Download Apoptosis in mouse J774 macrophages-a methodological study

1
Apoptosis in mouse J774 macrophages-a
methodological study
By : Md. Hasan Imam
Main Supervisor: Olle Stendahl
Co-supervisor: Veronika Brodin Patcha
ISRN: LiU-HU/MEDBIO/SP-A-2009/23-SE
Scientific Project Presented At the University of Linköping in Partial Fulfillment of The
Requirements for the Degree of Master of Medical Bioscience
This project was performed at the division of Medical Microbiology
Department of Clinical and Experimental Medicine
Linköping University. Sweden
June, 2009
2
1. Abstract:
The approach of studying apoptosis induced by UV ray and actinomycin treatment was done to
find a suitable positive control for that apoptosis study, with murine macrophage (J774 cell line).
This scientific project was done in order to develop a method with this cell line in our laboratory
condition for further apoptosis study in the presence of M.tuberculosis bacilli. It is well known
that Mycobacterium tuberculosis primarily targets the macrophages and use them as a critical
reservoir for the infection in the lung. Macrophages show several antimicrobial activities in order
to limit the spread of intracellular infection during an attack by tuberculosis bacilli and apoptosis
is one of the innate defense mechanism.
Our findings were important because it gives the necessary information’s regarding the possible
sites where care is to be taken during working with this cell line (J774). We investigated the
effect of UV exposure and actinomycin D treatment to find the suitable positive control for the
study of apoptosis in J774 cells. Moreover, we studied effect of yeast particles on apoptosis and
the phosphorylation of p38MAPK at two different time intervals in J774 cells. In order to
quantify the percentage of apoptotic cells, we stained the cells by FITCAnnexin V, Propidium
Iodide, and TMRE and evaluated the results by flow-cytometry.
Additionally, the phosphorylation of p38MAPK was assessed by western blot. The results
indicated steady increase in apoptosis, measured by both annexin-V and TMRE 18 hours after
UV exposure. Also we investigated phosphorylated p38MAPK after 18 hours from UV exposure
(result not given) but no band was found. Actinomycin on the other hand, had no effect on the
number of apoptotic cells and further study is needed to optimize its concentration. Non
opsonised yeast did not induce any increase in annexin-V staining compared to the control, but
did surprisingly induce a decrease in the mitochondrial membrane potential after 6hours, an
effect that was diminished after 18 hours, here also we didn’t find any band of that
phosphorylated p38MAPK during immunoblotting. Similar data were found with serum
opsonised yeast particles. Maybe the beta-glucan of yeast particle was involved here by
producing reactive oxygen species via NADPH oxidase and consequent mitochondrial
membrane potential loss but we couldn’t draw any conclusion from our result about apoptosis
occurring.
3
2. Popular summary
Our focus was to establish an experimental design of studying macrophage cell death in the
presence of Tubercular bacterium, because this macrophage cell death is one of an immune
mediated defense against M. tuberculosis. Two types of control was there to obtain validity of
the experiment with this J774 murine derived macrophage cell line. The positive control was
exposed to different stress conditions such as ultraviolet radiation and actinomycin (cytotoxic
agent), which will give the idea that the system is working. This positive control will prove the
comparative amount of cell death occurred by any other means. As a negative control we
recommended only cell without any treatment which was thought to have cells with only
spontaneous cell death.
We also used yeast particle both serum opsonised and non opsonised to observe the effect of the
yeast beta-glukan on cell death. This serum opsonisation prepares the macrophage to engulf the
yeast particle through an immune pathway.
The exposure of phosphatidyl serine (PS) on the outer surface of cell membrane and the
dispersion of TMRE dye through out the cytosol during mitochondrial resting membrane
potential loss are two symptoms of a dying cell we targeted. Fluorescent tagged Annxin-V which
is very specific to that PS and the TMRE dye were used to stain the cell and were measured by
flocytometry. Also to support the cell death identification, p38MAPK protein which is known to
have role on cell death, was measured too by Western blotting (a protein identification
technique) and every measurement was done at two different time intervals (at 6th and at 18th
hour) from the stress exposure.
Steady increase in the percentage of dying cells was found after 18 hours from the UV exposure
which indicates its usability as a positive control in our future work where as it was absent in
actinomycin treatment which needs further study to increase and optimize its concentration.
Both type (serum opsonised and non opsonised) yeast particle was found to decrease the
mitochondrial membrane potential at 6th hour of study which was abolished at 18th hour. Since
there was no result found from the annexin staining and Western blot analysis that is why we
couldn’t draw any conclusion about the cell death induction by yeast particle. About the loss of
mitochondrial membrane potential we extrapolated our result with similar types of study and
assumed the beta glucan of yeast particle was involved here by producing reactive oxygen
species via reduced form of nicotinamide adenine dinucleotide phosphate.
4
3. List of Contents
1. Abstract
2. Popular summery
2. List of Content
3. Abbreviations
4. Introduction
5. Aim
6. Materials and Methods
7. Results
8. Discussion
9. Acknowledgements
10. List of References
12. Fig1
13. Fig 2
14. Fig 3
15. Fig 4
16. Fig 5
Page NO
2
3
4
4
5-6
7
7-9
9-13
14
15
15-18
8
10
11
12
13
4. Abbreviations
TMRE- Tetramethylrhodamine ethylester perchlotare
FITC – Fluorescein isothiocyanate
MAPK- Mitogen-activated protein kinase
PI – Propidium iodide
SDS PAGE- Sodium sulphate polyacrylamide gel electrophoresis
PS- Phosphatidyl serine
5
5. Introduction
5.1. Macrophage
The macrophage is a monocyte derived cell that evolves when monocytes migrate to body
tissues. It is a type of white blood cell that displays a wide array of activities such as ingesting
debris, carrying antigen and presenting it to the T-cells during infection, digesting foreign
organisms and tumor cells etc. Some macrophages ingest necrotic cells in particular areas such
as macrophages in the lung, chuffer cells in the liver, and microglia in the neural tissue (Van
Furth et al., 1972).
5.2. Apoptosis
Apoptosis is also known as programmed cell death. Apoptotic signals can be generated both
from internal and external sources such as DNA damage or defects in DNA repair, cytotoxic
drug exposure/irradiation, death signals. Apoptosis can be recognized by a set of biochemical
and physical changes in nucleus, cytoplasm and plasma membrane (Schwartzman et al. 1993).
Disruption of contact with neighbors, becoming round and shrinking are the common features of
early apoptotic cells. In addition, apoptotic cells can display various signs of apoptosis e.g.
chromatin densing to form aggregates, fragmentation by endonucleases, dilation of endoplasmic
reticulum, forming vacuoles after swelling of cisternae in the cytoplasm, and disintegration of
cell junctions in the plasma membrane leading to membrane blebbing. The nucleus becomes
convoluted, turns into encapsulated fragments, and breaks apart forming apoptotic bodies.
Phagocytes can recognize the apoptotic cells by their loss of sialic acids (Falasca et al., 1996)
and by the asymmetry of phospholipids together with translocation of phosphatidylserin to the
outer membrane (Fadokl et al., 1992).
5.3. Initiation of apoptosis (Intrinsic Pathways)
The intrinsic pathway is triggered by cytotoxic stress, which induces the translocation of
proapoptotic Bcl-2 family members, such as Bax, to the mitochondria. This leads to the release
of mitochondrial chromosome c in to the cytosol, where it promotes the oligomerisation of the
pro apoptotic factor Apaf into a complex called the apoptosome. The apoptosome recruits and
activates caspase-9 which in turn promotes the activation of caspase-3 and lead to apoptosis.
5.4. Extrinsic Pathways
The extrinsic apoptotic pathway is triggered by ligand binding to cell-surface receptors, resulting
in the recruitment of various proteins to form the death-inducing signaling complex. This
complex promotes activation of caspase-8, which in turn activates caspase-3. Caspase-3 then
induces the cellular changes that characterize apoptosis.
5.5. Reactive Oxygen Species and Mitochondrial Dysfunction
Oxidative stress is a key factor in inducing apoptosis which is a consequence of having excess
levels of free radicals in the cell. Free radicals are highly reactive molecules which are formed
during different kinds of biochemical reaction take place in the cell. Many scavengers molecules
also known as antioxidants which are found in the cell are engaged to eliminate these free
radicals. This oxidative stress can cause damaging effect to cellular components such as cell
membrane, DNA, and proteins. Several mechanisms are involved in this cell death process
6
through this oxidative stress including the release of apoptosis-inducing factors (Bredensen et al.,
1996). Formation of excess reactive oxygen species can also lead to cell damage and death
through disrupting the function of mitochondrial membrane. It is well known that the
mitochondria store calcium and regulate the calcium levels in the cell which is particularly
crucial in neurons. Chemical communication between neurons are maintained by this calcium
level. Where as excess internal level can be toxic to neurons (Choi et al., 1995). Therefore, the
ability of the mitochondria to actively sequester calcium is very important to maintain neuronal
function and survival. This regulation of calcium level by mitochondria can also be interrupted
by oxidative stress. Mitochondrial dysfunction can lead to both apoptosis and necrosis (Kroemer
et al., 1995). Mitochondrial dysfunction is a cause of becoming whole in the membrane through
which mitochondria release their contents, including calcium and a molecule called cytochrome
c. Both calcium and cytochrome c can activate caspases and can play a role in apoptosis.
5.6. MAP Kinase
Mitogen-activated protein kinase (MAPK) cascades convey many signals from the cell surface to
nucleus. Upon activation this MAP kinase becomes phosphorylated. Five different groups of
MAP kinases have been described in mammals, p38 MAP kinase being one of them. The mostly
studied vertebrate MAPKs are ERK1/2(extracellular signal-regulated kinases), JNK (Jun-Nterminal kinase), and p38 kinases. It is well known that various kinds of stimuli including stress,
cytokines, mitogens etc can activate MAPK, where as JNK and p38 kinases mainly respond to
stress stimuli (Pearson et al., 2001). Many extracellular stimuli can activate p38 kinase in
macrophages, neutrophils, and T cells (Ono K et al., 2000). The activated p38 kinase takes part
in macrophage and neutrophil functional activities like respiratory burst, chemo taxis, granular
exocytosis, adherence, apoptosis, and in T-cell differentiation (Ono K et al., 2000).
5.7. Apoptosis and p38MAPK
Activation of p38MAPK pathway by a wide range of agents, such as NGF withdrawal and Fas
ligation (Kummer et al., 1997;Juo P et al., 1997) and apoptosis are often connected. The cysteine
proteases or the caspases play the central role, and are expressed as inactive zymogens (Henkart
et al., 1996; Alnemri et al., 1996). p38 MAPK activation can be blocked by caspase inhibitors
through Fas cross linking, where caspase activation has a down regulating effect (Juo P et al.,
1997). Caspase activation, on the other hand, is up regulatory by the over expression of dominant
active MKK6b(mitogen-activated protein kinase kinase) and hence the cause of cell death
(Cardone et al., 1997;Huang et al., 1997). According to this the p38 MAPK pathway can both
up and down regulate apoptosis but the mechanism is not clear. Some evidences suggests that,
p38MAPK activation induces cell type and stimulus dependent apoptosis. In Jurkat T-cells,
p38MAPK is activated during Fas ligation, however it is not required for apoptosis (Huang et al.,
1997; Juo P et al., 1997). On the other hand, in FS-4 fibroblasts the inhibitor of p38 MAPK,
SB203580 can block the apoptosis induced by sodium salicylate (Schwenger et al., 1997). In
PC12 cells, apoptosis is induced by NGF withdrawal (Kummer et al., 1997), and in bovine
pulmonary artery endothelial cell apoptosis can be induced by TLR-1(Yue TL et al., 1999).
7
6. Aim
The aim of this study was:
• To develop a method for studying apoptosis in J774 cell model. Moreover, the purpose was to
identify suitable positive controls in this cell-line.
• To see the effect of yeast particles (non opsonised and opsonised by serum components) on
apoptosis.
• To quantify apoptosis at different time intervals (after 6hours and 18 hours)
• To correlate apoptosis with phosphorylation of p38MAPK.
7. Materials and Methods:
7.1. Reagents: Hams F-10 GLUTA-maxTM-1 medium, fetal calf serum, penicillin, and
streptomycin were purchased from Life Technologies (United Kingdom), FACS FlowTM was from
BD Biosciences, TACSTM Annexin V-FITC was from R&D system (USA), HEPES,
Mercaptoethanol and Actinomycin were from Sigma, and TMRE were purchased from
Invitrogen. Sample buffer and running buffer were purchased from Bio Rad (CA). Phosphop38
MAPK antibody was obtained from Cell Signaling Technologies and p38 MAPK antibody came
from New England Biolabs Technologies. ECL system and Nitrocellulose membrane was
purchased from Amersham. Accutase was from Fisher Scientific. Triton X- 100 Sigma, Sodium
Dodecyl Sulfate (SDS) Sigma, Sodium Deoxicholate, Monobasic sodium phosphate, NaCl were
purchased from Sigma, Complete mini protease inhibitor cocktail came from Roche.
7.2. J774 Cell Culture: J774 cell are murine macrophages obtained from tumors of female
BALB/c mice. The J774 cells were cultured in F-10+GlutamaxTM-1 medium supplemented with
10% heat inactivated foetal bovine serum, penicillin(100 U/ml), and streptomycin (1000 µg/ml)
and 20mM HEPES in a humidified atmosphere of 5% CO2 at 37 o C. Cells were collected by
scraping at approximately 70% confluency and were seeded in 12 and 6 well plate at 0.5x10 6
cells / well for flowcytometry study, and 2x106 cells/ per well for western blotting respectively,
3days before experiments. Accutase was used to collect the cell duringflow cytometry study. The
cells used for western blotting experiments were collected by scrapping
7.3. Yeast Particle preparation: Heat inactivated baker’s yeast was homogenized 10 times in
KRG, washed (1000 x g, 3min, at room temperature) and diluted to 1x108/ml in KRG. The yeast
were opsonised in 25% human serum, for 30min at 37o C, washed and resuspended to 3x106 /ml.
Non opsonised yeast were treated in the same manner, with the exception in that no human
serum was added.
8
7.4. Staining and Analysis of Apoptosis by Flowcytometry.
A
B
Figure : 1. A. In side the circle the cell population is representing the healthy J774 cell
(excluding the debris) which was used to study the apoptosis. We know from the basic of flowcytometry that forward scatter reflect the size and shape whereas the reverse scatterreflects the
internal complexity of the cell. In case of similar types of cell we will get the cell population in
an specific region.
And Figure B was for healthy control in our experiment. Among the four different quadrants in
means
ans healthy live cell, where
Fig1.B, below left quadrant is negative for both FL1 and FL2 that me
apoptotic
optotic cell number.
as below right quadrant is positive for FL1 which representing the early ap
Quadrant of up right corner is positive for both FL1 and FL2 which representing the late
apoptotic or necrotic cell.
7.4.1. Annexin-V staining: After collection by pippeting, the cells were washed once in binding
buffer (1X).The samples were incubated with FITC-conjugated AnnexinV (15.5µl/ml in PBS)
for 16min on ice. The reaction was stopped by addition of cold binding buffer and the results
were evaluated by flow cytometry. In healthy cell, without the translocation of phosphatidyl
serine, it will not be annexin V positive and corresponding pic will be at the lower field in
histogram. In order to distinguish between apoptosis and nec
necrosis
rosis the samples were
counterstained with Propidium Iodide (PI) before evaluation.
7.4.2. TMRE staining: Collected cells were washed and incubated with TMRE (2µM) at 37oC
for 15 min and the results were evaluated by flow cytometry. In healthy cells with stable
membrane potential, this TMRE dye will enter into the mitochondria and the corresponding pic
9
will appear in higher field in the histogram, but in apoptotic cells with disrupted mitochondrial
membrane potential (∆ψm), this TMRE dye will disperse throughout the cytosol and
corresponding pic will shift left in the lower field in histogram, i.e. showing a decrease in the
mitochondrial membrane potential. The fluorescence of individual cells stained by FITCconjugated AnnexinV and TMRE was measured by flow cytometry, using a Becton Dickson
FACSCalibur (Becton Dickinson, San Jose, CA/USA) Mean fluorescence values were
determined, by counting 10,000 cells/sample. Gating was performed by using forward scatter
versus side scatter (excluding cell debris). Auto fluorescence of unstained cells was routinely
analyzed and subtracted from the fluorescence values of the stained cells.
7.5. Cell lyses and analysis of p38MAPK by western blotting: The J774 cells (2x106/well )
were treated with actinomycin D (0.025mg/ml) for 18 hours, UV radiation (10 min and 15 min),
or with serum opsonised and non opsonised yeast particles, (1:5) for 30 min. Cells were
resuspended in ice cold RIPA buffer (1% Triton X-100, 0.1% Sodium Dodecyl Sulphate(SDS),
0.5% Sodium Deoxycholate, 9.1mM monobasic sodium phosphate, 150mM NaCl, supplemented
with Complete mini protease inhibitor cocktail(Roche), 1 mM Naortovanadate, and 5 µM
dithiothretol ( Welin et al., 2008)) for 15 minutes. The cell were collected by scraping and
centrifugation (10000 x g,10min,at 4oC), where after the lysine was diluted with sample
buffer(3:1).The samples were subjected to electrophoresis and SDS-PAGE, and transferred to
nitrocellulose membrane(100 V,1Hour).The membrane was blocked in TBS-5%milk for 1hour at
room temperature. The membranes were incubated with a phospho-p38MAPK antibody (1:2000)
for one and half hour at room temperature, followed by a secondary HRP conjugated anti rabbit
antibody (1:2000). To visualize total amounts of p38MAPK proteins, the membranes were
stripped and reblotted with a rabbit polyclonal anti-p38MAPK antibody (1:2000).
7.6. Statistical Analysis: All experiments were repeated at least four times and each separate
experiment was run in duplicates or triplicates. Data are presented as Mean ± SD. Data from
each experiment were analyzed by Student’s t test and p< 0.05 was considered statistically
significant.
8. Results:
To induce apoptosis the J774 cells were exposed to UV for two different time points (10 min and
15 min) keeping fixed distance (10cm) from the UV source. In order to find a proper positive
control actinomycin D were used (0.025mg/ml) (data not shown). Two different approaches to
collect the cells were used, namely Accutase (300µl/well) followed by pipeting and cell
scrapping but finally we choose accutase using since this process protect the cell from
mechanical disruption. Apoptosis was evaluated by flow cytometry after 6 and 18 hours.
10
Fig.2. Effect of actinomycin and UV radiation on apoptosis after 6 hours. J774 cells (0.5x106
/well) were grown to 70% confluency. Apoptosis was induced by UV for 10 and 15 minutes at
the distance of 10 cm, or with actinomycin D (0.025mg/ml). 6 hours after the induction of
apoptosis, the cells were incubated with accutase (300µl, for 30 minutes), prior to collection by
vigorous pipetting. The cells were stained either by FITC conjugated annexin V(15.5µl/ml 16
minute on ice), or TMRE (2µM, 30 min at 37 o C ) where after apoptosis and mitochondrial
membrane potential was evaluated by flow cytometry. Gating was performed using forward
versus side scatter, counting 10000 cells per sample. None of the data was statistically
significant. Data are presented as the mean ± SD of four separate experiments. UV treated cells
were stained a very small percentage with TMRE after 6 hours study but not any one was
statistically significant. Actinomycin also induced the mitochondrial membrane potential loss to
a few percentages of macrophage but still it is statistically insignificant. No change was there
with annaxin V staining after 6 hour study. On the other hand we find an increased numbers of
cell stained by both annexin V and TMRE after 18 hour study. UV treated cells were found more
apoptotic and it was statistically significant for both annexin V staining and TMRE staining.
11
Fig.3. Effect of actinomycin and UV radiation on apoptosis after 18 hours. J774 cells (0.5x106
cells/well) were grown to 70% confluency. Apoptosis was induced by UV for 10 and 15 minutes
at the distance of 10 cm, or with actinomycin (0.025mg/ml). 18 hours after induction of
apoptosis, the cells were incubated with accutase (300 µl, for 30 minutes), prior to collection by
vigorous pipetting. The cells were stained either by FITC conjugated annexin V (15.5µl/ml 16
minute on ice), or TMRE (2µM, 30 minute, at 37oC) where after the apoptosis and mitochondrial
membrane potential was evaluated by flow cytometry. Gating was performed using forward
versus side scatter, counting 10000 cells per sample. Apoptotic cell percentage both for 10 and
15 UV exposure were statistically significant compared to control ( p=0.036 and 0.027 for
TMRE staining and p=0.03 and 0.007 respectively for annexin staining). Data represent the mean
± SD of four different experiments.
12
Fig.4. The effect of yeast on the induction of apoptosis. To study the effect of yeast particles
on apoptosis macrophages were incubated with yeast (1:5 or 1:10, NOY-non opnised yeast;
SOY- serum opsonised yeast) for 1 hour. Non-bound particles were washed away, and the cells
were incubated for additional five hours, prior to collection by the combination of accutase, and
pipeting. As a control, cells were incubated with actinomycin (0.025mg/ml). The differences in
apoptosis were statistically significant for TMRE for both serum opsonised (p=0.031), and non
opsonised yeast (p= 0.05), compared to control. Data represent the mean ± SD of four separate
experiments.
13
Fig. 5. Effect of yeast (both non opsonised and serum opsonised) on apoptosis after 18 hours
study. Macrophages were incubated with yeast both serum opsonised and non opsonised for one
hour. Non bound particles were washed away and incubated 17 more hours. After that it was
collected by accutase and pipeting. Actinomycin treated macrophages underwent apoptosis
significantly (p=0.047) for annexin compared to control and others are statistically insignificant.
Data represent the mean of four separate study ± SD.
Macrophages treated with serum opsonised yeasts, stained with TMRE showed more apoptosis
(18% and 30% for 1:5 and 1:10 respectively) after 6 hours (Fig 4) but the apoptosis was reduced
to 8% and 12% after 18 hours(Fig %). Non opsonised yeast induced a decrease in the
mitochondrial membrane potential after 6 hours (Fig 4), but only the higher MOI displayed a
mitochondrial damage 18 hours after induction (Fig 5).Similar results were obtained for nonopsonised yeasts (Fig 4 and 5).The yeast particles had no effect on the increase in annexin-V
staining, independently of serum opsonisation at any time point (Fig 4 and Fig 5).
Phosphorylation of p38 MAPK in J774 macrophages. Detection of phosphorylated
p38MAPK protein after 30 min, induced by yeast particle and detected using western-blot
analysis. Cells were incubated with yeast for 30 minutes, and lysed in RIPA buffer prior to
collection by a cell scraper and vigorous pipetting. The supernatant was kept in -70o C. Induction
of apoptosis by UV was done 10 and 15 min and incubated for 18hours. Also cells were treated
with actinomycin (0.025mg/ml) for 18 hours. After that both UV and actinomycin treated cells
were collected followed by lyses by RIPA buffer and vigorous pipeting. After that diluted
supernatant was separated by electrophoresis and transferred to nitrocellulose membrane. The
membrane was blocked with milk followed by incubation with antibodies. From left, first lane
was for ladder, followed by second, third, fourth, fifth, and sixth lane was for control,
actinomycin, non opsonised yeast, serum opsonised yeast, and UV respectively.
14
9. Discussion:
Actinomycin D- treated macrophages stained by TMRE, showed only a small increase after 6
hours (15%) and after 18 hours(16%) (Fig 2 and 3, black bars). Therefore, this concentration of
Actinomycin D (0.025 mg/ml) was not suitable one as a positive control. Since it showed
statistically significant increase at Fig 5 (p=0.047) but it was disappeared in other experiments.
More experiments investigating the effect of other concentrations should be executed.
Exposure of UV had no effect on neither mitochondrial damage, or on the number of apoptotic
cells after 6 hours (Fig 2). After 18 hours on the other hand UV induced a 15-20% increase in
TMRE staining, and 45-55% increase in the number of apoptotic cells (Fig 3). Results indicating
that it takes time to initiate apoptosis in J774 cell. According to the lack of induction apoptosis
by yeast particles in J774 cells (fig 4), we can conclude that the yeast particles can disrupt the
mitochondrial membrane potential (∆ψm).
The possible explanation of above nature is either that all apoptotic symptoms doesn’t go
together or that this mitochondrial membrane potential disruption by yeast particle is not an
apoptotic symptom at all, since it abolished with time. Mitochondria has control over apoptosis
by generation of ATP (Leist M et al., 1997) and maintaining membrane potential and also by
allowing some apoptogenic protein permeability to cytosol (Zoratti M et al., 1995; Kroemer G et
al., 2000). It may raise the possibility that membrane potential loss is a cause of apoptosis
induction perhaps the signal amplifier to convey further apoptotic process (Ly JD et al. 2003). In
order to become confirm about this apoptosis of mouse macrophage by yeast particle, we need to
do another kind of staining, such as TUNEL or we can study the morphology under microscope.
After 18 hours of study we didn’t find any phosphorylated p38MAPK but after 30 min of study
we found some weak bands (data not shown) which we unfortunately were not able to identify
due to handling mistakes. Upon re-blotting the membrane with p38MAPK antibody the blot
came out empty. This could be due to low protein content in the sample or that proteins were
stripped away during re-blotting. The time-kinetics for the intracellular signal generated by yeast
also need to be investigated in more detail. One possibility is that the phosphorylation of p38
MAPK has already taken place and that 18 hours on incubation no phosphorylaton of p38 can be
detected. Thus no conclusion can be drawn on the activation of p38MAPK, and further
experiments need to be done in future. Another possible explanation behind that empty
membrane could be that the incubation with the primary antibody was too short, antibody
concentration was too low, or that the antibody was too old.
Also from Fig-2 we find some loss (∆ψm) though it was not statistically significant and at 18
hour study (Fig 3) we find significant loss of mitochondrial membrane potential(∆ψm) along
with other apoptosis symptom (i.e. PS exposure). Again we found that, this yeast particle
induced the loss of mitochondrial potential (Fig 4) but that was abolished after 18 hour. The
possible reason behind the membrane potential loss may be the production of reactive oxygen
species via NADPH oxidase by yeast glucan. Study shows that yeast glucan increases the
production of hydrogen-peroxide as well as phagocytosis of macrophage ( S. Brattgjerd et al.,
1994). More investigation is needed to elucidate it. Also we found that, UV is good candidate to
become a positive control and for actinomycin we need to optimise the concentration for J774
macrophage.
15
10. Acknowledgements:
I would like to express my gratitude to those that have helped in completion of this project. First
I would like to thank Prof. Olle Stendahl for giving me the opportunity to work, also for his vast
experiences backed encouragement. Special thanks to Veronika for her unwavering support,
Anita for her teaching and love. I would also like to thank Eyler, Alex, Amanda, Daniel, Tommy,
Tony, Fredric, Marie, Atem, and many of my friends for making their resources and knowledge
available to me.
11. List of References:
Acehan D, Jiang X, Morgan DG, Heuser JE, Wang X, Akey CW. 2002. Threedimentional structur of the
apoptosome: implications for assembly, procaspase-9 binding, and activation. Mol Cell. 9(2):423-432.
Agace WW, Hedgas SR, Ceska M, Svanborg C. 1993.Interleukin-8 and the neutrophil response to mucosal gramnegative infection. J clin Invest. 92: 780-785.
Allen LA, Aderem A.1995. Protein kinase C regulates MARCKS cycling between the plasma membrane and
lysosomesin fibroblasts. EMBO J. 14:1109–20
Amanda Welin, Martin E Winberg, Hana Abdalla, Eva Särndahl, Birgitta Rasmusson, Olle Stendahl, Maria Lerm.
2008. Incorporation of Mycobacterium tuberculosis lipoarabinomannan in to macrophage membrane rafts is a
prerequisite for the phagosomal maturation block. Infect Immun.76(7), 2882-2887.
Ashkenazi A. 2002.Targetting death and decoy receptors of the tumour necrosis factor superfamily. Nat Rev Cancer.
2(6): 420-430.
Barnes PF et al.1993. Patterns of cytokine production by mycobacteriumreactive human T-cell clones.Infect
Immune. 61: 193-203.
Cardone MH, Salvesen GS, Widmann C, Johnson G, Frisch SM.1997.Cell. 90: 315-323.
Falasca L, Bergamini A, Serafino A, Balabaud C, Dini L. 1996. Human cupffer cell recognition and phagocytosis of
apoptotic peripheral blood lymphocytes. Exp Cell Res. 224: 152-162.
Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM.1992. Exposure of phosphatidylserine on
the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol.148:
2207-2216.
Fernandes-Alnemri T, Armstrong RC, Krebs J, Srinivasula SM, Wang L, Bullrich F, Fritz LC, Trapani JA,
Tomaselli KJ, Lit wack G, Alnemri ES.1996. Proc Natl Acad Sci USA. 93: 7464-7469.
Flynn JL.et al.1995. Tumor necrosis factor-α is required in the protective immune response against Mycobacterium
tuberculosis in mice. Immunity. 2: 561-572.
Flynn JL, Chan J. 2001. Immunology of tuberculosis. Annu Rev Immunol.19: 93-129.
Henkart PA.1996. Immunity. 4: 195-201.
16
Hirani N, Antonicelli F, Strieter RM.2001.The regulation of interleukin-8 by hypoxia in human macrophages—a
potential role in the pathogenesis of the acute respiratory distress syndrome (ARDS). Mol Med. 7: 685–697.
Huang S, Jiang Y, Li Z, Nishida E, Mathias P, Lin S, Ulevitch RJ, Nemerow GR, Han J.1997. Immunity. 6: 739-749.
Johanson LV, Walsh, Bockus BJ, Chen IB.1981. Monitoring of relative mitochondrial membrane potential in living
cells by fluorescence microscopy. J Cell Biol. 88: 526-535.
Juo P, Kuo CJ, Reinolds SE, Konz RF, Raingeaud J, Davis RJ, Biemann H-P, Blenis J.1997. J Mol Cell Biol. 17:
24-35.
Kaufmann SH. 2001. How can immunology contribute to the control of tuberculosis? Nature Rev Immunol. 1: 2030.
Kindler V, Sappino AP, Grau GE, Piguet PF, Vassalli P.1989. The inducing role of tumor necrosis factor in the
development of bactericial granulomas during BCG infection. Cell. 56: 731-740.
Kroemer G, Reed JC. 2000. Mitochondrial control of cell death. Nat Med. 6:513-519.
Kummer JL, Rao PK, Heidenreich KA.1997. J Biol Chem. 272: 20490-20494.
Ly LD, Grubb DR, Lawen A. 2003.The mitochondrial membrane potential(_ψm) in apoptosis; an update. Kluwer
Academic Publishers .8:115-128.
Liest M, Nicotera P.1997. The shape of cell death. Biochem Biophys Res Commun. 236: 1-9.
Mohan VP et al. 2001. Effects of tumor necrosis factor-α on host immune response in chronic persistent
tuberculosis: possible role for limiting pathology. Infect Immun. 69:1847-1855.
Oddo M, Renno T, Attinger A, Bakker T, MacDonald HR, Meylan PR.1998. Fas ligand induced apoptosis of
infected human macrophage reduces the viability of intracellular Mycobacterium tuberculosis. J Immunol. 160,
5448-5454.
Ono K, Han J. 2000. The p38 signal transduction pathway:activation and function. Cell Signal.12: 1-13.
Pearson G, Robinson F, Beers Gibson T, Xu BE, KArandikar M, Berman K, Cobb MH. 2001. Mitogen-activated
protein(MAP) kinase pathways:regulation and physiological functions. Endocrinol. Rev. 22: 153-183.
Reidl SJ, Shi Y. 2004. Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol. 5: 897907.
S Brattjerd, O Evensen, A Lauve. 1994. Effect of injected yeast glucan on the activity of macrophages in Atlantic
salmon. Immunology. 83: 288-294.
Scott A, Khan KM, Roberts CR, Cook JL, Duronio V. 2004. What do we mean by the term “inflammation”?A
contemporary basic science update for sports medicine. Br J Sports Med. 38:372–380.
Scanga CA et al. 2000. Deplation of CD4+T cells causes reactivation of murine persistent tuberculosis despite
continued expression of interferon-µ and nitric oxide synthase. J Exp Med. 192: 347-358.
Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME.1998.Two
CD95(APO-1/Fas) signalling pathways. Embo J. 17(6): 1675-1687.
Schwenger P, Bellosta P ,Vietor I,Basilico C, Skolnik EY, Vil-cek J. 1997. Proc Natl Acad Sci USA. 94, 2869-2873.
Schwartzman RA, Cidlowski JA.1993.The biochemistry and molecular biology of programmed cell death. Endocr
Rev.14: 133-151.
17
Serbina NV, Liu CC, Scanga CA, Flynn JL. 2000. CD8+ CTL from lungs of Mycobacterium tuberculosis infected
mice express perforin in vivo and lyse infected macrophages. J Immunol.165: 353-363.
Silk J, Vaux DL. 1998. Cell death. Curr Biol. 8: 528-531.
Stewart GR, Robertson BD,Young BD. 2003.Tuberculosis : a problem with persistence. Nature Rev Microbiol.1:
97-105.
Springer TA.1994.Traffic signals for lymphocyte recirculation and leukocyte emigration : The multistep
paradigm.Cell. 76: 301-314.
St Pierre BA, Tidball JG. 1994. Differential response of macrophage subpopulations to soleus muscle reloading after
rat hindlimb suspension. J Appl Physiol. 77: 290–297.
Turner J et al.CD8 and CD95/95L dependent mechanisms of resistance in mice with coronic pulmonary
tuberculosis. Respir Cell Mol Biol. 24: 203-209.
Turner J et al. 2002. In VIVO IL-10 production reactivates chronic pulmonary tuberculosis in C57BL/6 mice. J
Immunol.169: 6343-6351.
Turner J, Frank AA, Brooks JV, Marietta PM, Orme IM. 2001. Pentoxifylline treatment of mice with chronic
pulmonary tuberculosis accelerates the development of destructive pathology. Immunology.102: 248-253.
Van Fourth R, Cohn ZA, Hirch JG, Humphrey JH, Spector WG, Langevoort HL.1972.The mononeuclear phagocyte
system:a new classification of macrophages, and their precursor cells. Bull WHO. 46: 845-852.
Van Pinxteren LA, Cassidy JP, Smedegaard BH, Agger EM, Andersen P. 2000. Control of latent Mycobacterium
tuberculosis infection is dependent on CD8 T cells. Eur J Immunol. 30: 3689-3698.
Verbon A et al.1999. Cerum concentrations of cytokines in patients with active tuberculosis(TB) and after treatment.
Clin Exp Immunol.115: 110-113.
Yin XM. 2000. Signal transduction mediated by Bid,a pro death Bcl-2 family proteins, connects the death receptor
and mitochondria apoptosis pathways. Cell Res. 10: 161- 167.
Yue TL,Ni J, Romanic AM, Gu JL, Keller P, Wang C, Kumar S, Yu GL, Hart TK, Wang X, Xia Z, De Wolf WE,
Feuerstein GZ.1999. J Biol Chem. 274,1479-1486.
Zoratti M, Szabo I. 1995. The mitochondrial permeability transition. Biochem Biophys Acta. 1241: 139-176.