pH
7.4) and endogenous peroxidase activity was inhibited by
immersing the slides in 0.3% H2O2 in methanol for 15 min
at RT followed by washing with PBS. After incubation with
500 lg/ml normal goat IgG in 5% BSA in PBS for 60 min
at RT, the sections were incubated with HRP-goat anti-
biotin antibody (1:100) diluted with 5% BSA in PBS
overnight at RT. After washing with 0.075 % Brij 35 in
PBS, the HRP sites were visualized by DAB, H2O2, CoCl2,
and NiSO4 (NH4)2SO4 according to the method of Adams
[32]. As a negative control, some sections were subjected to
reaction without TdT.
Immunohistochemistry
Immunohistochemical staining was performed by the
indirect enzyme-labeled antibody method as described
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previously [27]. Briefly, lung paraffin-embedded sections
were deparaffinized with toluene and dehydrated with
serially graded ethanol solutions. For Bax staining, the
sections were autoclaved in 0.01 M citrate buffer (pH 6.0)
for 10 min at 120C. After inactivation of endogenous
peroxidase activity with 0.3% H2O2 in methanol for
15 min at RT, the sections were preincubated with
500 lg/ml normal goat IgG and 1% BSA in PBS for 1 h
at RT. In the case of SP-A staining, normal rabbit serum
was used as a blocking solution. The sections were then
reacted with the first antibodies for 2 h (Fas, FasL) or
overnight (Bid, Bax, Bcl-2, cytochrome c, activated cas-
pase-3) at RT. After incubation, the slides were washed
three times with 0.075% Brij 35 in PBS. Then the sec-
tions were incubated with HRP-conjugated goat anti-
rabbit IgG (1:200) for 1 h at RT, and washed three times
with 0.075% Brij 35 in PBS. The sites of HRP were
visualized with H2O2 and DAB. Except for Bax, the
sections were counterstained with methyl green. As a
negative control, normal rabbit IgG, normal rabbit serum
and normal goat IgG were used instead of the first anti-
body in each run. Serial sections were used to compare
the expression of these apoptosis-related proteins with
TUNEL-positive cells at the individual cell level. Double
staining for TUNEL and SP-A was performed as descri-
bed previously [13].
Western blot analysis
Lung tissues were homogenized in a lysis buffer containing
20 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 5 mM
EDTA, 1 mM PMSF, 10 lg/ml leupeptin, 10% glucerol,
and 1% Triton X-100 as described previously [25]. After
centrifugation at 13,000 · g for 10 min at 4 C, the
resultant supernatants was centrifuged a second time under
the same conditions. This supernatant was centrifuged at
28,000 · g for 10 min at 4C. The pellet was redissolved
in lysis buffer and represented the mitochondrial fraction.
The supernatant was frozen and stored at –80C. Protein
concentration was determined using a kit from Bio-Rad
laboratories according to the method of Bradford [33],
using BSA as a standard. Protein sample lysates (50 lg per
well) were mixed with the loading buffer containing
200 mM Tris-HCl (pH 8.0), 2.5% SDS, 0.5 M sucrose,
5 mM EDTA, 0.01% bromophenol blue, and 10% 2-mer-
captoethanol, boiled for 5 min, and separated by SDS-
PAGE with a 4–20% gradient gel according to the method
of Laemmli [34], and electrophoretically transferred onto
polyvinylidene difluoride membranes. The membranes
were blocked with 1% PBS containing 5% non-fat dry milk
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Apoptosis (2007) 12:1989–2001
for 1 h at RT, and then incubated with the first antibody
overnight using the following dilutions; Fas (1:800), FasL
(1:400), Bid (1:200), Bax (1:400) and Bcl-2 (1:400) in 1%
BSA in PBS. After washing, the membranes were incu-
bated with the second antibody, HRP-conjugated goat anti-
rabbit IgG (1:200) for 1 h at RT. The bands were visualized
with DAB and H2O2 in the presence of nickel and cobalt
ions, according to the method described above. To confirm
that the applied protein amounts were the same in each
line, the membranes were further reacted with mouse anti-
b-actin (1:8,000) as a loading control for 1 h, and then with
HRP-goat anti-mouse IgG (1:6,000). The staining density
of each band was quantified by a computer-assisted image
analyzer (DAB analysis system; Carl Zeiss, Göttingen,
Germany). We calculated the optical density of each band
in the lung as the sum of the gray values of all pixels
corresponding to the band.
Statistical analysis
More than 2,000 cells in lung tissue sections (alveolar
epithelium and stroma) per animal were counted and all
data were expressed as mean ± SEM. The experimental
groups were compared using One-way ANOVA with post
hoc Bonferrioni-Dunn. A P-value of less than 0.05
denoted the presence of a significant difference. All
analyses were preformed with a statistical software
package (StatView, version 5.0; Abacus Concepts,
Berkeley, CA, USA).
Results
Serial histological changes in rat lung after small
intestinal I/R
First, we assessed the histological changes in the lung
induced by small intestinal I/R, by examining HE-stained
sections of the lung. In the I/R groups, the thickness of the
alveolar septa was markedly increased, accompanied by
marked increase in infiltrating inflammatory cells after 1-h
ischemia (6.81 ± 0.44%, Fig. 1B) compared with the
control group (2.33 ± 0.09%, Fig. 1A). At 3 h of reperfu-
sion, the number of inflammatory cells was further
increased, which was associated with pulmonary conges-
tion, and alveolar bleeding was observed occasionally
within the alveolar spaces (12.98 ± 1.75%, Fig. 1C).
However, these changes were transient and the general
features of lung returned to the normal states at 24 h of
reperfusion (4.05 ± 0.09%, Fig. 1D).
Apoptosis (2007) 12:1989–2001 1993

Fig. 1 Lung histological changes in rat subjected to 1-h ischemia followed by reperfusion of the small intestine. (A) Sham-operation; (B) 1-h
ischemia; (C) 3 h of reperfusion; (D) 24 h of reperfusion. Hematoxylin and eosin staining. Scale bar: 50 lm

TUNEL staining of the lung during I/R of small Electron microscopic analysis of the rat lung during I/R
intestine of small intestine

To examine the involvement of apoptosis in transient lung
injury after I/R of small intestine, we performed TUNEL
staining of the lung tissues. In the sham-operated groups, a
few TUNEL-positive cells were observed in the lung
alveolar epithelium (Fig. 2A). After 1-h ischemia, TUNEL-
positive cells in the alveolar epithelium and septum, which
were identified as alveolar epithelial cells and inflammatory
lymphocytes, were significantly increased (Fig. 2B). At 3 h
of reperfusion, the number of TUNEL-positive cells was
increased markedly (Fig. 2C, E). However, the number
returned to almost the level of the normal and sham-oper-
ated groups by 24 h after reperfusion (Fig. 2D, E).
To further confirm the presence of apoptosis, we performed
electron microscopic (EM) examination of lung tissues at
3 h of reperfusion. EM study revealed typical apoptotic
alveolar epithelial type II cells (identified by the presence
of lamellar bodies) (Fig. 3A), and typical apoptotic lym-
phocytes in the alveolar septa (Fig. 3B). In addition,
macrophages phagocytozing many apoptotic bodies were
frequently found in the alveolar septa (Fig. 3C). TUNEL-
positive cells were simultaneously positive to type II
alveolar epithelial cell marker, SP-A (Fig. 3D), indicating
that the apoptotic cells of alveolar epithelium were type II
alveolar epithelial cells.

Fig. 2 TUNEL-positive cells in

the rat lung during ischemia-
reperfusion (I/R) of small
intestine. (A) Sham-operation;
(B) 1-h ischemia; (C) 3 h of
reperfusion; (D) 24 h of
reperfusion; (E) Quantitative
analysis of TUNEL-positive
cells in the lung at various time-
points of reperfusion after 1-h
ischemia of small intestine in
rats. More than 2,000 cells
(alveolar epithelium and stroma
of lung) were counted. The
number of TUNEL-positive
cells represents the
mean ± SEM (%) of five rats in
each group. *p \ 0.01. Note the
presence of TUNEL-positive
cells in alveolar epithelial cells
(arrows) and alveolar stromal
cells (arrowheads). Scale bar:
50 lm

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Fig. 3 Electron micrographs

(EM) of apoptotic cells in the
lung at 3 h of reperfusion in rats
with small intestinal ischemia-
reperfusion (I/R). (A) Note a
typical apoptotic alveolar
epithelial type II cell (identified
by the presence of lamellar
bodies); (B) typical apoptotic
lymphocyte; (C) typical
macrophages showing
phagocytosis of apoptotic body;
(D) double-staining for TUNEL
and SP-A. Arrows indicate
TUNEL-positive cells. Original
magnification ·4,000 (A–C).
Scale bar: 20 lm

Expression of apoptosis-related proteins in the rat lung markedly increased at 3 h of reperfusion in the alveolar
during I/R of small intestine epithelium (Figs. 4E, 5E).

Fas and FasL

For expressions of Fas and FasL proteins, fifty micrograms
of each sample were examined by Western blot analysis
(Figs. 4A, 5A, respectively). The membranes were further
reacted with monoclonal antibody for b-actin (42-kDa) as
a loading control. Densitometic analysis revealed
increased expression of Fas after ischemia alone (Fig. 4B),
while that of FasL was enhanced by reperfusion (Fig. 5B).
Both expressions reached maximum levels at 3 h of rep-
erfusion and then gradually returned to the normal levels.
When both expressions were examined by immunohisto-
chemistry, only a few alveolar epithelial cells were
positive to Fas, while FasL-positive epithelial cells were
more abundant in the sham-operated groups (Figs. 4C,
5C). In the I/R groups, however, the expression of Fas and
FasL was found in alveolar epithelial and stromal cells at
1 h of ischemia (Figs. 4D, 5D) and the expression was
Bcl-2 and Bax
Expression of Bcl-2 and Bax was analyzed by Western
blot and immunohistochemistry (Figs. 6, 7, respectively).
The expression of both proteins was detected in the
sham-operated groups. However, in the I/R groups,
transient opposite changes in the expressions were
observed; the expression of Bcl-2 was almost abolished,
while that of Bax increased and reached a maximum at
3 h of reperfusion. When the expression levels were
examined by immunohistochemistry, both expressions
were detected in the cytoplasm of alveolar epithelial
cells. As showing in Figs. 6C and 7C, we found abun-
dant Bcl-2 positive alveolar epithelial cells, while only a
few Bax positive cells were identified in the sham-
operated groups. In the I/R groups, in accordance with
the results of Western blotting, opposite changes in the

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Apoptosis (2007) 12:1989–2001 1995

Fig. 4 Western blotting (A–B)

and immunohistochemical
staining of Fas (C–F) in the rat
lung during ischemia-
reperfusion (I/R) of small
intestine. (A) Fas expression
was detected by Western blot
analysis; (B) staining density for
Fas was measured; (C) Sham-
operation; (D) 1-h ischemia; (E)
3 h of reperfusion; (F) 24 h of
reperfusion. Fas-positive cells
were markedly increased at 3 h
of reperfusion in alveolar
epithelial (arrows) and stromal
cells (arrowheads). Quantitative
analysis of the signal intensity
of Fas. Data were analyzed as
mean ± SEM of three rats.
*p \ 0.01. Scale bars: 20 lm

Fig. 5 Western blotting (A–B)

and immunohistochemical
staining of FasL (C–F) in the rat
lung during ischemia-
reperfusion (I/R) of small
intestine. (A) Specific band for
FasL in the lung at a various
time points; (B) staining density
for FasL was measured; (C)
Sham-operation; (D) 1-h
ischemia; (E) 3 h of
reperfusion; (F) 24 h of
reperfusion. FasL positive cells
were markedly increased at 3 h
of reperfusion in the alveolar
epithelial (arrows) and stromal
cells (arrowheads). Quantitative
analysis of the signal intensity
of FasL. Data were analyzed as
mean ± SEM of three rats.
*p \ 0.01. Scale bar: 20 lm

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Fig. 6 Western blotting (A–B)

and immunohistochemical
staining of Bcl-2 (C–F) in the
rat lung during ischemia-
reperfusion (I/R) of small
intestine. (A) Western blot
analysis shows the expression of
Bcl-2; (B) staining density for
Bcl-2 was measured; (C) Sham-
operation; (D) 1-h ischemia; (E)
3 h of reperfusion; (F) 24 h of
reperfusion. Arrows indicate
Bcl-2 positive cells in alveolar
epithelium. Quantitative
analysis of the signal intensity
of Bcl-2. Data were analyzed as
mean ± SEM of three rats.
*p \ 0.01. Scale bar: 20 lm

expression levels of Bcl-2 and Bax were found (Figs. 6 expression of cytochrome c was markedly increased in the
D–F, 7D–F). epithelium and stroma of alveoli at 3 h of reperfusion
(Fig. 9C). Similar to cytochrome c, activated caspase-3
was detected in only some epithelial cells of sham-operated
Bid
rats (Fig. 9E). In the I/R groups, cells positive to activated
caspase-3 were found in the epithelium and stroma of
To examine the involvement of Bid as a key component
alveoli after 1 h of ischemia (Fig. 9F) and their number
linking death receptor and mitochondrial pathways in the
further increased at 3 h of reperfusion (Fig. 9G).
induction of lung epithelial cell apoptosis, we analyzed the
expression of Bid by Western blotting and immunohisto-
chemistry. A specific band for Bid was detected in extracts
Effect of intravenous injection of anti-TNF-a antibody
from rat lungs (Fig. 8A). Furthermore, the amount of Bid in
on induction of apoptosis in the rat lung during I/R of
the cytoplasmic fraction gradually increased during I/R of
small intestine
small intestine, while that in the mitochondrial fraction
increased transiently at 3 h of reperfusion (Fig. 8A). The
To provide a direct evidence for the involvement of TNF-a
expression of Bid in the lung tissue was predominantly found
in the induction of apoptosis, we attempted to neutralize
in the cytoplasm of alveolar epithelial and stromal cells and
TNF-a by anti-TNF-a antibody at 3 h of reperfusion. As
was higher at 3 h of reperfusion relative to 0 h (Figs. 8C–F).
shown in Fig. 10, injection of anti-TNF-a antibody resulted
in significant reduction of the number of TUNEL-positive
cells in the epithelium and stroma of the lung.
Cytochrome c and activated caspase-3

To gain some insights into the role of death cascades in the
induction of apoptosis of rat lung during I/R of small
intestine, we examined the expression of cytochrome c and
activated caspase-3. In the sham-operated groups, staining
for cytochrome c was observed in the cytoplasm of only a
few alveolar epithelial cells (Fig. 9A). In the I/R groups,
Effect of intravenous injection of anti-TNF-a antibody
on gas exchange in the rat lung during I/R of small
intestine
Lung function was assessed by analyzing the pH, PO2,
PCO2, and HCO–3 in intestinal I/R and intestinal I/R plus

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Fig. 7 Western blotting (A–B)
and immunohistochemical
staining of Bax (C–F) in the rat
lung during ischemia-
reperfusion (I/R) of small
intestine. (A) Western blot
analysis shows the expression of
Bax; (B) staining density for
Bax was measured; (C) Sham-
operation; (D) 1-h ischemia; (E)
3 h of reperfusion; (F) 24 h of
reperfusion. Note the presence
of Bax-positive cells in
epithelial (arrows) and stromal
cells (arrowheads). Quantitative
analysis of the signal intensity
of Bax. Data were analyzed as
mean ± SEM of three rats.
*p \ 0.01. Scale bar: 20 lm
anti-TNF-a antibody treatment groups. As shown in
Table 1, the pH and HCO–3 were significantly lower, but the
PO2 was higher in the intestinal I/R groups compared to the
sham-operated groups. However, the treatment with anti-
TNF-a antibody restored significantly the function.
Discussion
Since lung injury is a well-known major complication after
intestinal I/R, our experimental model of lung injury,
though transient, should be useful for analyzing the
molecular events underlying the pathogenesis. Until now,
however, our understanding of the molecular mechanisms
of I/R-related lung injury has been limited except for the
involvement of apoptosis. In the present study, we con-
firmed first a transient increase in the number of apoptotic
cells in the lung epithelium after I/R of small intestine in
rats. The results of further analysis pointed to the role of
up-regulation of Fas and FasL expression, down-regulation
of Bcl-2 and up-regulation of Bax in the induction of
apoptosis, and indicated that the signaling cascades of the
two apoptotic pathways merged through a transient
increase of Bid in mitochondrial fraction. Moreover, our
present study provided a direct evidence for the involve-
ment of TNF-a as a possible serum mediator derived from
I/R intestine. Therefore, our findings provide important
1997
background for future strategies to combat lung injury after
I/R of small intestine.
The alveolar epithelium consists of alveolar type I and
type II cells. EM studies revealed that the apoptotic epi-
thelial cells were alveolar type II cells, as identified by the
presence of lamellar bodies. Furthermore, double-staining
showed that TUNEL-positive cells were mostly positive for
SP-A, confirming that the apoptotic cells were type II cells.
Simultaneously, we analyzed the percentage of apoptotic
epithelial and inflammatory cells in EM data and found that
apoptotic cells were primarily type II epithelial cells in the
rat lung during I/R of small intestine (data not shown).
Type II cells are functionally important and involved in
pulmonary response to various injuries, as well as surfac-
tant production [35]. The previous study revealed that
alveolar epithelial apoptosis was induced by intestinal I/R,
while the apoptotic cells were not distinguished between
alveolar type I and type II cells [36]. The present findings
are in agreement with the conclusion of the previous
studies using various experimental models on the role of
apoptosis of type II cells in the remodeling process that
accompanies tissues repair after lung injury [37–39]. Col-
lectively, these results indicate that the apoptotic cells
induced by I/R of small intestine were type II alveolar cells
in the lung.
TNF-a is a pleiotropic cytokine that triggers either cell
death or proliferation though TNFR1 and TNFR2,
123
1998 Apoptosis (2007) 12:1989–2001

Fig. 8 Western blotting (A–B)

and immunohistochemical
staining of Bid (C–F) in the rat
lung during ischemia-
reperfusion (I/R) of small
intestine. (A) Western blot
analysis shows specific band for
Bid in cytoplasm and
mitochondria; (B) staining
density for Bid in the cytoplasm
and mitochondria was
measured; (C) Sham-operation;
(D) 1-h ischemia; (E) 3 h of
reperfusion; (F) 24 h of
reperfusion. Note the presence
of Bid-positive cells in alveolar
epithelial (arrows) and stromal
cells (arrowheads). Quantitative
analysis of the signal intensity
of Bid. Data were analyzed as
mean ± SEM of three rats.
*p \ 0.01. Scale Bars: 20 lm

respectively. Since TNFR1 contains death cell domains
whereas TNFR2 lacks the death domain, the cellular
response to TNF-a depends to some extend on the level of
TNFR1 and TNFR2 expressions by the cell, as well as the
ratio of ‘‘soluble’’ and ‘‘membrane’’ bound TNFR [11, 40,
41]. Recently, Gushmann et al. has indicated that TNF-a
induced apoptosis in type II lung alveolar cells through
TNFR1 under hyperoxia [42]. Moreover, the previous
studies reported that intestinal I/R resulted in increases in
TNF-a level in systemic and portal circulations [6, 43, 44].
In agreement with these previous results, intravenous
administration of anti-TNF-a antibody markedly inhibited
apoptosis of type II alveolar cell after I/R of small intestine.
Thus, the accumulated evidence strongly indicates that
TNF-a, derived from I/R intestine, is a major mediator of
induction of lung cell apoptosis.
The Fas/FasL system is a key inducer of apoptosis in
various tissues such as liver, kidney, testis, small intes-
tine, and lung, especially under I/R conditions.
Previously, we reported that coexpression of Fas and
FasL was associated with lung injury after intestinal
transplantation [45]. In this study, we also found that
coexpression of Fas and FasL was induced in the rat
lung after I/R of small intestine. Therefore, these results
indicated that the Fas/FasL system plays more complex
roles in lung injury.
Bid, a member of the Bcl-2 family is a substrate of
caspase-8 and is activated by the TNFR1 pathway [20, 21,
46]. Importantly, Bid is presumed to communicate death
signals from the FADD-dependent extrinsic pathway to the
intrinsic mitochondrial pathway, through the induction of
cytochrome c release and subsequent activation of down-
stream caspases. In fact, our data strongly suggested that
the translocation of Bid from the cytoplasm to the mito-
chondria in the lung was transiently induced by I/R of
small intestine and that of Bid was inhibited by injection of
anti-TNF-a antibody (data not shown). Several recent in
vivo studies reported that Bid has a higher affinity for Bax
than Bcl-2 [47–49], and forms a complex with Bax that
triggers cytochrome c release from mitochondria [46].
Hence, Bid is a key molecule that induces lung epithelial
cell apoptosis after proteolytic cleavage by caspase-8. In
this context, it should be noted that the timing of up-reg-
ulation of Bax and cytochrome c expression in type II
alveolar cells coincided with that of Bid in the
mitochondria.

123
Apoptosis (2007) 12:1989–2001 1999

Fig. 9 Immunohistochemical staining of cytochrome c (A–D) and presence of cytochrome c and activated caspase-3-positive cells in
activated caspase-3 (E–H) in the rat lung during ischemia-reperfusion alveolar epithelial (arrows) and stromal cells (arrowheads). The
(I/R) of small intestine. (A, E) Sham-operation; (B, F) 1-h ischemia; highest expression levels of cytochrome c and activated caspase-3
(C, G) 3 h of reperfusion; (D, H) 24 h of reperfusion. Note the were noted at 3-h of reperfusion (C, G). Scale bars: 20 lm

Table 1 Effect of intravenous injection of anti-TNF-a antibody on

gas exchange in the rat lung during I/R of small intestine
Control I/R Anti-TNF a

Gas exchange
pH 7.29 ± 0.18 7.10 ± 0.03* 7.25 ± 0.10

PCO2 (mmHg) 37.34 ± 1.77 25.42 ± 1.01* 39.26 ± 2.26

PO2 (mmHg) 93.60 ± 2.31 134.80 ± 1.36* 105.20 ± 1.98

HCO–3 25.10 ± 0.86 16.02 ± 0.55* 22.88 ± 1.75

I/R, ischemia-reperfusion (SMA clamping for 1-h intestinal ischemia
followed by 3 h of reperfusion; Control, control group (sham-opera-
tion); Anti-TNF-a, intestinal I/R plus intravenous injection of anti-
TNF-a. Data are shown as mean ± SEM (n = 5)
Fig. 10 Quantitative analysis of TUNEL-positive cells in the rat lung
*p \ 0.01 compared to the control group
with intravenous injection of anti-TNF-a antibody after ischemia-

reperfusion (I/R) of small intestine. More than 2,000 cells (alveolar p \ 0.01 compared to the I/R group
epithelium and stroma of lung) were counted. The number of
TUNEL-positive cells represents the mean ± SEM (%) of five rats in
each group. *p \ 0.01, compared with I/R rats pretreated with the
apoptotic signaling pathways and mitochondrial translo-
0.9% normal saline cation of Bid.

Acknowledgments This study was supported in part by a Grant-in-

Conclusions
Our results indicated that I/R of small intestine resulted in
apoptosis of rat alveolar type II cells through a series of
events including systemic TNF-a, activation of two
Aid for Scientific Research from the Japan Society for the Promotion
of Science (nos. 16406005, 18390060 to T. Koji). We thank Mr.
Takashi Suematsu for this excellent EM techniques assistance and Dr.
Yorihisa Sumida, Dr. Shuichi Tobinaga, and Prof. Takeshi Nagayasu
(Division of Surgical Oncology, Department of Translational Medical
Sciences) for excellent technical support of assessment of arterial
blood gas exchange in this work.

123
2000
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