Analysis of Cellular Senescence Induced by Lipopolysaccharide in Pulmonary Alveolar Epithelial Cells Chang Oh Kim Department of Medicine The Graduate School, Yonsei University
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Analysis of Cellular Senescence
Induced by Lipopolysaccharide
in Pulmonary Alveolar Epithelial Cells
Chang Oh Kim
Department of Medicine
The Graduate School, Yonsei University
Analysis of Cellular Senescence
Induced by Lipopolysaccharide
in Pulmonary Alveolar Epithelial Cells
Directed by Professor June Myung Kim
The Doctoral Dissertation
Submitted to the Department of Medicine
The Graduate School of Yonsei University
In partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Chang Oh Kim
December 2010
This certifies that Doctoral
Dissertation of Chang Oh Kim is
approved.
------------------------------------ Thesis Supervisor : June Myung Kim
------------------------------------
Thesis Committee Member : Kwang-Gil Lee
------------------------------------ Thesis Committee Member : Joon Chang
------------------------------------
Thesis Committee Member : Sahng Wook Park
------------------------------------ Thesis Committee Member : Ae Jung Huh
The Graduate School
Yonsei University
December 2010
ACKNOWLEDGEMENTS
Although I had worked in the infection division, I have strong
interests in geriatrics. So, I learned in Dept. of Geriatrics, Mount Sinai
Hospital from Aug. 2005 to Dec. 2006.
This experience led me to this work and I daringly felt that infection
is very important component in geriatric field and there is closed
relationship. Therefore, I would like to perform further evaluation
about senescence research, especially immunosenescence. Then, I will
try to establish geriatrics in Korea.
I owe every piece of my work to my mentor, professor June Myung
Kim, and it may not be possible that this work has been performed
without his guidance. Professor Kwang-Gil Lee, Joon Chang, Sahng
Wook Park, and Dr. Ae Jung Huh, have served as committee member
and given me important advices to make this article better.
Finally, I would like to express my gratitude to my lovely wife and
two sons for their understanging and encouragement.
Chang Oh Kim
<TABLE OF CONTENTS>
ABSTRACT --------------------------------------------------------------------1
I. INTRODUCTION -----------------------------------------------------------3
II. MATERIALS AND METHODS --------------------------------------- 5
1. Cell culture and treatment of A549 cells with LPS ------------- 5
2. Determination of cellular viability and morphology ---------------- 5
3. Detection of cellular apoptosis ----------------------------------------5
4. Detection of cellular lysosomal content -----------------------------6
5. Senescence-associated β-galactosidase assay ----------------------6
6. Fluorescence-activated cell sorting analysis for telomere
length---------------------------------------------------------7
7. Determination of LPS-generated hydrogen peroxide -------------7
8. Effect of the antioxidant glutathione-----------------------------------8
III. RESULTS -------------------------------------------------------------------9
1. Cellular morphologic findings by LPS treatment ------------------9
2. Effect of LPS on cellular viability and apoptosis ------------------10
3. Effect of LPS on cellular lysosomal content ------------------------ 12
4. Expression of senescence-associated β-galactosidase ------------- 13
5. Telomere length analysis by LPS treatment------------------------- 14
6. Concentration of hydrogen peroxide by LPS treatment -----------15
7. Effect of GSH on LPS-induced cellular viability ---------16
IV. DISCUSSION---------------------------------------------------------------17
V. CONCLUSION---------------------------------------------------------------22
VI. REFERENCES------------------------------------------------------------23
ABSTRACT (IN KOREAN) ------------------------------------------------- 27
LIST OF FIGURES
Figure 1. Effect of LPS on A549 cellular morphology------9
Figure 2. Effect of LPS on A549 cellular viability ----------11
Figure 3. Caspase cascade activity by LPS treatment---------11
Figure 4. Senescence-associated increase in cellular
lysosomal content------------------------------------12
Figure 5. Senescence-associated increase in β-galatosidase
activity -------------------------------------------------13
Figure 6. Shortened telomere length by LPS------------------14
Figure 7. Hydrogen peroxide formed on A549 cells which
were exposed to LPS --------------------------------15
Figure 8. GSH inhibits reduction in cellular viability -------16
1
ABSTRACT
Analysis of cellular senescence induced by lipopolysaccharide
in pulmonary alveolar epithelial cells
Chang Oh Kim
Department of Medicine
The Graduate School, Yonsei University
(Directed by Professor June Myung Kim)
There have been few studies on the relationship between senescence and
acute infection, although immunosenescence has been recently introduced. In
this work, it was examined the possibility of lipopolysaccharide (LPS)
causing cellular senescence in lung alveolar epithelial cells. The morphologic
and biochemical characteristics of senescence induced by LPS were analyzed.
Then, it was clarifed how this cellular senescence phenomenon is associated
with oxidative stress effect induced by LPS and whether antioxidants could
inhibit reduced cellular viability by oxidant stress effect of LPS.
Human-like type II lung epithelial cells (A549) were used. In cell viability
using cell counting kit-8, exposure to LPS decreased cellular viability and
induced growth arrest in a concentration-dependent manner. The pre-
apoptotic concentration of LPS was determined by caspase activation using a
Caspase-Glo 3/7 luminescence assay kit. This concentration of LPS caused
morphologic characteristics shown in senescent cells and elevated senescence-
associated β-galactosidase activity. In addition, lysosomal content associated
with senescence was increased by LPS at the pre-apoptotic concentration.
However, this concentration of LPS did not shorten the telomere length.
Exposure to LPS resulted in the formation of hydrogen peroxide in a
2
concentration-dependent manner. The ability of LPS to reduce cellular
viability was inhibited by the presence of glutathione.
This study revealed that LPS could induce cellular senescence in lung
alveloar epithelial cells, and these phenomena were closely associated with
hydrogen peroxide production by LPS. Taken together, it is suggested that
LPS-induced cellular senescence may play an important role in limiting the
tissue repair response after sepsis.
--------------------------------------------------------------------------------------
Key words: lipopolysaccharide, cellular senescence, infectious diseases,
reactive oxygen species
3
Analysis of cellular senescence induced by lipopolysaccharide
in pulmonary alveolar epithelial cells
Chang Oh Kim
Department of Medicine
The Graduate School, Yonsei University
(Directed by Professor June Myung Kim)
I. INTRODUCTION
Aging is a complex natural process potentially involving every molecule,
cell and organ in the body. However, this definition is not always accurate and
usually does not affect an indiviual’s viability. To differentiate simple aging
changes from those that increase the risk of disease, disability, or death,
gerontologists usually use a more precise term, senescence1.
Senescence is progressive deterioration of many bodily functions over time.
Loss of such functions is accompanied by decreased fertility and increased
risk of mortality as an individual gets older. Nevertheless, senescence is one
of nature’s least understood biological processes. Many theories have been
proposed to explain the senescence process in nature. These theories are not
mutually exclusive, because senescence is viewed as a combination of many
processes, which are interactive and interdependent, determining an
individual’s lifespan and health1, 2
.
Cellular senescence is originally described as the finite replicative lifespan
of human somatic cells in culture. Senescent cells enter an irreversible growth
arrest, exhibit a flattened and enlarged morphology, and express a different set
of genes, including negative regulators of the cell cycle such as p53 and p163.
This hypothesis of cellular aging was first described by Hayflick in the 1960s4.
Cellular senescence is one key element that is tightly linked to aging-related
4
and degenerative diseases5. However, the role of cellular senescence in acute
and subacute diseases (e.g. acute respiratory distress syndrome and sepsis) has
not attracted much attention in the past.
Lipopolysaccharide (LPS) is an endotoxin released mainly from gram
negative bacteria during sepsis. LPS causes acute lung injury and acute
respiratory distress syndrome. In LPS-induced lung injury, the
polymorphonuclear leukocyte is a major causative agent and is responsible for
excess production of superoxide anion in the lung6, 7
. Also, macrophages of
the lungs can produce the reactive oxygen materials8-10
.
Several studies have shown that exposure of mammalian cells to oxidants
can produce diverse results with respect to cell growth that are dependent on
oxidant concentration. Thus, low levels of reactive oxygen species can be
mitogenic rather than growth inhibitory11, 12
. Multiple sublethal exposures to
oxidants such as hydrogen peroxide or tert-butyl hydroperoxide can induce
cells to enter a state of premature cellular senescence13, 14
. Exposure to higher
concentration of reactive oxygen specieis can result in cell death due to
apoptosis or necrosis.
Lung alveolar epithelial cell replication, particularly the replication of type
II pneumocytes, is a critical step in the tissue repair process leading to
restoration of lung function following an injury to the epithelial lining15, 16
.
Hydrogen peroxide inhibited alveolar epithelial wound repair by inducing
apoptosis in an in vitro wound repair assay17
. Only few studies have shown
that LPS induces the production of reactive oxygen species lung alveolar
epithelial cells.
Therefore, we first analyzed the effects of LPS in lung epithelial cells. In
this work, we examined the possibility of LPS causing cellular senescence in
lung alveolar epithelial cells. Then, we studied how this cellular senescence
phenomenon is associated with oxidative stress effect induced by LPS and
whether antioxidants could inhibit LPS-induced cellular viability reduction.
5
II. MATERIALS AND METHODS
1. Cell culture and treatment of A549 cells with LPS
Human type II epithelial-like (A549) cells were used. Cell culture was
performed in RPMI 1640 (RPMI; Welgene, Daegu, Korea) containing 10%
fetal bovine serum (FBS; Welgene, Daegu, Korea), 100 U/ml penicillin and
100 μg/ml streptomycin (Welgene, Daegu, Korea). The cells were incubated
for 24 h to allow cell adherence, and then the medium was removed and
replaced with RPMI 1640 containing 1% FBS, 100 U/ml penicillin and 100
μg/ml streptomycin. To determine the effect of LPS on A549 cells, cells were
plated at 2.2×104
cells/well into 6-well tissue culture plates or at 1.0×103
cells/well into 96-well plates. After overnight incubation, LPS (Sigma, St
Louis, MO, USA) at an appropriate concentration was added to cell plates.
2. Determination of cellular viability and morphology
Cellullar viability was checked using a cell counting kit-8 (CCK-8)
(Dojindo, Rockvile, MD, USA). On days 2, 3, 5 and 7 after LPS treatment,
1/10 of media volume of CCK-8 was added to cell culture plates. After 4 h,
cell proliferation was determined by measuring the absorbance at 450 nm
using an ELISA reader Spectra max 340 (Molecular Devices, Sunnyvale, CA,
USA). Cellular morphology was examined on post-LPS treatment days 2, 5
and 7 by a phase-contrast microscopy OLYMPUS IX-71 (Olympus, Center
Valley, PA, USA).
3. Detection of cellular apoptosis
The presence of apoptotic cells was assessed by determining caspase
activation using a Caspase-Glo 3/7 luminescence assay kit (Promega, Sydney,
Australia). Cells were plated at 1×104 (100 μl/well) into clear bottom, opaque
wall 96-well tissue culture plates and incubated for 24 h. The medium was
removed and the cells were then treated with LPS for 24 h. Caspase activity
6
was then assayed. The luminescence of plates was read with a luminometer
(MicroLumat LB 96 P).
4. Detection of cellular lysosomal content
Early-passage cells were washed three times with phosphate-buffered saline
(PBS) followed by incubation for 15 min in 5 μg/ml acridine orange (Anaspec,
San Jose, CA, USA) with HBSS (Welgene, Daegu, Korea) at 37°C. Cells
were subsequently washed with 1% PBS and examined by confocal
microscopy LMS510 (Carl Zeiss, Oberkochen, Germany).
5. Senescence-associated β-galactosidase assay
The presence of senescence-associated β-galactosidase (SA β-gal) activity
was detected according to a previously described method18
. SA β-gal is
known as a biochemical marker associated with senescence, because β-
galatosidase accumulates in senescent cells18
. All reagents for SA β-gal
staining solution were purchased from Sigma (Sigma, St Louis, MO, USA).
Cells were prepared as above and then washed twice with 1% PBS and fixed
for 5 min in 2% formaldehyde/0.2% glutaraldehyde. The cells were incubated
at 37°C with freshly prepared SA β-gal staining solution containing citric acid
(40 mM), sodium phosphate (40 mM), potassium ferrocyanide (5 mM),
potassium ferricyanide (5 mM), sodium chloride (150 mM), magnesium
chloride (2 mM), and 5-bromo-4-chloro-3-indolyl β-D-galactosidase (X-Gal,
1 mg/ml). Then cells were stained for 16 h for full color development and
subsequently examined under phase-contrast microscopy OLYMPUS IX-71
(Olympus, Center Valley, PA, USA) and the number of SA β gal-positive
cells was counted.
7
6. Fluorescence-activated cell sorting analysis for telomere length
Telomere serves as a molecular meter of cellular division. A shortened
telomere length may be considered as an indication for senescent cell19
.
Telomere length analysis was performed using a TeloTAGGG telomere
length assay kit (Roche, Mannheim, Germany) according to the
manufacturer’s instructions. After treatment with LPS, genomic DNA was
prepared and digested by a Hinf 1/Rsa I enzyme mixture. Each digested DNA
was transferred in nylon membranes (Millipore, MA, USA) using SSC
transfer buffer (3 M NaCl and 0.3 M sodium citrate) followed by loading in
0.8% agarose gel and fixed by Spectro Linker XL-1500 UV-crosslinker (120
mJ) (Spectronics Corp. Westbury, NY, USA). After adding a hybridization
solution, telomere probe was incubated at 42°C for 3 h, and the solution was
treated with Anti-DIG-AP. The membrane was incubated in detection buffer
for 2-5 min, and about 3 ml substrate solution was applied. The squeezed
hybridization was exposed to X-ray CP-BU new 100NIF (Agfa, Morsel,
Belgium).
7. Determination of LPS-generated hydrogen peroxide
Hydrogen peroxide production was determined by plating cells at 1.0×103
cells/well into 96-well tissue culture plates. The medium was removed and the
cells were washed twice with PBS. Hydrogen peroxide production was then
assessed using an Amplex Red Hydrogen Peroxide/Peroxidase assay kit
(Molecular probes, Eugene, Oregon, USA). A Krebs-Ringer phosphate
(KRPG) solution was prepared with NaCl (145 mM), Na3PO4 (5.7 mM), KCl
(4.86 mM), CaCl2 (0.54 mM), MgSO4 (1.22 mM), and glucose (5.5 mM, pH
7.35). A reaction mixture was made by mixing KRPG with Amplex Red
solution (50 μl) and HRP (0.1 U/ml). After LPS treatment, cells were
replaced with KRPG (20 μl). The reaction mixture (100 μl) was added to
each well and the medium were incubated at 37°C. Fluorescence was
8
determined at 560 nm using an ELISA reader Spectra max 340 (Molecular
Devices, Sunnyvale, CA, USA)
8. Effect of the antioxidant glutathione
A549 cells were plated at 1.0×103 cells/well into 96-well tissue culture plate.
After incubating the cells for 24 h, the cell medium was replaced with RPMI
1640 containing 1% FBS. After overnight, the cells were treated by
glutathione (GSH) (Sigma, St Louis, MO, USA) (0 to 10 mM). Cells were
treated with an appropriate concentration of LPS on days 3, 4 and 5, and then
cellular viability was determined using a cell counting kit-8 as described
above.
9. Statistics
Data were analyzed by Student’s t test or ANOVA using SPSS 18.0 (SPSS
Inc, Chicago, IL, USA). A p value less than 0.05 was considered significant.
9
III. RESULTS
1. Cellular morphologic findings by LPS treatment
At all time points there was no distinguishable morphological difference, as
determined by phase-contrast microscopy, between control cells and cells
treated with LPS at below 10 μg/ml. There was a slight morphological
difference between control cells and cells treated with 10 to 20 μg/ml of LPS
on day 2. On day 5, LPS at 10-20 μg/ml caused an increase in cell size and
caused all cells to exhibit flattened and atrophic morphology (Fig. 1).
However, after day 5, control cells and LPS-treated cells grew and became
confluent (Fig. 1)
Day 2
0 ㎍/ml 1 ㎍/ml 10 ㎍/ml 20 ㎍/ml
Day 5
10.0 um 10.0 um 10.0 um 10.0 um
Day 7
10.0 um 10.0 um 10.0 um 10.0 um
A CB
E
D
L
F
KGI
HG
Fig. 1. Effect of LPS on A549 cellular morphology. Control cells (I) and cells
treated with LPS (G, K, L) grew and became confluent by day 7. LPS (10-20
μg/ml) inhibited cell replication and induced the formation of enlarged and
flattened cells (C, D, G, H) on days 2 and 5. Scale bar = 100 μm
10
2. Effect of LPS on cellular viability and apoptosis
After day 7 of LPS treatments, as concentration of LPS increased, cellular
viability decreased as determined by CCK-8 assay (Fig. 2). CCK-8 data
revealed that the cellular viability with LPS treatment was 2.048 ± 0.134 at 0
μg/ml, 2.086 ± 0.096 at 1 μg/ml, 1.928 ± 0.150 at 5 μg/ml, 1.792 ± 0.103 at 10
μg/ml, 1.657 ± 0.094 at 15 μg/ml, and 1.491 ± 0.161 at 20 μg/ml. Cellular
viability decreased significantly (p<0.05) at 15 μg/ml or greater concentration
of LPS.
To investigate whether the apoptotic effect of LPS treatment is determined
by a direct relation of concentration and the LPS concentration that induces
cellular apoptosis, the apoptotic effect induced by LPS was measured. The
apoptosis data revealed that caspase-3 and -7 activities were 44.11 ± 5.33 at 0
μg/ml, 43.42 ± 4.87 at 5 μg/ml, 45.72 ± 5.41 at 10 μg/ml, 52.31 ± 4.18 at 15
μg/ml, 64.34 ± 8.97 at 20 μg/ml. There was a significant (p<0.05) increase of
caspase-3 and -7 activities at 20 μg/ml of LPS, confirming activation of the
apoptotic pathway (Fig. 3). In this result, the apoptosis significantly occurred
at 20 μg/ml of LPS in A549 cells.
11
0
0.5
1
1.5
2
2.5
0 1 5 10 15 20
OD
45
0
LPS (㎍/ml)
**
Fig. 2. Effect of LPS on A549 cellular viability. Cells were plated at 2.2×104
cells/well and were incubated for overnight followed by a 24 h exposure to
LPS (0-20 μg/ml). The results represent the mean ± standard deviation
(*p<0.05; n=4).
0
10
20
30
40
50
60
70
80
0 5 10 15 20
LPS (㎍/ml)
Ca
pa
se-3
/-7
Acti
vit
y (
RL
U)
*
Fig. 3. Caspase cascade activity by LPS treatment. A540 cells were treated
with LPS (5 to 20 μg/ml) for 24 h and caspase-3/-7 activities were determined
(RLU, relative light units). The results represent the mean ± standard
deviation (*p<0.05; n=4).
12
3. Effect of LPS on cellular lysosomal content
According to the results of CCK-8 and caspase activity, the pre-apoptotic
concentration of LPS was determined as 15 μg/ml. We then investigated
whether this concentration of LPS increases the lysosomal mass using the
acridine orange staining methods. When examined by fluorescence
microscopy following staining with acridine orange, orange fluorescence was
highly expressed in cells treated with LPS (15 μg/ml) compared to control
cells, indicating lysosomal uptake of the dye (Fig. 4).
A B
Fig. 4. Senescence-associated increase in cellular lysosomal content. Acridine
orange was used to stain lysosomes in control (A) and LPS (15 μg/ml)-treated
cells (B) on day 7. Orange color is indicated uptake of the fluorochrome by
lysosomes. Each was performed in duplicates.
13
4. Expression of senescence-associated β-galactosidase
Consistent with the development of an increased lysosomal content, A549
cells exposed to LPS (15 μg/ml) resulted in the expression of SA β-gal
activity (Fig. 5). Control cells expressed low SA β-gal activity, which did not
exceed 0.77 ± 0.51% of total cell counted. On day 1, 8.67% of cells treated
with LPS (15 μg/ml) were positive for of SA β-gal activity. By days 5 and 7,
the positive expression of SA β-gal activity increased to approximately 10%
and 20%, respectively.
0
5
10
15
20
25
Day 1 Day 3 Day 7
Control LPS 15 /ml
Time (day)
SA
-β-G
al
Po
siti
ve
cell
s (%
)
Fig. 5. Senescence-associated increase in β-galatosidase activity. Control and
LPS (15 μg/ml)-treated cells were cultured for 1, 3, and 7 days and stained to
measure SA β-gal activity. This experiment was performed in duplicates, and
the number of total and SA β-gal-positive (blue staining) cells was counted.
14
5. Telomere length analysis by LPS treatment
As another senescence phenomenon, telomere length was analyzed using
Southern blots. A549 cells were treated with various concentrations of LPS,
and a slight decrease in telomere length was observed in a concentration-
dependent manner (8.4 kb at 0 μg/ml and 6.7 kb at 20 μg/ml of LPS) (Fig. 6).
Fig. 6. Shortened telomere length by LPS. Telomere restriction fragments
were shown by Southern blots. Genomic DNA was digested with Hinf 1/Rsa I
enzyme, blotted, and probed with a radiolabeled telomere-specific
oligonucleotide primer.
15
6. Concentration of hydrogen peroxide by LPS treatment
Exposure of A549 cells to LPS slightly increased the concentration of H2O2
in a dose-dependent manner (Fig. 7). At 3 hours, the concentration of H2O2 in
untreated cells was determined to be 3.36 ± 0.48 μM, and this increased in the
presence of LPS to a maximum of 6.93 ± 0.38 μM at the concentration of LPS
(15 μg/ml at 9 h). Except higher concentration of LPS (20 μg/ml at 9 h) (data
not shown), LPS treatment elevated the concentration of H2O2 in a
concentration-dependent manner.
4
4.5
5
5.5
6
6.5
7
7.5
3 6 9
0 5 10 15
Time (hr)
Hy
dro
gen
Per
oxid
e (μ
M)
Fig. 7. Hydrogen peroxide formed on A549 cells which were exposed to LPS.
A549 cells were exposed to LPS (0 to 20 μg/ml) in growth medium and the
formation of hydrogen peroxide was monitered every 3 h. The results
represent the mean of three individual experiments.
16
7. Effect of GSH on LPS-induced cellular viability
The presence of GSH (0.1 to 10 mM) showed protection in A549 cells from
cellular viability reduced by LPS. This result was observed at 1 mM of GSH
and above in a concentration-dependent manner (Fig. 8). The significant
effect of GSH was observed at 5 mM and 10 mM (*p<0.05).
Fig. 8. GSH inhibits reduction in cellular viability. Cells were plated 1.0×103
cells/well and incubated for 24 h followed by a single 24 h exposure to LPS
(15 μg/ml) with or without the antioxidant, GSH (0.1 to 10 mM). Cell number
was quantified 24 h later. The results represent the mean ± standard deviation
(*p<0.05; n=4).
17
IV. DISCUSSION
Although senescence theories are very complex, the characteristics of
senescence phenotype are relatively well known. The senescent phenotype
was characterized by an altered morphology, including an enlarged and
flattened cellular appearance, increased lysosomal content, and an inability to
respond to mitogenic stimuli4. In this work, LPS at various concentrations
increased A549 cells size and caused all cells to exhibit atrophic morphology.
In addition, A549 cells exposed to high concentration of LPS showed
decreased cellular viability and growth arrest. Since apoptosis significantly
occurred at higher concentration of LPS, the pre-apoptotic concentration was
decided at LPS 15 μg/ml. This concentration of LPS significantly reduced
cellular viability but did not cause apoptotic cellular change. So, this
concentration could be deemed as sublethal stimulation concentration.
When examined by lysosomal uptake for senescence phenotype, lysosomal
contents were highly increased in cells treated with the pre-apoptotic LPS
concentration (15 μg/ml) compared to control cells. As mentioned above, the
senescent phenotype was characterized by the presence of increased
lysosomal content determined by the lysosomtropic fluorochrome, acridine
orange. Acridine orange is a nucleic acid selective fluorescent cationic dye
useful for cell cycle determination. It enters acidic compartments such as
lysosomes and become protonated and sequestered. In low pH conditions, the
dye emits orange fluorescence. Acridine orange staining revealed an incresed
in lysosomal content with replicative senscence20
. Similarly, elevated
activities of lysosomal enzymes have been associated with in vitro
senescence21, 22
. Although lung alveolar epithelial cells presented an enlarged,
flattened appearance and increased lysosomal content when stimulated with
LPS, it was difficult to differentiate these findings between cellular
senescence and apoptosis. These phenomena could be also shown in cellular
apoptosis. However, a recent study has shown a strong correlation between
18
elevated cellular lysosomal mass, β-galactosidase protein mass, β-
galactosidase activity under various intracellular pH conditions, and SA β-
galactosidase cytochemical staining with replicative senescence23
.
The β-galatosidase activity is explained by overexpression and
accumulation of the endogenous lysosomal β-galatosidase specifically in
senescent cells21
. It is widely used as a biomarker for senescent and aging
cells, because it is easy to detect and reliable both in situ and in vitro. In this
study, exposure of A549 cells to LPS (15 μg/ml) increased the expression of
SA β-gal activity. Particularly, SA β-gal activity was decreased in a higher
concentration (20 μg/ml) LPS than in the pre-apoptotic LPS on day 7 (data
not shown).
Although the pre-apoptotic concentration of LPS produced morphological
and biochemical characteristics consistent with cellular senescence, it did not
shorten the telomere length. However, shortened telomere length is not always
necessary to indicate cellular senescence.
Three separate mechanisms promote oxidant-induced cellular senescence24
.
Persistent phosphorylation of p38MAPK
following acute exposure to H2O2 was
shown to result in a senescent phenotype25
, while a chronic exposure to
oxidants has been demonstrated to induce telomere dysfunction leading to
senescence26
. Alternatively, oxidative stress resulting in nonspecific damage
to DNA may have an impact on cell cycle progression leading to senescence27
.
Therefore, according to our telomere length analysis, phosphorylation of
p38MAPK
or damage to DNA may be associated with LPS induced cellular
senescence. Because of acute exposure to LPS, these results may be
particularly related to phosphorylation of p38MAPK
. In fact, a previous study
showed that LPS stimulates a rapid and sustained phosphorylation p38MAPK
in
rat lung pericytes28
. Therefore, further evaluation is needed to examine
whether phosphorylation p38MAPK
in lung epithelial cells are induced by
sublethal exposure to LPS.
19
Oxidants are generated as a result of normal intracellular metabolism in
mitochondria and peroxisome, as well as in a variety of cytosolic enzyme
systems1, 29, 30
. In addition, a number of external agents can trigger production
of reactive oxygen species. A sophisticated enzymatic and non-enzymatic
antioxidant defense systems including catalase, superoxide dismutase and
glutathione peroxidase counteract and regulate overall reactive oxygen species
levels to maintain physiological homeostasis31
. Lowering the levels of
reactive oxygen species below the homeostatic set point may interrupt the
physiological role of oxidants in cellular proliferation and host defense.
However, reactive oxygen species may cause extensive damage to proteins
membranes and DNA. Increased reactive oxygen species may also be
detrimental and lead to cellular senescence and eventually may trigger
apoptosis, a form of programmed cell death1, 29, 30
. In addition, oxydative
damage accumulates in cells and tissues, triggering many of the bodily
changes that occur as aging progresses. Reactive oxygen species have been
implicated not only in senescence but also in degenerative disorders,
including cancer, atherosclerosis, cataracts, and neurodegeneration32
.
In these results, exposure of A549 cells to LPS resulted in the formation of
H2O2 in a concentration-dependent manner. The presence of GSH provided
concentration-dependent protection for A549 cells from senescence-induced
concentration of LPS. GSH is the most physiologically relevant antioxidant in
the epithelial lining fluid of the lung and thus, this antioxidant was examined
for its ability to inhibit LPS-induced cellular viability reduction24, 33
. So, this
antioxidant was examined for its ability to inhibit LPS-induced viability
reduction. In vivo, the average concentration of GSH in the epithelial lining
fluid of the lungs is lower in cystic fibrosis patients than in normal healthy
subjects33
. Recent clinical trials have shown that GSH levels in epithelial
lining fluid of patients can be markedly increased by the use of aerosolized
GSH with consequent improvement in lung function34, 35
. Therefore, the
20
concentration dependent production of hydrogen peroxide by LPS and GSH
inhibition of reduced cellular viability by LPS could be explained as that the
pathogenesis of cellular senescence is associated with oxidant stress effect of
LPS.
These results showed that cellular senescence occurred at the pre-apoptotic
concentration of LPS in lung alveolar epithelial cells. There have been few
studies on the relationship between cellular senescence-mediated cell
replication and bacterial toxin. However, a recent study revealed pyocyanin-
induced premature cellular senescence in lung epithelial cells24
. Pseudomonas
aeruginosa is an important cause of acute nosocomial infection and chronic
infection for patients with cystic fibrosis. Pyocyanin is a major Pseudomonas
aeruginosa toxin that induces oxidative stress in endothelial and epithelial
cells36, 37
. This premature cellular senescence induced by pyocyanin could
play an important role in limiting the lung repair response from chronic
infection.
Because sepsis is a very important example of acute infection, it could be
very difficult to apply LPS-induced cellular senescence in clinical setting.
However, although sepsis is a deadly, acute disease, survivors also suffer
long-term consequences38
. Clinical data underscore subsequent high mortality
rates associated with patients who are long-term survivors of the acute septic
episode38
. For patients who survived severe sepsis for 1 year, the mortality
rate is predicted to be 26%, and many patients succumb to lung complication38,
39. This long term consequences may be caused by immune dysregulation or
immunosuppresion following sepsis. It may be suggested that these results
could explain another pathogenesis of chronic consequences of sepsis.
However, there must be more laboratory and clinical evidences to clarify the
pathogenesis of chronic consequences of sepsis.
There were a few limitations in this research. First, in this study, apoptosis
was significantly activated in LPS at concentration higher than 15 μg/ml.
21
Therefore, we considered 15 μg/ml of LPS as the pre-apoptotic concentration.
However, this concentration level was relatively higher than that of LPS used
in other apoptosis in vitro study40
. Although morphological and biochemical
changes which are characteristics of senescence were shown at the pre-
apoptotic concentration level in multiple trials, it must be produced in other
study settings to examine whether senescence changes are induced by the
similar concentration of LPS. Second, although reduced cellular viability by
LPS on A549 cells were inhibited by the addition of GSH, it was not
absolutely shown whether the presence of GSH protected the LPS-induced
cellular senescence. Accordingly, further studies are needed to elucidate
whether GSH does not increase SA β-gal activity and lysosomal content.
Third, because A549 cell line was used in this study, further study may need
to be conducted with other cell lines and other study models.
Despite a few limitations, this study revealed that LPS might induce
cellular senescence in lung alveolar epithelial cells and that these phenomena
were assoicated with hydrogen peroxide production induced by LPS. In
addition, GSH might inhibit a reduction in cellular viability. However, further
study trials may need to be conducted in sepsis animal model or in vivo study
like sepsis survival patients.
22
V. CONCLUSION
There have been few studies on the relationship between senescence and
acute infection. In this work, it was examined the possibility of
lipopolysaccharide (LPS) causing cellular senescence in lung alveolar
epithelial cells.
1. Senescent morphologic phenomena were induced by LPS on A549 cells.
And cellular lysosomal content in A549 cells and senescence associated
β-galatosidase activity was increased at the pre-apoptotic LPS
concentration.
2. Exposure of A549 cells to LPS resulted in the formation of hydrogen
peroxide in a concentration-dependent manner. In addition, the presence
of GSH inhibited a reduction by LPS in cellular viability.
3. It is suggested that lung alveolar cellular senescence induced by LPS
could explain the cause of subsequent high mortality in long-term
survivors of sepsis. However, more laboratory and clinical evidences
are needed to clarify the pathogenesis of chronic consequences of sepsis.
4. These results may hold some clues to the relationship between
senescence and infectious diseases.
In conclusion, lung alveolar epithelial cells exposed to sublethal
concentration of LPS showed cellular senescence by reactive oxygen species
effect.
23
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27
ABSTRACT (IN KOREAN)
폐포상피세포에서 내독소에 의해 유발된 세포노화현상 분석
<지도교수 김 준 명 >
연세대학교 대학원 의학과
김 창 오
면역노화라는 개념이 최근에 알려지고 있지만, 아직까지
노화현상과 급성 감염질환과의 연관성에 대하여 알려진 바가 많지
않다. 이에 저자는 패혈증에서 분비되는 내독소를 이용하여 실제
급성 폐손상 이후 회복기전에서 중요한 역할을 하는
폐포상피세포에서 노화현상이 일어나는 지에 대하여 연구하였다.
그리고 내독소에 의한 세포노화현상의 기전에 반응성산화물질이
작용하는 지에 대하여 규명하고자 하였으며, 또한 폐포상피세포의
변화에 대하여 항산화제가 미치는 영향을 분석하였다.
폐포상피세포 (A549 세포)를 배양하고 적절한 농도의 내독소를
주입 후 1, 3, 7일에 세포의 형태학적 변화를 관찰한 결과,
내독소의 농도에 따른 세포증식의 저하를 확인하였다. Caspase-Glo
3/7 실험을 이용한 세포자멸사 분석에서 세포자멸사가 일어나기 전
단계의 내독소 농도에서 형태학적으로 세포노화현상을 관찰할 수
있었다. 노화현상을 관찰하기 위하여 아크리딘오렌지 염색법을
이용하여 용해소체 용량의 정도를 알아보고, 노화와 관련된 베타
갈락토시다아제 활성도 검사를 한 결과, 용해소체 용량 및
베타갈락토시다아제 활성도가 유의하게 증가하였다. 텔로미어길이
분석법에서는 상대적으로 유의한 결과를 볼 수 없었지만, 내독소의
28
농도에 따른 과산화수소의 생성을 확인할 수 있었으며, 항산화제인
글루타티온에 의하여 내독소에 의한 세포증식의 감소가 억제되는
것을 알 수 있었다. 본 연구의 결과에 따르면 내독소에 의하여
세포자멸사가 일어나기 전 단계에서 세포노화현상이 일어날 수
있으며, 이는 반응성산화물질의 생성에 의한 것으로 생각된다.
패혈증과 같은 급성감염질환에서 회복된 생존자의 장기예후가
불량한 것은 패혈증에 의하여 손상된 세포의 회복기능이 저하된
것에 기인할 가능성이 있다. 내독소에 의하여 유발된
세포노화현상이 이러한 손상된 세포의 회복기능의 저하에 중요한
역할을 할 수 있을 것으로 생각되며, 향후 이에 대한 추가 연구가
필요할 것이다.
--------------------------------------------------------------------------------------
핵심되는 말: 내독소, 세포노화, 감염질환, 반응성산화물질
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