Pulmonary Health Effects I:
Inside the lungs
University of California Los Angeles
University of Southern California
Michigan State University
University of California Irvine
University of Wisconsin-Madison
Photochemical smog Los Angeles-type
Prevalence of Allergic Disease
Since the Industrial Revolution
Saxon et al
The Impact of Air Pollution on Asthma in Children
Source: Southern California Environmental Health Sciences Center, 2007
1. Why are pollutant particles dangerous?
2. Is it the particles themselves or their
chemical components that cause the
damage?
3. What are the potential mechanisms
DEP as a model particulate pollutant
Diesel Exhaust
Diesel Exhaust Particles
Carbon
core
Organic
chemical
& metal
coating
Cell death
Mouse Macrophage Studies
1. DEP toxicity is significantly reduced
after the removal of organic chemicals
2. Antioxidant, NAC, could effectively
inhibit DEP-induced oxidative stress
as determined by the level of
intracellular reactive oxygen radicals
Oxygen radical production
3. NAC could effectively inhibit DEP
toxicity
The effect of antioxidant on DEP toxicity
Dose-dependent DEP toxicity
Human Bronchial Epithelial Studies
•Organic extract of DEP induced
dose- and time-dependent toxicity
in human bronchial epithelial cells
Time-dependent DEP toxicity
•Polycyclic aromatic hydrocarbonenriched aromatic fraction and quinoneenriched polar fraction are more potent
in inducing oxidative stress
Induction of oxidative stress by
organic DEP chemicals
What is oxidative stress?
Normal
Redox
equilibrium
Oxygen radical
Production
Oxygen radical
Inactivation
Oxygen radical
Inactivation
Oxidative
Stress
 GSH
 GSSG
Oxygen radical
Production
Oxidative Stress Induces a Stratified Cellular Response
Level of
oxidative stress
High
GSH/GSSG
Ratio
Cell response :
Low
GSH/GSSG
Ratio
Tier 3
Tier 1
Normal
Tier 2
AntiInflammation
oxidant
Defense
Nel, Loo, et al. J. Biol. Chem., 278:50781-90, 2003
Toxicity
(Apoptosis)
DEP Study Summary
Organic DEP chemicals induces adverse
biological effects through the generation
of stratified oxidative stress response
Which particle type in the ambient air
is most dangerous and why?
….. PM10 ?
…... PM2.5 ?
……Ultrafine particles
Transmission Electronmicrograph of a PM2.5 Particle
PM2.5 = aerodynamic
diameter < 2.5 micron
Main sources are
soil, crustal elements,
sea salt etc.
May contain some
emission particles
EPA regulated
(mass standard)
(Courtesy Dr Sheldon Friedlander, UCLA)
Nel. Science, 308: 804-06, 2005
Transmission Electronmicrograph of Ultrafine Particle
Main sources are
emission particles or
condensation of
vapors
Diesel exhaust particles
Not EPA regulated
(will require a numbers
standard)
(Courtesy Dr Sheldon Friedlander, UCLA)
Nel. Science, 308: 804-06, 2005
Characteristics of Harmful
Inhaled Particles
1. Small size
2. Large surface area
3. Ability to be taken up
4. Toxic chemicals
Oxidant Potential and PAH Content
of Different Size Particles
Fold increase compared to coarse
Oxidant potential
PAH content
110
Coarse
90
70
Fine/Ultrafine
Ultrafine
50
35
30
25
20
15
10
5
0
Correlation
Cl-10/11/01
Cl-11/27/01
Cl-1/15/02
Site/date
Li et al. Environmental Health Perspectives. 2003
SC-1/15/02
Control
UFP
Coarse
Control
The Cellular Effects of Different Size Particles
HO-1 luciferase reporter
transgenic mice
UFP
Fine
Filtered air PM2.5 Ultrafine
Summary of Particle Size Comparison
Ultrafine particles are more dangerous than coarse
and fine particles due to their ability to carry more
pro-oxidative organic chemicals and stronger oxidant
potential
How do ambient particles impact
allergic airway inflammation
such as asthma?
Experimental DEP Effects on Asthma
Allergen
PM
Allergen
Adjuvant
Sensitization
PM
Allergen
 Airway
inflammation
Allergic
inflammation
PM
Airway
Reactivity
Wheezing
DEP Augments Allergen-specific IgE in Humans
160
Ragweed
Ragweed + DEP
IgE (U/ml)
120
80
40
0
Day 0
Day 1
Diaz-Sanchez et al. J. Immunol., 158:2406 -2413, 1997
Day 4
Day 8
Animal Model to Study PM Effect
on Allergic Sensitization
Allergic sensitization protocol
Intranasal
PM
PM + OVA
PM + OVA
PM + OVA.
PM + OVA
1% OVA
1% OVA
Necropsy
Inhalation
Day: 1
2
4
7
9
22 23
25
Li et al, Environ Health Perspect, 2009
Ambient UFP enhanced OVA-induced allergic inflammation
30
BAL Differential Cell Count
Cell number (x104)
25
Ctrl
OVA
UF#1
UF#1/OVA
20
15
#
*
10
5
0
Total
Mono
OVA-IgG1
2500
Eosino
600
#
*
Neutro
OVA-IgE
#
*
500
2000
1500
1000
*
500
0
Lympho
OVA-IgE (U/ml)
OVA-IgG1 (pg/ml)
3000
*
400
300
200
100
Ctrl
OVA
UF#1
Li et al, Environ Health Perspect, 2009
UF#1+OVA
0
*
Ctrl
OVA
UF#1
UF#1+OVA
Ambient Fine/UF particles failed to exert an adjuvant effect
on OVA sensitization
35.0
Ctrl
OVA10
Endo+OVA
UF #1+OVA
F/UF #1+OVA
P < 0.01
30.0
25.0
P < 0.01
P < 0.05
5.0
P < 0.01
10.0
P < 0.05
15.0
P < 0.01
20.0
P < 0.01
Cell number (x10e4)
40.0
P < 0.01
45.0
0.0
Total
Mono
Eosino
Lympho
Neutro
UFP-induced eosinophil infiltration
was accompanied by
increased IL-5 in BAL
IL-5 (pg/ml)
400
P < 0.01
300
P < 0.01
200
100
VA
F/
U
F
#1
+O
#1
+O
VA
U
F
VA
10
En
do
+O
VA
O
Sa
l in
e
0
200
F
F/
U
F
U
#1
+O
#1
+O
VA
VA
0
VA
VA
F/
U
F
#1
+O
#1
+O
VA
U
F
VA
En
do
+O
O
VA
10
0
400
En
do
+O
200
P < 0.05
600
10
400
P < 0.05
800
VA
600
1000
O
800
P < 0.01
Sa
l in
e
P < 0.05
OVA-IgE (pg/ml)
1000
Sa
l in
e
OVA-IgG1 (pg/ml)
Ambient UFP increased IgG1 and IgE productionIn OVA-sensitized mice
UFP-induced Allergic Inflammation in Nasal Maxilloburbinates
Nasal morphometry
0.6
Mucus
#
*
Saline
OVA/UF#1
0.5
0.4
0.3
0.2
0.1
0.0
Eosinophils/mm basal lamina
Major basic Protein
AB/PAS
(Mucosubstances)
(Eosinophils)
Li et al, Environ Health Perspect, 2009
OVA/UF
Volume Density
(nl/mm2 basal lamina)
OVA
H&E
(Morphology)
Saline
50
OVA
Eosinophils
*
40
30
20
10
0
Saline
OVA/UF#1
OVA
UFP-induced allergic inflammation in conducting airways
Major basic Protein
AB/PAS
(Eosinophils) (Mucosubstances)
H&E
(Morphology)
Saline
Li et al, Environ Health Perspect, 2009
OVA
OVA/UF
Schematic drawing of the murine respiratory tract highlighting the intrapulmonary sites
where pro-oxidative UFP exert its adjuvant effect on OVA sensitization
Li et al, Environ Health Perspect, 2009
Morphometric Analysis of Mucosubstances and Eosinophils
in Proximal and Distal Axial Airway
Proximal Axial Airway
Distal Axial Airway
Generation 5
Generation 11
#
*
8
4
2
Saline
OVA/UF
4
3
2
40
*
30
20
10
ND
Saline
OVA/UF
Li et al, Environ Health Perspect, 2009
0
OVA
50
0
5
*
1
Eosinophils/mm basal lamina
0
Eosinophils/mm basal lamina
*
6
#
*
6
Volume Density
(nl/mm2 basal
lamina)
Volume Density
(nl/mm2 basal
lamina)
10
OVA
Saline
30
OVA/UF
OVA
*
25
20
15
2
0
ND
Saline
OVA/UF
OVA
OC
Analyses of PM chemical
composition and redox activity
47.3
UF#1
EC
Chloride
240
Phosphate
200
Sulfate
Total PAH content
(pg/g PM)
Nitrate
18.9
Sodium
F/UF#1
Ammonium
Potassium
*
*
160
120
80
40
Unknown
0
64.6
0.06
DTT consumption
(nmol/g/min)
UF#2
UF#1

1
5 8.3 1
F/UF#1
UF#2
5
5
8.3 10 1
UF #1
*
UF #2
*
0.05
0.04
0.03
0.02
0.01
0.00
8.3 10 g/ml
F/UF #1
UF#1
F/UF#1
UF#2
HO-1
Li et al, Environ Health Perspect, 2009
Single intranasal administration of ng quantities of UFP is
sufficient to enhance allergic sensitization to co-administered OVA
p < 0.009
13
OVA-IgG1 (pg/ml)
1
Necropsy
UFP/OVA
Day: 0
OVA
challenge
UFP
4000
14
15
16
p < 0.009
3000
2000
1000
0
Saline
*
12
10
500 ng of UFP
Saline
OVA
OVA+UFP
*
8
6
*
4
2
0
*
Mono
Eosino
Lympho
*p < 0.05 compared to Saline and OVA
Neutro
OVA+UFP
p < 0.02
400
300
200
100
0
Total
OVA
p < 0.02
500
OVA-IgE (U/ml)
Cell number (x104)
14
Saline
OVA
OVA+UFP
Animal Model to Study PM Adjuvant Effects
through Real-time Inhalation of Ambient UFP
i. Primary immune response
ii. Secondary immune response
Relevance of the Secondary IR:
Asthma flares in atopics after a sudden surge in ambient PM levels
PM Exposure
in Mobile Laboratory
near 110 Freeway
UFP
4
18 19
22
Day 1
2
18 19
22
UFP
4
18 19
22
Day 1
2
18 19
22
UFP
4
18 19
23
24
23 24
26
23
24
26
OVA+FA
23 24
26
OVA+UFP
22
26
OVA+FA
OVA+UFP
FA
OVA+UFP
26
OVA+UFP
FA
OVA
23 24
Nec
2
22
Nec
Day 1
18 19
Nec
Saline
OVA+FA
Nec
FA
Nec
Inhalation
23
24
Nec
Sensitization (i.n.)
26
Ambient UFP enhance secondary allergic response
in already-sensitized mice
1400
1000
800
OVA-IgE
2o
Eosinophils
14000
Challenge:
OVA/FA
OVA/UFP
Eosinophil No/ml
OVA-IgE (u/ml)
1200
#
*
*
600
400
200
0
OVA
OVA/UFP
1000
OVA-IgG1 (pg/ml)
12000
10000
8000
6000
*
4000
2000
Saline
*
OVA-IgG1
800
600
*
400
200
0
#
*
Saline
OVA
OVA/UFP
0
Saline
OVA
OVA/UFP
UFP inhalation during the secondary immune response
further increased cytokine gene expression in the lung
8
Fold Change Relative to Saline/OVA/FA
a
b
c
Eotaxin
7
2o Challenge:
6
OVA/FA
5
OVA/UFP
a
b
4
50
45
40
35
30
25
20
15
10
5
0
3
2
1
0
Muc5ac
Saline
OVA
OVA/UFP
a
b
c
a
b
Saline
OVA
OVA/UFP
8
IL-5
7
a
b
c
6
5
a
b
4
3
2
1
0
Saline
OVA
OVA/UFP
100
90
80
70
60
50
40
30
20
10
0
a
b
c
IL-13
a
b
Saline
OVA
OVA/UFP
The target sites of ambient UFP
during the primary immune response
1°
The distal lung is the target of ambient UFP
during the secondary immune response
Distal lung
A
B
C
D
OVA/FA
OVA/UFP
Challenge
Proximal axial airway (G5)
alveolar
ducts &
alveoli
UFP Inhalation-enhanced allergic inflammation in the
distal lung is accompaned by oxidative stress
Sensitization
OVA
Ym1 (Chi3l3)
OVA + UFP
a
b
c
15
OVA/FA
OVA/UFP
Challenge
Fold Change Relative
to Saline/OvaFA
B
A
10
5
1
0
D
C
a
b
Saline
OVA OVA/UFP
Sensitization
1.5%
1.0%
3.2%
1.6%
6.8%
Organic compounds
Elemental carbon
Nitrate
Sulfate
Ammonium
Total metals
Unidentified
4.3%
81.6%
The target sites of ambient UFP
during the secondary immune response
1°
2°
Summary of Animal Studies
 Ambient UFP are capable of acting as an adjuvant to enhance
immune response to experimental allergen
 UFP are able to interfere with both primary and secondary immune
response
 There is a close correlation between the adjuvant effect of PM and
particulate redox-active organic chemical content
 Real-life inhalation of pro-oxidative UFP during allergen challenge
could lead to a profound allergic inflammation deep in the lung in
already-sensitized animals
Overall Summary
 Induction of oxidative stress by particle-associated organic chemicals
plays an important role in the adverse health effects of particulate pollutants
 UFP are more dangerous than coarse and fine particles
 UFP are capable of enhancing both primary and secondary immune
response to an experimental allergen
 The adjuvant effect of PM is closely correlated to particles’ organic
chemical content and oxidant potential
 Real-life inhalation of UFP during secondary allergen exposure could
lead to a profound allergic inflammation deep in the lung in prior
sensitized animals
 Relevance to human: The boosting effect of UFP inhalation on the
secondary immune response may provide an explanation for the asthma
flares after a sudden surge in ambient PM level
Acknowledgements
UCLA (Chemistry, toxicology, immunology)
UC Irvine (Inhalation)
Dr. John Froines
Dr. Arthur Cho
Dr. Andre Nel
Dr. Arantza Eiguren
Debra Schmitz
Emma Di Stefano
Meiying Wang
Dr. Michael Kleinman
Dr. Dianne Meacher
Glenn Gookin
USC (Environmental engineering)
Dr. Constantinos Sioutas
Dr. Zhi Ning
Payam Pakbin
MSU (Inhalation, pathology)
Dr. Jack Harkema
Dr. Jim Wagner
Lori Bramble
Ryan Lewandowski
UW-Madison (Chemistry)
Dr. James Schauer