Transcript H 2 O 2

1
PHM142 Metabolic Biochemistry and Immunology
WHITE CELLS
CHEMICAL WARFARE lecture outline
1) Molecular mechanisms of leukocyte function (live 2 days)
• Rheumatoid arthritis
• Chronic Granulomatous Disease CGD
2) Eosinophils-live 8-12 days when activated (live 8-12 days when
not activated). Contain peroxidase, RNase, DNase, lipase
3) Macrophages (greek:big eaters; 21um), amoeboid movement
Phagocytose foreign cells AND produce growth factors and
cytokines
• But macrophages also induce tissue injury
• Cause multiple organ failure, Acute Resp.Distr.Syndr., sepsis
2
White (Immune) Cells
Type of cell
Main function
Red blood cells (erythrocytes)
Transport O2 and CO2
White blood cells (leukocytes)
polymorphonuclear/granular leukocytes
Neutrophils (50  60%)
Eosinophils (1  4%)
Destroy invading bacteria
Basophils (0.5  2%)
Phagocytose & kill invading antigens
Destroy parasites and modulate allergic
inflammatory reactions.
Release histamine, serotonin, bradykinin,
heparin, and cytokines; converts arachidonic
acid to prostaglandins & leukotrienes
Mononuclear leukocytes:
(T & B) mediate cytokine release
Lymphocytes (20  40%)
Phagocytose & kill ingested microbes
Monocytes (2  9%)
( macrophages in extravascular tissue)
Killer cells
Megakaryocytes ( platelets)
Kill virally infected cells and tumor cells
(offer “natural” immunity as well as
adaptive)
Initiate blood clotting; also release
histamine and serotonin
3
1
2
3
Macrophage
Thin sections of the major types of white blood cells (leukocytes) found
in the circulation, showing the variety of internal structures observed.
4
1 Molecular mechanisms of neutrophil function
Amer J Med. 109, 33-44 (2000)
Neutrophil membrane electron transport
I
G
n
l
t
r
CYTOSOL
H2O2
+
G
NADP
g
S
g
G
u
l
d
H 2O
l
H
NADPH S
u
e
6
h
S
G
P
PHAGOSOME MEMBRANE
2
H2O 2
O2.
NADP+
2
N
o
O2
NADPH
-
G
l
u
A
x
D
i
G
l
u
PHAGOSOME VACUOLE
H2O 2
+
Cl m
-
Fe2+
HOCl
B
y
a
e
5
Inflammation or infection
MPO(myeloperoxidase), HOCl(hypochlorite), SOD (superoxide dismutase).
HOCl + red cell GSH = GSH cyclic sulfonamide (biomarker for HOCl formation in
vivo that reflects inflammation (oxidant formed by neutrophils))
Living with a killer: The effects of hypochlorite/hypobromite on mammalian cells
Chem.Res.Toxicol.21,1011-6 (2008)
6
Activation of leukocyte NADPH oxidase (Nox)
showing assembly of the enzyme and fusion of the oxidase-containing vesicle with the
phagosomal membrane.
Babior, 2000. American Journal of Medicine. 109:33  44.
7
(phosphorylation
by kinase)
Proton Channel
Function
+ H+
membrane
b558
b558
Proton
Channel
Vacuolar space
phox is “phagocyte oxidase”
NADPH oxidase (Nox) gp91phox is b558 with proton channel (or K+?)
Activation DON’T MEMORISE!
Medicine 79, 170-200 (2000)
8
NADPH OXIDASE DEFICIENCY
Chronic Granulomatous Disease (CGD)
White Cell
bact.
0.1 M Chloride
in blood
• CGD can kill Bacteria that produce H2O2 and have low catalase activity, e.g., streptococci.
• But can’t kill bacteria which have high catalase activity or low H2O2 production, e.g., serratia,
nocardia, and aspergillus; fungal, staphylococcus, burkholderia (pneumonia, sepsis).
NADPH oxidase family - makes antibacterial H2O2
• NOX1 – colon > prostate, uterus, breast,macrophage
• NOX2 - phagocyte H2O2 for MPO >> hepatocyte,
B lymphocyte, cardiomyocytes, endothelial cells
• NOX3 inner ear, fetus
• NOX4 kidney, bloodvessels,cardiomyocytes,endoth.
• NOX5 lymphoid tissue,testis
Rheumatoid Arthritis is an autoimmune
inflammatory disease that may affect
(skin, blood vessels, heart, lungs,
muscles-- but principally attacks joints
(proliferative synovitis progressing to
cartilage destruction/joint ankylosis.
Pathogenesis (unknown etiology):genetic susceptibility;
joint damage mediated by leukocytes or exogenous
arthritogen (virus, mycobacteria).
10
leukocytes
Synovium
(synovial fluid)
Babior, 2000. American Journal
of Medicine. 109:33  44.
From Am J Med. (2000). 190:33-44
11
NSAID therapy for RA
IBUPROPHEN COX-1 inhib. 300mg 3 times perday
Cardiovascular & GI bleeding risk,inhib platelets, kidney/liver tox.
ALEVE (Naproxen) 200mg otc ; lasts 12h
• Inhibits inflammation , COX-I and COX-2 inhibitor
• Decrease pain, temp, muscle pain, menstrual cramps
• Lower stroke risk than ibuprofen; high dose risk GI bleed.
VIOXX COX-2 inhibitor for OA,RA 1999 introd.
• 2004 (withdrawn due to heart attack, stroke)
CELEBREX (celecoxib)1998 COX-2 inhib.400mg
• low cardiorenal tox, platelet effects & GI bleeding
Side effects of COX-1 inhibitors
12
• e.g. Aspirin may cause stomach bleeding and risk GI
ulcer formation as a result of stomach cell
mitochondrial uncoupling and acidosis.
• inhibits COX-1 activity thereby increasing tissue
unsat. fatty acid levels and causing acidosis.
• decreases PGE2 levels that protect stomach membr.
• inhibits thromboxane formn. and platelet aggreg.
• unsat.fatty acid + PGS(prostaglandin synthase) attacks protective
mucous layer
13
Mechanism of NSAID induced GI toxicity
• Stage I NSAIDs decrease intestinal mucosal
prostanoids (PGE,TXB2, 6-keto-PGF)
• Mitochondrial uncouplers (e.g.indomethacin,DNP)
compromised intestinal barrier). Resp.inhibited
• Stage 2 mild inflammation + aspirin
• Stage 3 histopathology, ulcers and bleeding
• Rat model: ulcers, intestinal inflammation or gastric
permeability induced by indomethacin or DNP +
Cox inhib (aspirin).
Aliment Pharmacol. Ther. (2000) 14,639-650
Rheumatoid Arthritis Therapy
Repetitive hypoxia in
joints, and
endothelial cells
Monosodium
urate crystals
in joint
ATP
hydrolysis
14
 Hypoxanthine
from ATP
Reperfusion O2 +
endothelial cell
xanthine oxidase
Uric acid
ALLOPURINOLinhib.
ROS
endothelial cell
nitric oxide
Destruction of joint
peroxynitrite
15
16
Anti-Inflammation therapy
• ROS Scavenger Therapy
SOD, selenomethionine/Vit E, 5-aminosalicylate, penicillamine:Cu
• Macrophage inhibitor therapy:
Gold thiomalate or auranofin or zinc or copper salicylate
• Prostaglandin Synthetase Inhibitors:-(NSAIDS)
COX-1 many cells e.g.aspirin,ibuprofen, but GI bleeding,kidney
COX-2 inflammatory cells e.g. VIOXX (withdrawn), CELEBREX
but cardiovascular problems.
• “Biologics” antibodies that inhibit inflammatory cytokines e.g.TNF-α
• Diet: decrease arachidonate intake (meat), increase omega 3 fatty
acids (fish) decreases bad prostaglandins, decrease Fe intake
References: Semin. Arthritis Rheum. 27: 366-70 (1998).
Autoimmunity Reviews 7,1-7(2007) Anti-inflammatory Biologics
17
2 EOSINOPHILS: (chemical warfare cont.)
Accumulate in parasite infection, asthma, rheumatoid diseases,
Hodgkin’s lymphoma and allergic or inflammatory diseases
1)destroy parasitic worms, tumor cells, fungi and bacteria by forming
hypobromite
H2O2 + Br- + H+
HOBr (hypobromite) + H2O
2) cytokine production e.g. PAF, LTC4 unlike neutrophils (leukocytes) .
Biochem J. 358, 233-239 (2001),
Journal of Biological Chemistry270 (7) 2905-2913(2000).
18
• White blood cells
within tissue, have a
role in innate and
adaptive immunity
• They engulf
pathogens and debris
via phagocytosis, and
move around via
amoeboid movement
19
3
MACROPHAGES - chemical warfare function
1) Endocytosis and exocytosis via specific receptors for IgG and C3
coated in bacteria
2) H2O2 production by NADPH oxidase to kill mycobacteria
3) Arachidonate oxidation to prostaglandin
4) Cytokine production - upon activation by
PDGF
a) lipopolysaccharide (endotoxin)
TNF-a
b) immune system activation BCG infection
IL-1
c) inflammation or interferon (IFN-g)
PAF
TGF a and b
arginine
nitric oxide
kill tumor cells
5) endocytosis and delivery to lysosomes (via scavenger receptor) of
oxidised LDL (low density lipoprotein) - can result in transformation
to foam cell (the basis of the formation of atherosclerotic plaque)
20
Tumor Necrosis Factor (TNFα) as the primary trigger
for inflammatory response
Macrophages,monocytes,lymphocytes,keratinocyte
• TNFα incr in chronic inflammatory diseases:
rheumatism,arthritis,encephalitis,tumors
 Rheumatoid arthritis , psoriasis,Crohn’s disease
• Proinflammatory > antiinflammatory cytokines
• DRUG THERAPY: NSAIDs, GC glucocorticoids, Disease
Modifying Antirheumatic Drugs (DMARDs).
21
Drug induced hepatocyte cytotoxicity caused by activated immune
cells releasing cytokines and reactive oxygen species
(Kupffer cells , macrophages ,neutrophils) i
1) Toxic doses of drugs or chemicals injure hepatocytes.
Injured hepatocytes release factors that attract Kupffer cells to specific
regions of the liver.
2) Additional mononuclear phagocytes are also recruited from blood and
bone marrow precursors.
3) Once localised in the liver, the macrophages become activated by
hepatocyte-derived factors of endothelial cells.
4) Activated macrophages and endothelial cells release cytokines
e.g.TNFα & platelet activating factor prime & activate Kupffer cells
which release Reactive Oxygen Species and more cytokines.
5) Some chemoattractants and cytokines can attract and activate
neutrophils that also contribute to hepatocyte injury.
22
Macrophages and Tissue Injury
Toxicant
Target tissues
Gold thiomalate
Gadolinium chloride
(macrophage inhibitor)
Activated macrophages
e.g. Kupffer cells
Amplification
Cytotoxicity
Mediators
Tissue Injury
Model for the role of macrophages in tissue injury by
generating inflammatory mediators.
23
Macrophage killing mechanism
• Macrophage oxid.LDL which are then endocytosed
by the oxid LDL receptor.
Present antigens to T cells as foreign substances
• Exocytose (via specific receptors) IgC and C3
coated bacteria
• H2O2 kills mycobacteria and form cytokines
PDGF,PAF,TNFα & β, IL1
• Unsaturated fatty oxidation to aldehydes causes foam cell
formation & plaque formation from macrophages.
Inflammatory mediators implicated in toxicity
Toxicant
Mediator
1) Reactive oxygen intermediates
(H2O2, .OH)
Reactive nitrogen intermediates
(peroxynitrite)
Lung
Liver
Ozone
Asbestos
Amiodarone
Bleomycin
Endotoxin
Acetaminophen
Corynebacterium parvum
Carbon tetrachloride
1,2-dichlorobenzene
Phenobarbital
Endotoxin
2) Hydrolytic enzymes
(collagenase, elastase)
Endotoxin
Silica
3) Lipids
(leukotrienes, prostaglandins,
thromboxanes)
Hyperoxia
4) IL-1
Cigarette smoke
5) TNF-a (mitochondrial toxin,
reactive oxygen species)
Cadmium chloride
Toxicology 160, 111  8 (2001).
Ann. Rev. Pharmacol. Toxicol. 35, 655 (1995).
Alcohol
24
4. Biologics: anti-cytokine antibodies
25
• TNFa blockers 1)Etanercept for RA
• 2) Infliximab (remicade)Crohns disease,
• drug antigen?, cancer a chimeric human Fab.
3) Adalimumab (Humira) human monoclonal for
macular degen.
4)Stelera for psoriasis, 5 shots p.a.$50K
• Rituximab kills ANCA-vasculitis NEJM363,221-232(2010)
& CD20 B cell,NH lymphoma,RA, Wegeners ?
• Abatacept,fusion protein inhib.T cell costim.RA
• Avastin (bevacizumab) inhib.VEGF-A (vascular
endoth.growth factor,eye mac.degen, breastcancer).Withdrawn.
26
B cells named from bursa of fabricius(birds)
• Plasma cells are large B cells exposed to antigens
which produce antibodies that bind to microbes. In
tissues not plasma.
• Contain rough e.r. & cell rapid apoptosis (short life)
• Memory B cells formed from activ.B cells(long life)
• 23% B-1 cells IgM>IgG in peritoneal & pleural
cavities
• B-2 cells
27
Immunosuppressants and
anti-inflammatories RA
• B cell depleting agents
like rituximab
• human monoclonal
antibody against IL-6R
cytokine
28
Biomarkers for inflammation disease
Pro-inflammatory cytokines
• Elevated cytokines CRP,IL-1B,IL-1B,IL-6,IL-8
• Tumor necrosis factor alpha
• c-ANCA anti-neutrophil cytoplasmic stain
Myeloperoxidase is the target antigen,Wegeners
• p-ANCA anti-neutrophil perinuclear stain
Proteinase 3 is the target antigen
• x-ANCA chronic inflammatory bowel disease
29
Biomarkers for determining oxidative stress
associated with inflammation diseases
•
•
•
•
•
•
•
•
•
1) Plasma lipid hydroperoxide
2) Oxidative DNA damage DNA 8-OHdQ
Urinary 8-OHdQ
Thymine oxidation HMdU
DNA strand breaks (Comet assay)
3) Protein carbonyls using dinitrophenylhydrazine
Protein oxidation - cysteine, methionine, histidine
Protein AGEs
4) Fructose oxidation forms toxic glyoxal
30