hasan bayram 1,2 , bülent göğebakan 2 , Öner dikensoy 1 , erhan ekinci 1
DESCRIPTION
Effects of Diesel Exhaust Particles on Cell Viability and Release of Inflammatory Cytokines from Human Lung Epithelial Cells *. Hasan Bayram 1,2 , Bülent Göğebakan 2 , Öner Dikensoy 1 , Erhan Ekinci 1 . - PowerPoint PPT PresentationTRANSCRIPT
Effects of Diesel Exhaust Particles Effects of Diesel Exhaust Particles on Cell Viability and Release of on Cell Viability and Release of Inflammatory Cytokines from Inflammatory Cytokines from Human Lung Epithelial CellsHuman Lung Epithelial Cells**
Hasan Bayram1,2, Bülent Göğebakan2, Öner Dikensoy1, Erhan Ekinci1.
Department of Respiratory Medicine1, Respiratory Cell Culture Laboratory2, School of Medicine, University of Gaziantep, Gaziantep
*Supported by the Scientific Research Fund of Gaziantep University.
Introduction-1Introduction-1• Association between particulate matter
10µm (PM10) pollution and respiratory morbidity and cardiopulmonary mortality(McConnell R et al, 2003, Pope CA et al, 2004 )
• Airway epithelial cells may play role in PM10-induced respiratory morbidity
• Diesel exhaust particles (DEP) induce release of inflammatory mediators from airway epithelial cells(Bayram H et al, 1998)
Introduction-2Introduction-2• DEP, under serum free condition, increase
A549 cell viability by inducing cell cycle and suppressing apoptosis of these cells
• DEP exert these effects by inducing oxidative stress, JNK and NF-B pathways, while inhibiting p21CIP1/WAF1 expression(Bayram H et al, 2006)
• DEP-induced A549 cell proliferation may be associated with IL-8 and GM-CSF release from these cells
HHypothesisypothesis
ObjectivesObjectives
• To investigate effects of DEP on A549 cell viability
• To investigate effects of N-acetylcysteine, JNK inhibitor (SP600125) and ERK inhibitor (PD 98059) on this phenomenon
• To investigate whether there is an association with IL-8 and GM-CSF release
MetMethodshods• A549 cell culture• Incubation for 24, 48 and 72 hrs with DEP (0, 5, 10,
50, 100, 200, 400, 1000 and 2000g/ml)
• Incubation for 48 hrs with 50g/ml DEP in the
absence or presence of N-acetylcysteine (3.3-10mM), JNK inhibitor (SP600125, 3.3-33 M) and ERK inhibitor (PD 98059, 3.3-33M)
• MTT Staining: A549 cell viability• ELISA: IL-8 and GM-CSF analysis
ResultsResults
Effects of Effects of DDEP on EP on A549 Cell ViabilityA549 Cell Viability
24h
***p<0.0001 vs 0g/ml DEP0.0
0.5
1.0
1.5
2.0
Diesel Exhaust Particles (g/ml)(10%)
* *
*
*
Opt
ical
Den
sity
0.0
0.5
1.0
1.5
2.0
Diesel Exhaust Particles (g/ml)
*
* * **
*
(10%)
Opt
ical
Den
sity
0.0
0.5
1.0
1.5
2.0
(10%) Diesel Exhaust Particles (g/ml)
* **
Opt
ical
Den
sity
72h48h
Effect of N-Acetylcysteine (NAC) on Viability of A549 Cells Following 48 hrs’ incubation with DEP (50g/ml)
FCS 0 3.3 10 33 0 3.3 10 330.0
0.5
1.0
1.5
2.0
2.5
###
**
10%DEP(50g/ml)+NAC(mM)
SF+NAC(mM)
###
***
###p<0.0001 vs SF**p<0.001; ***p<0.0001 vs DEP
Op
tica
l De
nsi
ty
FCS 0
DMSO 3.
3 10 33 0
DMSO 3.
3 10 330.0
0.5
1.0
1.5
2.0
(10%)DEP(50g/ml)+SP600125(JNKinhM)
SF+SP600125(M) (JNK inh.)
######
#***
#p<0.05 vs SF###p<0.0001 vs SF***p<0.0001 vs DEP
Op
tical
Den
sity
Effect of JNK inh. (SP600125) on Viability of A549 Cells Following 48 hrs’ incubation with DEP (50g/ml)
Effect of ERK inh. (PD98059) on Viability of A549 Cells Following 48 hrs’ Incubation with DEP (50 g/ml)
0.0
0.5
1.0
1.5
2.0
(%10)DEP(50g/ml)+PD98059
SF+PD98059(M)
######
***
###p<0.0001 vs SF**p<0.001 vs DEP
***p<0.0001 vs DEP
**
Op
tical D
en
sity
Effects of Effects of DDEP on IL-8 EP on IL-8 Release from A549 CellsRelease from A549 Cells
*p<0.05***p<0.0001 vs 0g/ml DEP
48h 72h
24h
Serum 0 5 10 50 10
020
040
010
0020
000
2000
4000
6000
8000
Diesel Exhaust Particles (g/ml)
* *
IL-8
(p
g/m
l)
Serum 0 5 10 50 10
020
040
010
0020
000
100
200
2000400060008000
10000
Diesel Exhaust Particles (g/ml)
* * **
IL-8
(p
g/m
l)
Serum 0 5 10 50 10
020
040
010
0020
000
200
400
600
800
1000
Diesel Exhaust Particles (g/ml)
*** ***
IL-8
(p
g/m
l)
Effects of DEP on Release of GM-CSF from A549 Cells Following 72hrs’ Incubation
Serum 0 10 50 10
020
040
010
000
5
10
15
*
*
Diesel Exhaust Particles (g/ml)
GM
-CS
F (
pg
/ml)
*p<0.05 - 0g/ml DEP
Summary-1Summary-1• DEP at doses of 200-400µg/ml induced A549
cell viability following 24hrs’ incubation, whereas higher doses (1000-2000µg/ml) decreased cell viability.
• DEP induced A549 cell viability after 48hrs’ (10-400µg/ml) and 72hrs’ (50-400µg/ml) incubation
• 10-33mM NAC, 33µM JNK inhibitor and 3.3-10µM ERK inhibitor inhibited DEP-induced cell viability
Summary-2Summary-2
• Although 50-400µg/ml DEP inhibited IL-8 release following 48hrs’ incubation, 5µg/ml DEP induced release of this cytokine after 72hrs.
• 400µg/ml DEP increased release of GM-CSF following 72hrs’ incubation.
ConclusionConclusion• DEP induce airway epithelial cell viability• DEP exert this effect by inducing oxidative
stress and cell signalling pathways (JNK and ERK) known to be sensitive to the oxidative stress
• The role of DEP-induced cytokine release in cell proliferation need to be investigated further.
AcknowledgementsAcknowledgements
AcknowledgementsAcknowledgements
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