Physiological Fluxes of Reactive Oxygen Species During Inflammation: Reality Check

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Sunrise Free Radical School Society for Free
Radical Biology and Medicine Washington, DC
November 2007
Physiological Fluxes of Reactive Oxygen Species
During Inflammation Reality Check
Matthew Grisham, Ph.D. LSU Health Sciences
Center Dept. of Physiology1501 Kings
HighwayShreveport, LA 71130 USA
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Physiological Fluxes of Reactive Oxygen Species
During Inflammation Reality Check

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Inflammation is a protective response designed to
destroy invading pathogens

• Redness (rubor)
• Heat (calor)
• Swelling (tumor)
• Pain (dolar)

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Inflammation is a Double-Edge Sword
? Defects in mounting an inflammatory response
may lead to grave illness or death. ? Excessive
inflammation leads to tissue injury, pain and
loss of function.
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Location Tissue Microcirculation
Arterioles
Veins
Arteries
Venules
Capillaries
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PMN-Endothelial Cell Interactions In Response to
Infection Post-Capillary Venules
BLOOD
TISSUE
Circulating PMNs
Adhesion
Extravasation
Phagocytosis and Destruction of Bacteria
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OXIDATIVE BURST
NADPH
NADP
CYTOPLASM
O2-
O2
2O2- 2H O2 H2O2 H2O2 Cl- H
H2O HOCl HOCl RNH2 H2O
RNHCl
PHAGOLYSOSOME
MPO
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Large Numbers of PMNs Invade Tissue During an
Inflammatory Response
Venule
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Chronic Gut Inflammation is Characterized By the
Infiltration of Large Numbers of PMNs
Grisham 2006
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ROS Production by Activated PMNs
MPO
RNH2
RNHCl
H2O2 Cl- HOCl
O2-
O2
NADPH Oxidase
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Oxygen Consumption by PMA-Activated PMNs in
Suspension
200
Vmax 20 µM/min Km (O2) 5-10µM 4 x 106
PMNs/ml
Oxygen (µM)
100
PMAphorbol myristate acetate
0
20
Time (minutes)
Grisham et. al. 1983
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Stoichiometric Conversion of Oxygen to
Superoxide By Activated Neutrophils
250
200
PMA
200
150
150
O2 Concentration (µM)
O2- Production (µM)
100
100
50
50
PMA
0
0
6
8
2
4
Time (minutes)
From Makino JBC 1986
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Reality Check 1
PMA-Stimulated ROS Production by PMNs in
Suspension Is this Physiological?
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Inflammatory Mediators (not PMA) Interact with
PMNs Associated with Endothelial Cells and the
ECM
APC
T-Cell
PMNS
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Biologically-Relevant Inflammatory Mediators Do
NOT Activate PMNs in Suspension
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PMN-Endothelial Cell and PMN-ECM Interactions In
Response to Infection
BLOOD
TISSUE
Circulating PMNs
Adhesion
Extravasation
Phagocytosis and Destruction of Bacteria
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Plastic Surfaces Activate PMNs
Uncoated Plastic Wells PMNs
H2O2 Production
Time (minutes)
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Coating Plastic Surface with Extracellular
Matrix Proteins (Serum, Fibronectin or Laminin)
Eliminates Spontaneous Activation of PMNs
Uncoated Plastic
H2O2 Production
Coated Plastic
Time (minutes)
C.F. Nathan, 1986-90
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TNF-a or FMLP Induce Large and Prolonged Release
of H2O2 When PMNs are Plated on Serum,
Fibronectin or Laminin-Coated Plastic Surfaces
PMA
TNF
FMLP
2
H2O2 (nmol)
Rate H2O2 Production 2 - 4 nmol/min/106 PMNs
1
none
0
120
0
60
Time (minutes)
Nathan JCI 1989
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PMNs use a Variety of Adhesion Molecules to
Interact with Endothelial Cells and the ECM CD18
L-Selectin
CD11a/CD18 (LFA-1) CD11b/CD18 (Mac-1)
VLA-4 (a4ß1)
P-Selectin
VCAM-1
ICAM1
E-Selectin
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Human and Canine Leukocyte Adhesion Deficiency
Disease CD18 Deficiency
Lymph Node Infection (Adenitis) Incision to
drain abscess took 4 months to heal
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TNF-Induced ROS Production is Dependent Upon the
Interaction of the ß2 Integrin (CD18) With ECM
Proteins
Healthy
CD18-Deficient 2
CD18-Deficient 1
PMA
PMA
TNF
PMA
TNF
None
TNF
Fibronectin-Coated Wells
Nathan JCB 1989
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TNF-Induced ROS Production is Dependent Upon
Interaction of Extracellular Matrix Proteins with
PMN-associated CD18
CD18 Ab (ug/ml)
Nathan JCB 1989
Fibronection-Coated Wells
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Endothelial Cell-
PMN Interactions
BLOOD
TISSUE
Circulating PMNs
Adhesion
Extravasation
Phagocytosis and Destruction of Bacteria
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Physiological Levels of TNF Activate PMNs
Associated With Endothelial Cell Monolayers
Nathan et. al. 1990
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Reality Check 2
PMN-Mediated ROS Production at Ambient
O2 Concentrations Is this Physiological?
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Not Really Consider the Following
? pO2 is the partial pressure of O2 in air,
solution or in tissue and is expressed in mm of
Hg. Air contains 21 O2 which corresponds to a
pO2 of 100 mmHg or 5.2 mM. ? The pO2 in
air-equilibrated buffer (pH 7.4 37C) is also
100 mmHg (100 Torr) but because of its low
solubility in water, O2 concentration in solution
is 200 µM.
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Tissue pO2 is a function of O2 delivery,
diffusion distance to nearby capillaries and O2
consumption
Except for the lung, skin epidermis and
cornea, tissue pO2 ranges from 5-40 mmHg or from
10-80 µM (assuming an O2 of 100 mmHg or 200 µM
in solution)
Tissue pO2 approximates venous pO2
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Inflammation, Tissue Microcirculation and pO2
Arterioles
Veins
Arteries
Venules
Capillaries
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Perivascular and Tissue pO2
Hamster Cheek Pouch
Air-equilibrated buffer 100 mmHg
pO2 (mmHg)
Duling and Berne
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Tissue pO2 Values
Tissue pO2 (mm Hg)
Kidney (cortex) Kidney (medulla) Skeletal
muscle Intestine Brain Liver (Periportal
zone) Liver (Perivenous zone)
45 23 25 25 15 45 22
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Physiological pO2 of the Intestine at
Rest, Following Hemorrhage and During Nutrient
Absorption
Suffusion solution
Penetration of Villus Apex
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Begin Glucose Solution
30
pO2 (mmHg)
Hemorrhage
20
Infuse Blood
Stop Glucose
10
0
Time (minutes)
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Oxygen Gradient within the Liver
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Metabolic Heterogeneity of Hepatic Parenchymal
Cells along the Sinusoid
Tissue pO2
Mitochondrial volume Oxidative energy metabolism
Oxidative xenobiotic metabolism Glucose
production
PV
PP
pO2 45 mmHg (90 µM)
pO2 22 mmHg (48 µM)
Glucose uptake Glycolysis
Xenobiotic biotransformation
TY Aw
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Why are these considerations important in
the context of ROS generation?
Because most cells and tissue are not exposed to
ambient O2 tension (i.e. 100 mmHg 200µM). In
reality, physiological pO2 for the vast majority
of cells and tissue is far below ambient oxygen
tension.
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Cultured Cells Grown at Ambient pO2 are
Subjected to Hyperoxia
dO2-/dt k O2 electron donor
Doubling of the pO2 will double O2- production
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pO2 (mmHg)
Oxygen Modulates The Growth of Fibroblasts
Balin and Pratt In Vitro Cell Dev Biol 2002
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Respiration-Deficient Cells are Resistant to
Damaging Effects of Hyperoxia
Resp- 20 O2
Resp- 80 O2
Li et. al. FRBM 2004
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Cultured Cells Grown at Ambient pO2 are
Subjected to Hyperoxia
Natarajan 2003
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What are the Rates of PMN-Derived ROS
Generation at Physiological pO2?
UNKNOWN. However one would predict that 106
fully-activated PMNs would be functional at pO2
values as low as 10 mmHg (20 µM) assuming a KmO2
of 5-10µM for NADPH oxidase. However
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Reality Check 3
Rates of ROS Formation by PMNs Would Predict That
all Tissue O2 Would be Consumed Within A Matter
of a Few Minutes
Rate H2O2 Production 2-4 nmol/min/106 PMNs
Nathan JCI 1989
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Not Necessarily Inflammatory Vasodilators
(PGE2, NO, adenosine) Will Promote the Delivery
of O2 to the Inflammatory Site via Enhancing
Tissue Blood Flow (O2 Delivery Blood Flow x
O2)
Poiseuilles Law Relates pressure (P), flow (Q)
and resistance (R) P Q X R where R8L?/pr4 Q
(P)(pr4)
Llength, ?viscosity, rradius
Note that 2-fold increase in vessel diameter
results in a 16-fold increase blood flow
8L?
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Inflammation-induced hyperemia is a good thing
when responding to a bacterial infection or
tissue injury.
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But what about sustained increases in blood flow
associated with chronic inflammation?
Rheumatoid Arthritis
Crohns Disease
Crohns Disease
Adding Fuel to the Fire Potential for Hyperoxic
Insult
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Summary and Conclusions
? Inflammatory mediator (TNF-a, IL-1ß)-induced
ROS production by PMNs is dramatically enhanced
by the interaction of PMN-associated CD18 with
extracellular matrix (ECM) proteins. ? Except
for the lung, tissue pO2 is far below ambient pO2
and approximates venous oxygen tension ( 40
mmHg). ? Blood flow affects tissue pO2 and thus
may regulate the magnitude and duration of
PMN-derived ROS generation during times of
infection and inflammation.
Physiological fluxes of PMN-derived ROS are
dependent upon the physical association of these
phagocytes with endothelial cells and ultimately
the ECM as well as tissue pO2
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Susceptibility of Periportal vs Perivenous Zones
to Hypoxia/Reoxygenation
Whole liver
Periportal vs perivenous
240
360
Reoxygenation
Reoxygenation
Hypoxia
Hypoxia


180
PP
240
Perfusate ALT (IU/L)

120


120

60
PV

0
0
0 40 60 80
0 40 60 80
Time, min
Time, min
AW et. al.
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ROS involvement in HR-induced Periportal injury
SOD 2/3
NAC
240
240
Reoxygenation
Hypoxia
Reoxygenation
Hypoxia
180
5 mmol/L NAC
180
Perfusate ALT (IU/L)
120
120
1 unit/g body SOD bolus iv
60
60
SOD 2/3




0
0
0 40 60 80
0 40 60 80
Time, min
Time, min
Aw et. al.