Dalle Lucca And George C. Tsokos Tong Shi, Vaishali R. Moulton ...
Tong Shi, Vaishali R. Moulton, Peter H. Lapchak, Guo-Min Deng, Jurandir J.
Dalle Lucca and George C. Tsokos
Am J Physiol Gastrointest Liver Physiol 296:339-347, 2009. First published Dec 18, 2008;
doi:10.1152/ajpgi.90607.2008
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Am J Physiol Gastrointest Liver Physiol 296: G339–G347, 2009.
First published December 18, 2008; doi:10.1152/ajpgi.90607.2008.
Ischemia-mediated aggregation of the actin cytoskeleton is one of the major
initial events resulting in ischemia-reperfusion injury
Tong Shi,1 Vaishali R. Moulton,1 Peter H. Lapchak,1 Guo-Min Deng,1 Jurandir J. Dalle Lucca,2
and George C. Tsokos1
1Division of Rheumatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts;
and 2Walter Reed Army Institute of Research, Silver Spring, Maryland
Submitted 22 October 2008; accepted in final form 16 December 2008
Shi T, Moulton VR, Lapchak PH, Deng G, Dalle Lucca JJ, Tsokos
in human myocardial tissue and C5- or C6-deficient mice and
GC. Ischemia-mediated aggregation of the actin cytoskeleton is one of
rabbits are protected from IR injury strongly suggests that
the major initial events resulting in ischemia-reperfusion injury. Am J
complement is a major mediator of IR injury (15, 36, 51).
Physiol Gastrointest Liver Physiol 296: G339 –G347, 2009. First pub-
Studies in C3- and C4-deficient mice and factor B- or factor
lished December 18, 2008; doi:10.1152/ajpgi.90607.2008.—Ischemia-
D-knockout mice suggest that all three complement pathways
reperfusion (IR) injury represents a major clinical challenge, which
mediate IR injury (39, 41, 44, 48). Although the significance of
contributes to morbidity and mortality during surgery. The critical role
complement has been well established, the cellular mecha-
of natural immunoglobulin M (IgM) and complement in tissue injury
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has been demonstrated. However, cellular mechanisms that result in
nisms leading to its activation in tissues are not clear.
the deposition of natural IgM and the activation of complement are
In addition to the contribution of complement, natural anti-
still unclear. In this report, using a murine intestinal IR injury model,
bodies are also shown to be involved in the pathogenesis of IR
we demonstrated that the
-actin protein in the small intestine was
injury (20, 44, 48). Recombination activation gene (RAG)1/2-
cleaved and actin filaments in the columnar epithelial cells were
deficient mice lacking natural antibodies and CR1/2-null mice
aggregated after a transient disruption during 30 min of ischemia.
with defects in T-dependent B-2 B cell responses to foreign
Ischemia also led to deposition of natural IgM and complement 3
antigens undergo less IR injury (14). Furthermore, natural
ajpgi.physiology.org
(C3). A low dose of cytochalasin D, a depolymerization reagent of the
immunoglobulin (Ig) M has been identified as one of culprits of
actin cytoskeleton, attenuated this deposition and also attenuated
IR injury (3, 48, 50). Recently, nonmuscle myosin type II
intestinal tissue injury in a dose-dependent manner. In contrast, high
(NM-II) heavy chain A and C have been identified as self-
doses of cytochalasin D failed to worsen the injury. These data
targets of natural IgM and IR injury in both the small intestine
indicate that ischemia-mediated aggregation of the actin cytoskeleton,
rather than its disruption, results directly in the deposition of natural
and in the skeletal muscle of mice (49). Thus it has been
IgM and C3. We conclude that ischemia-mediated aggregation of the
suggested that neoantigens or modified epitopes presented on
actin cytoskeleton leads to the deposition of natural IgM and the
the cell surface in ischemic tissues may trigger complement
on March 20, 2010
activation of complement, as well as tissue injury.
activation via natural IgM deposition (49). However, the mech-
anisms that lead to the exposure of these neoantigens need
cytochalasin D; complement; deposition
further investigation.
The actin cytoskeleton is known to play a crucial role in
ISCHEMIA-REPERFUSION (IR) injury, resulting in damage to local
maintaining the functional and structural integrity of cells (21).
and remote organs after periods of ischemia, is a major con-
The three-dimensional network of the actin cytoskeleton inter-
tributor to morbidity and mortality during myocardial infarc-
acts with selected plasmalemmal proteins and ATP depletion
tion, transplantation, stroke, surgery, and trauma (6, 8, 9, 25,
or ischemia disrupt the actin cytoskeleton in vascular smooth
47). However, currently there are no effective therapies be-
muscle cells, endothelial cells, as well as epithelial cells (21–
cause the mechanisms that result in tissue injury are not fully
23, 32, 40). Multiple cleavage of actin protein has been
understood.
observed in apoptotic cells (19, 28), and one of these cleaved
Although IR injury causes a strong inflammatory response
fragments leads to apoptosis-like morphological changes in
(18, 44, 45, 48), accumulated evidence has shown that com-
cultured cells (29).
plement plays a critical role in IR injury. The finding that
We hypothesized that alteration of the actin cytoskeleton
chemotactic complement (C)3-cleavage products are found in
mediated by ischemia is one of the major initial events that
damaged heart tissue indicates that IR injury is complement
result in the deposition of natural IgM, activation of comple-
dependent (17, 42). Additionally, the myocardium is protected
ment, and further tissue injury. In this paper we establish a
from necrosis by cobra venom factor, which depletes C3
definite link between ischemia-mediated alteration of the actin
activation via the activation of the alternative pathway (12, 17,
cytoskeleton and the deposition of natural IgM, the activation
25, 27), and by recombinant soluble human complement re-
of complement, and IR injury. We show in a murine intestinal
ceptor (CR)1, which promotes inactivation of C3 by factor I
IR injury model that ischemia induces aggregation of the actin
(45). Soluble CR1 also reduces cerebral infarct volume (18)
cytoskeleton in columnar epithelial cells in small intestinal villi
and intestinal injury (13). The observation that C5b-9 deposits
after a period of transient disruption. We also show that ische-
mia-mediated aggregation of actin filaments and deposition of
Address for reprint requests and other correspondence: T. Shi or G. C.
Tsokos, Division of Rheumatology, Beth Israel Deaconess Medical Center,
The costs of publication of this article were defrayed in part by the payment
330 Brookline Ave. CLS 928, Boston, MA 02215 (e-mail: tshi@bidmc.
of page charges. The article must therefore be hereby marked “advertisement”
harvard.edu or gtsokos@bidmc.harvard.edu).
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
http://www.ajpgi.org
0193-1857/09 $8.00 Copyright © 2009 the American Physiological Society
G339
G340
ACTIN CYTOSKELETON AGGREGATION IN IR INJURY
IgM and C3/C3d fragment are diminished by an optimal dose
nus) and horseradish peroxidase-conjugated secondary antibodies and
of cytochalasin D. In addition, low doses of cytochalasin D
images were captured using a Fujifilm LAS-40000 luminescent image
attenuate IR injury in a dose-dependent manner whereas high
analyzer (Fujifilm, Valhalla, NY).
doses do not worsen the injury. Taken together, these data
Confocal microscopy. Jejunal segments were rinsed with cold PBS
and snap frozen in frozen tissue embedding media. The tissues were
reveal that ischemia-mediated aggregation of the actin cy-
sectioned transversely (6
M), fixed with 10% phosphate-buffered
toskeleton plays a crucial role in mediating the deposition of
formalin for 10 min, and permeabilized with 0.1% Triton X-100-PBS
IgM and C3, as well as IR injury.
for another 10 min. Sections were blocked with 1% BSA-PBS at room
temperature for 1 h and then incubated with fluorochrome-labeled
MATERIALS AND METHODS
primary or secondary antibodies at room temperature for 1 h. After
being washed and mounted on slides, sections were analyzed by
Materials. Cytochalasin D was purchased from Sigma-Aldrich (St.
confocal microscopy (Nikon Eclipse Ti, Nikon Instruments,
Louis, MO). Hoechst 33342, Alexa Fluor 546-labeled phalloidin, and
Melville, NY).
Alexa Fluor 488-labeled DNase I were bought from Invitrogen (Carls-
To evaluate the ratio of global to filamentous (G/F)-actin using
bad, CA). Microvascular clips were obtained from Biomedical Re-
quantitative fluorescence image analysis (35), tissue sections were
search Instruments (Silver Spring MD). FITC-anti-mouse IgM and C3
stained with fluorochrome-labeled DNase I and phalloidin, which bind
antibodies were purchased from Immunology Consultant Laboratory
to global actin and filamentous actin, respectively (24). Fluorescence
(Newberg, OR). Goat anti-mouse C3d was purchased from R & D
from the columnar epithelial cells of small intestinal villi was obtained
Systems (Minneapolis, MN). Mouse anti- -actin NH2-terminus anti-
by confocal microscopy under identical conditions. Intensity of fluo-
body was bought from Abcam (Cambridge, MA), and mouse anti- -
rescence was analyzed by Nikon EZ-C1 FreeViewer 3_20_615 Gold
actin COOH-terminus antibody was obtained from Santa Cruz Bio-
(Nikon Instruments, Melville, NY). The ratio of G/F-actin was cal-
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technology (Santa Cruz, CA).
culated by dividing DNase I fluorescent intensity by phalloidin fluo-
Animal model of murine intestinal IR. All mice used in this study
rescent intensity. Increased ratios of G/F-actin reflect disruption of
were maintained in specific pathogen-free conditions in the animal
actin filaments, and decreased ratios of G/F-actin indicate aggregation
research facility at the Beth Israel Deaconess Medical Center
of actin filaments.
(BIDMC). All experiments were performed in accordance with the
Statistical analysis. Data are expressed as means
SD. Differ-
guidelines and approval of the Institutional Animal Care and Use
ences between groups were evaluated by Student’s t-test. P
0.05
Committee of the BIDMC. Male C57BL/6 mice aged 8 to 10 wk were
was considered statistically significant. A two-tailed distribution and
ajpgi.physiology.org
purchased from Jackson Laboratory (Bar Harbor, ME) and acclimated
paired t-test were used to evaluate the differences in the ratios of
for 1 wk. Mice were anesthetized by intraperitoneal injection of a
G/F-actin. A two-tailed distribution and unpaired t-test were em-
combination of ketamine-xylazine-acepromazine (100:20:3 mg/kg)
ployed for the differences in injury scores.
(1) and subjected to IR as described previously (14) with some
modifications that included ischemia for 20 to 30 min and reperfusion
RESULTS
for 10 min to 2 h. At various times after ischemia and reperfusion,
mice were euthanized and tissues were harvested. Sham mice were
Ischemia induces cleavage of
-actin protein. On the basis
subjected to an identical surgical protocol aside from artery clamping.
of a previous study showing that a 15-kDa fragment of actin
on March 20, 2010
In inhibition experiments, food, but not water, was withdrawn for 24 h
leads to morphological changes of cells undergoing apoptosis
before anesthesia. All procedures were performed while maintaining
(29) and our interest to find potentially cleaved proteins in the
mouse body temperature at 37°C using a controlled heating pad. Mice
small intestine, we conducted experiments to compare the
subjected to inhibition experiments underwent small intestine intralu-
minal injection 0.5–1.0 ml of cytochalasin D dissolved in dimethyl
differential expression of proteins in sham-, ischemia-, or
sulfoxide (DMSO)-PBS immediately after arterial clamping. Vehicle-
IR-treated small intestine. The superior mesenteric artery
treated mice were given an equal volume of DMSO.
(SMA) was clamped with a microvascular clip to generate
Histology. About 15 cm of jejunal segment was rinsed and fixed
ischemia in the jejunum for 30 min followed by reperfusion
immediately with cold 10% phosphate-buffered formalin. The tissues
2 h. Jejunal segments harvested without clip clamping were
were then embedded in paraffin, sectioned transversely (5
m), and
designated as sham samples, before clip removal as ischemia
stained with hematoxylin and eosin (H & E). Villus damage was
samples, and after reperfusion as IR samples. Jejunal segments
scored according to the severity of injury. The complete destruction of
were processed and subjected to 2-DIGE. One spot with a
the villus was scored “6” and no injury in the villus was scored “0”
molecular weight of 30 kDa identified in the ischemia samples
(14). The scores of injury were calculated by the following equation:
(Fig. 1, A and B) was isolated and sequenced. The abundance
injury score
¥(score
Ni)/N. Ni represents the number of villi with
the same injury score. N represents the total number of villi counted
of this protein was increased 11.43-fold after ischemia and
(250 villi).
0.78-fold after IR compared with sham samples. The MAS-
Proteomic analysis. Jejunal segments obtained from mice (n
COT search engine analysis identified this protein to be
-ac-
3/group) subjected to sham, 30-min ischemia, and 30-min ischemia-2
tin.
h reperfusion were harvested and sent on dry ice directly for two-
Mouse anti- -actin COOH-terminus antibody identified a
dimensional difference gel electrophoresis (2-DIGE) and mass spec-
band of 40 kDa in the ischemia (Fig. 1C, lanes 2 and 3) and IR
trometry analysis provided by Applied Biomics (Hayward, CA).
samples (Fig. 1C, lanes 4 and 5) but not from sham samples
Western blot assay. Jejunal segments from individual mice sub-
(Fig. 1C, lane 1), indicating that -actin protein (42 kDa) was
jected to various durations of ischemia and reperfusion were homog-
cleaved during ischemia and reperfusion. A faint 30-kDa band
enized in buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM
in 20-min ischemia samples (Fig. 1D, lane 2) and a strong band
KCl, and 1 mM DTT) with complete protease inhibitor cocktail
in 30-min ischemia sample were also visualized in Western
(Roche, Mannheim, Germany). Concentrations of protein extracts
were determined using Micro BCA protein assay kit (Pierce, Rock-
blot (Fig. 1D, lane 3). This band was also seen in 10-min
ford, IL). Protein extracts were processed under reducing conditions
reperfusion samples (Fig. 1D, lane 4). However, it disappeared
and subjected to standard Western blot analysis. Blots were developed
in 2-h reperfusion samples (Fig. 1D, lane 5). These results are
with mouse anti- -actin antibodies (NH2 terminus and COOH termi-
consistent with the 2-DIGE results above, indicating that ische-
AJP-Gastrointest Liver Physiol • VOL 296 • FEBRUARY 2009 • www.ajpgi.org
ACTIN CYTOSKELETON AGGREGATION IN IR INJURY
G341
mia induces cleavage of
-actin and the cleaved fragment is
Ischemia with or without reperfusion results in alteration of
destroyed by reperfusion.
the actin cytoskeleton. Disruption of the actin cytoskeleton
Mouse anti- -actin NH2-terminus antibody failed to identify
induced by ATP depletion or ischemia have been reported in
this 30-kDa band (data not shown), indicating that the cleaved
various cell types (21–23, 32). However, it has also been
fragment represents the COOH terminus of
-actin. In this
demonstrated that the actin cytoskeleton polymerizes at 2 h and
experiment, 25-kDa bands of the Ig light chain were consid-
then depolymerizes in a camptothecin-induced apoptotic
ered as loading controls since the primary antibody used in the
model in HL-60 cells (35). In addition,
-actin is cleaved to
Western blot assay was a mouse immunoglobulin (Fig. 1D).
yield 40- and 30-kDa bands during ischemia in our intestinal
IR model (Fig. 1). Therefore, it is possible that ischemia may
alter the actin cytoskeletal network in tissues. We conducted
experiments focusing on the alteration of the actin cytoskeleton
to address this hypothesis. The SMA was clamped with a
microvascular clip to generate jejunal ischemia for 20 or 30
min and reperfused for 10 min or 2 h. Jejunal segments were
processed and then stained with fluorochrome-labeled phalloi-
din and DNase I, Hoechst 33342 or H & E.
H & E staining revealed that 30-min ischemia caused minor
injury in small intestinal villi (injury score: 1.54
0.58, n
5; P
0.003) whereas 2-h reperfusion following 30-min
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ischemia resulted in severe injury (injury score: 4.18
0.44,
n
8; P
0.0001) compared with sham segments (injury
score: 0.18
0.07, n
4).
Confocal microscopic images showed that phalloidin stain-
ing of the actin cytoskeleton in the columnar epithelial cells of
the villi is lighter and less dense after 20-min ischemia (Fig. 2B)
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whereas staining becomes brighter and more dense in 30-min
ischemia tissues (Fig. 2C) compared with sham-treated tissues
(Fig. 2A). This indicates that ischemia results in an initial
disruption of the actin cytoskeleton, followed by aggregation.
This was confirmed by a quantitative analysis of G/F-actin; the
ratio of G/F-actin was 2.08
1.34 (n
6, Fig. 3) in the
columnar epithelial cells from sham mice, which significantly
increased to 4.93
3.62 (n
6, P
0.038, Fig. 3) after
on March 20, 2010
20-min ischemia. However, after 30-min ischemia the ratio of
G/F-actin was significantly decreased to 2.54
2.52 (n
6,
P
0.007, Fig. 3) consistent with the observation above that
the actin cytoskeleton aggregates after 30-min ischemia (Fig.
2C). These results indicate that ischemia results in aggregation
of actin filaments after an initial transient disruption. However,
the actin cytoskeleton was disrupted again when the small
intestine was reperfused for 10 min (Fig. 2D) and was de-
stroyed completely after reperfusion for 2 h (Fig. 2E). The
disruption induced by reperfusion was also demonstrated by an
increase in the ratio of G/F-actin to 4.79
4.58 (n
6) or
4.87
3.69 (n
6) after 10 min or 2 h reperfusion, respec-
tively. These results indicate that the restoration of blood
supply damages the actin cytoskeleton.
Fig. 1. Ischemia induces multiple cleavages of -actin. Jejunal segments were
harvested and homogenized in buffer. Proteins, pooled from 3 mice per group,
were labeled with various CyDyes and subjected to 2-dimensional difference
gel electrophoresis (2-DIGE). A: sham-treated sample was labeled in red;
30-min ischemia sample was labeled in green. B: 30-min ischemia sample was
labeled in red; 2-h reperfusion sample was labeled in green. Arrows indicate
the dots that were sequenced. In separate experiments, proteins were subjected
to standard Western blot. Blots were developed with mouse anti- -actin
antibodies and horseradish peroxidase-conjugated anti-mouse antibody. Lane
1, sham; lane 2, 20-min ischemia; lane 3, 30-min ischemia; lane 4, 30-min
ischemia/10-min reperfusion; lane 5, 30-min ischemia/2 h reperfusion in C and
D. Arrows indicate 40-kDa protein in C and 30-kDa protein in D. Western blot
images represent 3 independent experiments.
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G342
ACTIN CYTOSKELETON AGGREGATION IN IR INJURY
Fig. 2. Ischemia induces disruption and aggregation of the actin cytoskeleton. Jejunal segments were processed and then stained with fluorochrome-labeled
phalloidin (red) and Hoechst 33342 (blue). A: sham. B and F: 20-min ischemia. C and G: 30-min ischemia. D and H: 30-min ischemia/10-min reperfusion. E
and I: 30-min ischemia/2 h reperfusion. Images represent more than 3 independent experiments.
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Ischemia with or without reperfusion induces natural IgM
In contrast, IgM deposits were only detected in blood vessels
and C3 deposition. To investigate whether ischemia with or
of sham samples (Fig. 4A).
without reperfusion results in natural IgM and C3 deposition,
Both C3 and C3d deposits were observed on the microvilli
we evaluated this deposition in small intestinal villi from mice
of the columnar epithelial cells from mice subjected to 30-min
ajpgi.physiology.org
subjected to various durations of ischemia with or without
or 2-h IR (Fig. 4, J and O). Both C3 and C3d deposits were also
reperfusion by confocal microscopy. Jejunal segments were
visualized on the villi from mice subjected to 30- or 10-min IR
processed as described above and stained with FITC-anti-IgM
(Fig. 4, I and N). Deposits of C3 and C3d were clearly detected
or anti-C3 antibodies, or anti-C3d antibody/FITC-secondary
in tissues from mice subjected to 30-min ischemia (Fig. 4, H
antibody, Alexa Fluor 546-phalloidin, and Hoechst 33342.
and M) but barely from those subjected to 20-min ischemia
Confocal microscopy showed IgM deposits on the membranes
(Fig. 4, G and L). This indicates that ischemia results in
of villus epithelial cells, which are in close proximity to blood
deposition of C3 and more specifically C3d. These results
vessels and also on the microvilli of the columnar epithelial
on March 20, 2010
reveal that not only reperfusion but also ischemia without
cells from mice subjected to 10 min (Fig. 4D) or 2 h reperfu-
reperfusion activates complement in the small intestine.
sion (Fig. 4E). IgM deposits were clearly visualized (Fig. 4C)
Ischemia-mediated aggregation of the actin cytoskeleton
in 30-min ischemia samples although minor IgM deposits were
results in IR-mediated injury. We next hypothesized that ische-
observed on cores of the villi in 20-min ischemia samples (Fig.
mia-mediated alteration of the actin cytoskeleton eventually
4B), indicating that ischemia efficiently induces deposition of
leads to IR tissue injury. Therefore, we used cytochalasin D, a
natural IgM. The IgM deposits on the villi from mice subjected
reagent that disrupts and blocks polymerization of actin fila-
to 30- or 10-min IR confirmed that the deposition of IgM in
ments (37), to alter the actin cytoskeletal network in small
ischemic tissues is due to specific binding of IgM to the tissues.
intestinal villi to test this hypothesis. Mice were treated as
described above. Various doses of cytochalasin D in DMSO-
PBS ( 0.1% in vol/vol) were injected into the small intestinal
lumen immediately after the clip was applied to the SMA.
After 30-min or 2-h IR, jejunal segments were processed and
stained with H & E. Results of the cytochalasin D dose-
response study showed that 1 M of cytochalasin D effectively
inhibited small intestinal injury (injury score: 1.38
0.55, n
12, P
0.0001, Fig. 5), compared with the injury score in
tissues from vehicle-treated mice (3.83
0.97, n
16, Fig. 5).
This inhibition was dose dependent when doses of cytochalasin
D used were less than 1 M. This indicates that cytochalasin D
dose-dependent alteration of the actin cytoskeleton protects
mice from the small intestinal injury. Although cytochalasin D
lost its inhibitory effect with gradually increased doses (Fig. 5),
Fig. 3. Kinetic analysis of the ratio of G- to F-actin ratio (G/F-actin) columnar
epithelial cells of small intestinal villi during ischemia (I) and reperfusion (R).
higher doses (30 and 100 M) of cytochalasin D did not induce
■, Vehicle-treated samples;
, cytochalasin D-treated samples. Ischemia re-
additional injury than that from vehicle-treated mice (P
0.75
sults in aggregation of actin filaments after an initial transient disruption.
and P
0.86, respectively). This indicates that cytochalasin
However, the actin cytoskeleton was disrupted again when the small
D-induced disruption of the actin cytoskeleton does not con-
intestine was reperfused for 10 min and was destroyed completely after
reperfusion for 2 h.
tribute to the injury. All experimental mice died after 200
M
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ACTIN CYTOSKELETON AGGREGATION IN IR INJURY
G343
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ajpgi.physiology.org
Fig. 4. Ischemia results in deposition of natural IgM and C3/C3d. Jejunal segments were stained with fluorochrome-labeled phalloidin (red), anti-C3 (green),
anti-C3d (green), and Hoechst 33342 (blue). A, F, K: sham. B, G, L: 20-min ischemia. C, H, M: 30-min ischemia. D, I, N: 30-min ischemia/10-min reperfusion.
E, J, O: 30-min ischemia/2 h reperfusion. Images represent 3 independent experiments.
of cytochalasin D was injected whereas sham-treated mice died
effects of cytochalasin D on IR-induced morphological
within several minutes after 1
M of cytochalasin D was
changes of the actin cytoskeleton. Mice were treated with 1
injected so that we were unable to obtain results from cytocha-
M of cytochalasin D or vehicle and subjected to various
lasin D-treated sham samples. These findings suggest that
durations of ischemia and reperfusion. Frozen sections of
on March 20, 2010
aggregation of actin filaments ultimately leads to IR tissue
jejunal segments were stained with fluorochrome-labeled phal-
injury.
loidin and DNase I, as well as Hoechst 33342. Confocal
Optimal concentration of cytochalasin D attenuates the alter-
microscopic results showed that the brightness, thickness, and
ation of the actin cytoskeleton. To further investigate how cy-
integrity of the actin cytoskeleton in the villus columnar
tochalasin D alters the actin cytoskeleton, we observed the
epithelial cells from mice subjected to 20-min ischemia (Fig.
2F) were comparable to those from sham-treated mice without
cytochalasin D treatment (Fig. 2A) and were brighter, more
dense than those in samples from 20-min ischemia-treated
mice treated with vehicle (Fig. 2B). These data indicated that
the optimal dose of cytochalasin D blocks the disruption of the
actin cytoskeleton. In samples from 30-min ischemia-treated
mice treated with cytochalasin D (Fig. 2G) or vehicle (Fig. 2C)
the integrity of the actin cytoskeleton was comparable, indi-
cating that the actin cytoskeleton does not undergo aggrega-
tion. The actin cytoskeleton network is highly organized in the
columnar epithelial cells from mice subjected to 10-min or 2-h
reperfusion (Fig. 2, H and I), compared with those from
vehicle-treated mice subjected to 10 min or 2 h of reperfusion
(Fig. 2D and E). These images demonstrate that cytochalasin D
blocks reperfusion-mediated damage of the actin cytoskeleton.
Fig. 5. Cytochalasin D attenuates tissue injury in a dose-dependent manner.
The findings above are supported by changes in the ratio of
Jejunal segments from mice undergo ischemia-reperfusion and injection of
G/F-actin. The ratio of G/F-actin in the columnar epithelial
various doses of cytochalasin D were processed and stained with hematoxylin
and eosin. The inhibition of tissue injury indicates that cytochalasin D
cells of small intestinal villi from mice subjected to 20-min
dose-dependent alteration of the actin cytoskeleton protects mice from the
ischemia (2.19
1.24, n
6, P
0.89) was comparable to
small intestinal injury. That higher doses of cytochalasin D did not induce
that from sham mice without cytochalasin D treatment (2.08
additional injury than that from vehicle-treated mice indicates that cytochalasin
1.34, n
6, Fig. 3) and was significantly lower than that from
D-induced disruption of the actin cytoskeleton does not contribute to the
injury.
vehicle-treated mice subjected to 20-min ischemia (4.93
AJP-Gastrointest Liver Physiol • VOL 296 • FEBRUARY 2009 • www.ajpgi.org
G344
ACTIN CYTOSKELETON AGGREGATION IN IR INJURY
3.62, n
6, P
0.04, Fig. 3), consistent with the observation
Ischemia-mediated aggregation of the actin cytoskeleton
above (Fig. 2F) indicating that 1
M of cytochalasin D
leads to deposition of IgM and C3. Ischemia-mediated depo-
attenuates the disruption of the actin cytoskeleton. The ratio of
sition of natural IgM and C3 in small intestinal villi has been
G/F-actin from mice subjected to 30-min ischemia increased,
shown here and by others (48), and ischemia-mediated aggre-
rather than decreased, slightly to 2.60
2.40 (n
6, P
0.54,
gation of the actin cytoskeleton is demonstrated above. On this
Fig. 3), compared with those from mice subjected to 20-min
basis, we hypothesized that ischemia-mediated aggregation of
ischemia. This result also agrees with the observation above in
the actin cytoskeleton directly results in the deposition of
which cytochalasin D blocks the aggregation of actin filaments
natural IgM and C3. To test this hypothesis, we determined
(Fig. 2G). This may also mean that the aggregation of actin
whether cytochalasin D can attenuate ischemia-mediated dep-
filaments may be a result of the initial disruption and actin
osition of IgM and C3/C3d. Mice treated with 1
M of
filaments may be unable to aggregate owing to the blockade of
cytochalasin D or vehicle were subjected to various durations
the disruption by cytochalasin D as in tissues from vehicle-
of ischemia and reperfusion. Frozen sections of jejunal seg-
treated mice. Thus it can be concluded that the disruption of the
ments were stained for IgM and C3/C3d plus phalloidin and
actin cytoskeleton initiates the aggregation. The ratios of G/F-
Hoechst 33342. Confocal images demonstrate decreased dep-
actin increased significantly (n
6, P
0.02, Fig. 3) and went
osition of IgM and C3/C3d on the villi from cytochalasin
down to initial levels (n
6, P
0.64, Fig. 3) after 10 min or
D-treated mice subjected to various durations of ischemia
2 h of reperfusion, respectively, compared with that from mice
and reperfusion (Fig. 6) compared with the villi from vehi-
subjected to 20-min ischemia (Fig. 3). The ratios were much
cle-treated mice (Fig. 4). However, cytochalasin D does not
Downloaded from
lower than those from vehicle-treated mice subjected to 10-min
interfere with the natural deposition of IgM or C3/C3d in
or 2-h reperfusion, respectively (Fig. 3), supporting the con-
blood vessels (Fig. 6). These results clearly indicate that the
clusion above that cytochalasin D protects the actin cytoskel-
optimal concentration of cytochalasin D attenuates the dep-
eton from reperfusion-mediated disruption (Fig. 2, H and I).
osition of IgM and C3, and this strongly supports the
These data strongly support the hypothesis that cytochalasin D
hypothesis that ischemia-mediated aggregation of the actin
alters ischemia and reperfusion-mediated disruption and aggre-
cytoskeleton directly leads to the deposition of natural IgM
gation of the actin cytoskeleton.
and C3.
ajpgi.physiology.org
on March 20, 2010
Fig. 6. Cytochalasin D blocks deposition of natural IgM and C3/C3d. Jejunal segments from mice subjected to ischemia-reperfusion and injection of optimal
dose of cytochalasin D were stained with fluorochrome labeled-phalloidin (red), anti-C3 (green), anti-C3d (green), and Hoechst 33342 (blue). A, E, I: 20-min
ischemia. B, F, J: 30-min ischemia. C, G, K: 30-min ischemia/10-min reperfusion. D, H, L: 30-min ischemia/2 h reperfusion. Images represent 3 independent
experiments.
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ACTIN CYTOSKELETON AGGREGATION IN IR INJURY
G345
DISCUSSION
These data support our observation of actin cytoskeletal aggre-
gation in the small intestine. Ischemia-induced consumption of
In the present study, we investigated the alteration of the
ATP in the epithelial cells may lead to the aggregation too (22,
actin cytoskeleton during ischemia with or without reperfusion.
32, 35).
We demonstrated that ischemia results in multiple cleavages of
Several studies have shown that IR results in the deposition
-actin (Fig. 1) and aggregation of the actin cytoskeleton after
of natural IgM and IgG, the activation of complement (3, 9, 12,
a transient disruption (Figs. 2 and 3). The aggregation of the
17, 18, 27, 36, 44, 45, 48, 51), and the subsequent formation of
actin cytoskeleton mediated by ischemia induces deposition of
the membrane attack complex that results in tissue necrosis (2,
natural IgM and C3 (Fig. 4). Furthermore, cytochalasin D
36, 51). It was suggested that natural IgM against NM-II
attenuates the aggregation of the actin cytoskeleton (Fig. 2), the
directly causes tissue injury after IR and a peptide mimicking
subsequent deposition of natural IgM and C3 (Fig. 6), as well
an epitope on NM-II blocks complement activation and pre-
as IR injury (Fig. 5).
vents tissue injury (49, 50). These studies clearly indicate that
It has been reported that the actin protein is cleaved by
the deposition of natural IgM and the activation of complement
interleukin-1
converting enzyme (ICE) family proteases,
during IR is due to a turnover of antigens on cell membranes,
which are active players of programmed cell death (apoptosis)
which are not exposed to the innate immune system under
(30), to produce three major peptides (40 – 41, 30 –31, and
physiological conditions (49). Our observations in this report
14 –15 kDa) in vitro and in cultured apoptotic cells (19, 28).
support these findings. We also observed that actin filaments
We demonstrated, in vivo, employing 2-DIGE and Western
aggregate after a transient disruption. So we hypothesized that
blot analysis in this report, that ischemia-mediated cleavage of
the alteration of the actin cytoskeleton results in the deposition
Downloaded from
-actin in mouse small intestine yields bands with molecular
of natural IgM and complement. We used cytochalasin D, a
weights of
30 and 40 kDa (Fig. 1). Our results reveal that
fungal metabolite that binds to the barbed end of actin fila-
ischemia-mediated cleavage of
-actin occurs, in vivo, in
ments (37), to test this hypothesis. The fact that cytochalasin D
tissues.
does not inhibit glucose transport (34) makes it acceptable for
Ischemia-mediated disruption of vascular smooth muscle
our ischemia-mediated actin cytoskeletal alteration study. It
cells and epithelial cells in the kidney has been reported (21,
has been reported that cytochalasin D inhibits the rapid poly-
23). However, ATP depletion induced by antimycin A in the
merization of actin (10, 46). We developed a novel model that
ajpgi.physiology.org
proximal tubule-derived LLC-PK1 cell line results in disrup-
makes it possible to study the effects of cytochalasin D on cell
tion of the cortical cytoskeleton, and at same time actin
function in vivo. In this model, various doses of cytochalasin D
monomers significantly convert into filamentous actin to form
were injected immediately into the small intestinal lumen after
large cytoplasmic aggregates (32). Furthermore, energy deple-
the SMA was clamped. We believe that SMA clamping blocks
tion leads to disintegration of filamentous actin and formation
the absorption of cytochalasin D into the blood circulation
of numerous small clumps of filamentous actin in the cyto-
since all animals died several minutes after its injection without
plasm of cultured aortic endothelial cells (22). Using an intes-
SMA clamping. A 1- M dose of cytochalasin D is optimal to
on March 20, 2010
tinal IR injury model in which small intestinal villi are vulner-
block both disruption and aggregation of actin filaments (Figs.
able to IR insult, we demonstrated in vivo by phalloidin
2 and 3). This finding is consistent with the observation that
staining that the actin cytoskeleton in the columnar epithelial
cytochalasin D treatment of cells induces actin aggregation
cells of the small intestinal villi is disrupted initially and then
while simultaneously depolymerizing preexisting actin cy-
aggregates during a short term of ischemia (Fig. 2). We also
toskeletal components (33). However, higher doses of cytocha-
demonstrated this result quantitatively by calculating ratios of
lasin D, resulting in disruption of actin filaments (43) and
G/F-actin (Fig. 3). We were unable to measure concentrations
detachment of the epithelial cells from the intestinal villi (data
of global actin and filamentous actin using the assay of DNase
not shown), failed to worsen tissue injury (Fig. 5). This
I inhibition described previously (5) because of very high
indicates that the disruption does not contribute to IgM- or
endogenous DNase I activity in intestinal samples, which may
complement-mediated tissue injury (Fig. 4). We conclude that
come from the pancreas. Our finding of the initial disruption of
ischemia-mediated aggregation after an initial disruption in-
the actin cytoskeleton is supported by observations that high
duces deposition of natural IgM and C3 (Figs. 5 and 6). It is
cytosolic Ca2
concentrations, which increase rapidly after
also possible that the subsequent aggregation results from the
2-min exposure to a metabolic inhibitor, result in depolymer-
initial disruption. Under physiological conditions, polymeriza-
ization of the actin cytoskeleton (22). Thus it is possible that
tion and depolymerization may be well balanced whereas this
during early ischemia in our study a rapid increase in free
balance is biased to either disruption or aggregation during
cytosolic Ca2
results in the disruption of filamentous actin.
ischemia.
Free Ca2
also helps villin, one of the major actin-associated
It has been suggested that NM-II heavy chains, which were
proteins (11), to break or sever filamentous actin (16). The
identified as targets of natural IgM, may be exposed to the
cleavage of actin fragments by ICE family proteases demon-
innate immunity during ischemia (49). NM-II binds to actin
strate impaired capability to block DNase I activity and to
filaments, playing a critical role in regulating cell motility and
polymerize normally (19). Ectopic expression of a 15-kDa
polarity (7). It has also been reported that the injection of
fragment of actin, which is cleaved from actin filaments by ICE
anti- 2-glycoprotein I ( 2-GPI) antibodies and anti-phospho-
family proteases during apoptosis, induces morphological
lipid antibodies into Rag1- and CR2-deficient mice restore
changes, very similar to those of apoptotic cells (29). Although
intestinal injury (14). 2-GPI, a plasma protein, was reported to
we were unable to visualize the 15-kDa fragment of actin by
bind to phosphatidylserine (PS) on the outside of the mem-
Western blot analysis, the accumulation of these fragments
brane of apoptotic cells (4, 26). These observations indicate
may contribute to the later aggregation of actin filaments.
that anti- 2-GPI and anti-PS antibodies mediate tissue injury
AJP-Gastrointest Liver Physiol • VOL 296 • FEBRUARY 2009 • www.ajpgi.org
G346
ACTIN CYTOSKELETON AGGREGATION IN IR INJURY
due to the formation of complexes of PS/
teric ischemia/reperfusion-induced injury in complement receptor 2/com-
2-GPI/antibodies or
PS/antibodies on the cell membrane, which lead to the activa-
plement receptor 1-deficient mice. J Immunol 173: 7055–7061, 2004.
15. Fleming SD, Mastellos D, Karpel-Massler G, Shea-Donohue T, Lam-
tion of complement. It is possible that ischemia-mediated
bris JD, Tsokos GC. C5a causes limited, polymorphonuclear cell-inde-
aggregation of actin filaments results in the turnover or expo-
pendent, mesenteric ischemia/reperfusion-induced injury. Clin Immunol
sure of NM-II, PS, and/or other antigens to the outer surface of
108: 263–273, 2003.
cell membrane so that they can be recognized by natural IgM
16. Glenney JR Jr, Kaulfus P, Weber K. F actin assembly modulated by
and IgG. Although it is challenging technically for us to test the
villin: Ca
-dependent nucleation and capping of the barbed end. Cell 24:
471– 480, 1981.
turnover hypothesis directly using anti-NM-II or anti- 2-GPI
17. Hill JH, Ward PA. The phlogistic role of C3 leukotactic fragments in
antibodies, the blockade of ischemia-mediated deposition of
myocardial infarcts of rats. J Exp Med 133: 885–900, 1971.
natural IgM and C3 by cytochalasin D clearly supports this
18. Huang J, Kim LJ, Mealey R, Marsh HC Jr, Zhang Y, Tenner AJ,
notion that needs further investigations. Future studies should
Connolly ES Jr, Pinsky DJ. Neuronal protection in stroke by an sLex-
focus on how ischemia-mediated aggregation leads to the
glycosylated complement inhibitory protein. Science 285: 595–599, 1999.
19. Kayalar C, Ord T, Testa MP, Zhong LT, Bredesen DE. Cleavage of
turnover of antigens in the members.
actin by interleukin 1 beta-converting enzyme to reverse DNase I inhibi-
In conclusion, we revealed that the deposition of IgM and
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complement during IR is a direct result of ischemia-mediated
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ACKNOWLEDGMENTS
21. Kellerman PS, Clark RA, Hoilien CA, Linas SL, Molitoris BA. Role of
The authors thank Dr. K. Frank Austen for critical reading of the manu-
microfilaments in maintenance of proximal tubule structural and func-
Downloaded from
script.
tional integrity. Am J Physiol Renal Fluid Electrolyte Physiol 259: F279 –
F285, 1990.
GRANTS
22. Kuhne W, Besselmann M, Noll T, Muhs A, Watanabe H, Piper HM.
Disintegration of cytoskeletal structure of actin filaments in energy-
The work was supported by Grant no. W81XWH-07-1-0286 from Medical
depleted endothelial cells. Am J Physiol Heart Circ Physiol 264: H1599 –
Research and Materiel Command.
H1608, 1993.
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