PHM226 II WHITE CELLS

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PHM226 II WHITE CELLS CHEMICAL WARFARE
lecture outline

• Molecular mechanisms of leukocyte function
• Rheumatoid arthritis
• CGD disease
• 2) Eosinophils
• Macrophages
• Function
• Growth factors and cytokines
• But macrophage - induced tissue injury
• Multiple organ failure, ARDS, sepsis

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Blood Cells
Main function Transport O2 and CO2 Destroy
invading bacteria Phagocytose kill invading
antigens Destroy parasites and modulate allergic
inflammatory reactions Release histamine,
serotonin, bradykinin, heparin, and cytokines
converts arachidonic acid to prostaglandins
leukotrienes (T B) mediate cytokine
release Phagocytose kill ingested microbes
Kill virally infected cells and tumor cells
(offer natural immunity as well as
adaptive) Initiate blood clotting also release
histamine and serotonin
Type of cell Red blood cells
(erythrocytes) White blood cells (leukocytes)
polymorphonuclear/granular
leukocytes Neutrophils (50 ? 60) Eosinophils (1
? 4) Basophils (0.5 ? 2)
mononuclear leukocytes Lymphocytes (20 ?
40) Monocytes (2 ? 9) (? macrophages in
extravascular tissue) Killer cells Megakaryo
cytes (? platelets)
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2
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Macrophage
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Thin sections of the major types of white blood
cells (leukocytes) found in the circulation,
showing the variety of internal structures
observed.
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Molecular mechanisms of neutrophil function
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Amer J Med. 109, 33-44 (2000)
Neutrophil membrane electron transport
CYTOSOL
PHAGOSOME MEMBRANE
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2
PHAGOSOME VACUOLE
Fe2
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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
on mammalian cells IUMB Life 50,259-266 (2000)
Chem.Res.Toxicol.21,1011-6 (2008)
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(No Transcript)
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A CGD disease clinical test for the lack of
leukocyte respiratory burst.
Leukocyte Respiratory burst by phagosome membr.
elect. transport.
Leukocytes (from normal patient)
Opsonized bacteria or N-formyl methionine peptide

O2 level in solution
Oxygen uptake is insensitive to cyanide
Time
Oxygen in solution is measured by an O2 electrode
superoxide detected by NBT reduction to formazan
(blue precipitate).
Made by bacteria recognized by mammalian cells
as foreign.
? No O2 consumption by CGD patients leukcoytes.
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Trends in Biochem. Sci. 2, 63 (1977)
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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.
10933 ? 44.
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(phosphorylation by kinase)
Proton Channel Function
H
Proton Channel
membrane
b558
b558
Vacuolar space
phox is phagocyte oxidase
NADPH oxidase (Nox) activation
gp91phox is b558 with proton channel (or K?)
Medicine 79, 170-200 (2000)
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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 cant kill bacteria which have high catalase
  activity or low H2O2 production, e.g., serratia,
  nocardia, and aspergillus fungal,
  staphylococcus, burkholderia (pneumonia, sepsis).

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LEUKOCYTE ELECTRON TRANSPORT Chronic
granulomatous disease (CGD)

• Two-thirds have mutations in gp91phox gene ?
  Unable to form H2O2.
• i.e., no cyanide resistant respiratory burst
  dont live beyond childhood
• Have X-linked chromosomal deficiency
• Have no cyt. b558 function
• b-subunit 91kD glycosylated X chromosome
  DEFECT
• a-subunit 22kD normal chromosome 16
• CGD patients have mutation in p47phox gene
  (reach adulthood)
• Have autosomal recessive deficiency
• CGD patients have no cytosol activating protein
  that phosphorylates and activates NADPH oxidase
  which causes translocation of the oxidase from
  the endoplasmic reticulum to the plasma membrane.

Medicine 79, 170 ? 200 (2000) J Leukocyte Biol.
69, 191 ? 6, 2001
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THERAPY FOR CGD DISEASE

• Prophylaxis with trimethoprim-sulfamethoxazole
  and interferon gamma
• Bone marrow transplantation or granulocyte
  transfusion (with severe infections)
• Hemopoietic stem cell gene therapy
  (retrovirus-mediated expression of gp91phox to
  reconstitute NADPH oxidase Dinauer et al.,
  1999. Blood. 94(3)914-22)..
• Possible Pharmaceutical Therapy?

Opsin Chemoattractant
Polystyrene Beads
Glucose oxidase (Fungal)
Glucose O2
H2O2
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NADPH oxidase family tissue locationJ.Biol.Chem
283,16961-16965(2008)

• NOX1 colongtprostate,uterus,breast,macrophage?
• NOX2 phagocyte H2O2 for MPO gtgt hepatocyte,B
  lymphocyte, cardiomyocytes,endothelial cells
• NOX3 inner ear,fetal
• NOX4 kidney,blood vessels,cardiomyocytes,endoth.
• NOX5 lymphoid tissue,testis
• Duox1 2 (dual oxidase) makes thyroxine hormone
  and H2O2 for thyroid peroxidase TPO gtlung,GI

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• 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).

leukocytes
Synovium (synovial fluid)
Babior, 2000. American Journal of Medicine.
10933 ? 44.
From Am J Med. (2000). 19033-44
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Rheumatoid Arthritis Therapy
Repetitive hypoxia in joints, and endothelial
cells
ATP hydrolysis
? Hypoxanthine from ATP
Reperfusion O2 endothelial cell xanthine oxidase
Monosodium urate crystals in joint
Uric acid
ALLOPURINOL
ROS
endothelial cell nitric oxide
peroxynitrite
Destruction of joint
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Anti-Inflammation therapy

• ROS Scavenger Therapy
• SOD, selenomethionine/Vit E,
  5-aminosalicylate, penicillamineCu (cuprimine)
• 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-a
• 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

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• EOSINOPHILS
• Accumulate in parasite infection, asthma,
  rheumatoid diseases,
• Hodgkins lymphoma and allergic or inflammatory
  diseases
• 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) .

2
MPO
Biochem J. 358, 233-239 (2001).
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MACROPHAGES - 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)
3
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GROWTH FACTORS FORMED BY MACROPHAGE AND
LYMPHOCYTE - regulate the growth, replication and
differentiation of cells Cell biologists -
growth factors regulate the differentiation of
tissues Immunologists - interleukins formed by
lymphocytes and macrophages govern immunity
and inflammation Hematologists -
colony-stimulating factors or cytokines formed by
hematological cells to drive proliferation
and to differentiation of hematological cells
in the bone marrow Function - inflammatory and
immune responses - embryogenesis -
wound healing and fibrosis
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Cellular signalling by growth factors and
cytokines
Cytokine Original MW (kDa) amino
Beneficial Toxic
source from cDNA acids effects
effects clone TNFa
macrophage 17.3 233
Antiviral Endotoxic

shock TNF b lymphocytes
Antiparasitic Tumour necrosis M?CSF
a macrophages 45 256
Proliferation and M?CSF b monocytes 90
554 differentiation of G?CSF
lymphocytes macrophages IL?1b 15
146 Bone marrow recovery
after chemotherapy IFN viral infected
17 146 Lymphocyte
growth/differ- cells entiation
suppress tumour growth inhibit
viral replication
Formation stumulated by bacteria, bacterial
products, eg. Lipopolysaccharide (endotoxin)
immune system activators, C5a, immune complex,
interferon (IFNg), BCG infection used to
activate host immunity to suppress tumour growth
or antimicrobial functional disorders eg CGD,
leprosy
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SURGERY TRAUMA e.g. if intestinal surgery
causes prolonged interrupted blood flow
(reperfusion injury) EARLY ORGAN FAILURE if
endotoxin from Gram ve cell wall of Gram-ve
bacteria in the intestine crosses gut barrier
Endotoxin releases immune cell growth factors
humoral/cellular mediators (now too late!) e.g.
TNF, IL, arachidonic oxidation production Attract
PMN, macrophages, endothelium cells which
further release TNF,NO,H2O2 anaphylactic shock
mast cells release histamine (e.g. ARDS (adult
respiratory distress syndrome),
hepatitis Multiorgan failure, sudden death
Harrison 2007 Principles of Medicine
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Tumor Necrosis Factor (TNFa) as the primary
trigger for inflammatory response

• ?Macrophages,monocytes,lymphocytes,keratinocyte
• TNFa incr in chronic inflammatory diseases
  rheumatism,arthritis,encephalitis,tumors
• Rheumatoid arthritis , psoriasis,Crohns disease
• Proinflammatory gt antiinflammatory cytokines
• Drug therapy NSAIDs, GC glucocorticoids,DMARDs
  disease modifying antirheumatic drugs
• Biologic therapy Enbrel (TNF receptor
  fusion),antibodies humira or remicade

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Model for the potential role of nonparenchymal
cells (Kupffer cells , macrophages ,neutrophils)
in hepatotoxicity
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.TNFa 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.
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Macrophages and Tissue Injury
Toxicant Target tissues Activated
macrophages e.g. Kupffer cells Mediators
Gold thiomalate Gadolinium chloride (macrophage
inhibitor)
Amplification
Cytotoxicity
Tissue Injury
Model for the role of macrophages in tissue
injury by generating inflammatory mediators.
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Macrophage killing mechanism

• Macrophages endocytose present antigens to T
  cells as foreign substances
• Exocytose (via specific receptors) IgC and C3
  coated bacteria
• Produce H2O2 to kill mycobacteria
• Produce cytokines PDGF,PAF,TNFa ß, IL1 convert
  arginine to NO
• Prevents foam cell formation by endocytosis of
  oxid. LDL by macrophage oxid LDL fusion with
  lysosomes and digestion by cathepsins Plipases.
  

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INFLAMMATION MODEL FOR IDIOSYNCRATIC DRUG
TOXICITY (Liver injury, bone marrow
agranulocytosis, lung toxicity)
OBrien et. al. Drug Discovery Today 2005 10,
617-625
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Table 2 Inflammatory mediators implicated in
toxicity
Toxicant
Mediator Lung Liver 1) Reactive oxygen
intermediates Ozone Endotoxin (H2O2,
.OH) Asbestos Acetaminophen Amiodarone Co
rynebacterium parvum Reactive nitrogen
intermediates Bleomycin Galactosamine
(peroxynitrite) Carbon tetrachloride
1,2-dichlorobenzene Phenobarbital En
dotoxin 2) Hydrolytic enzymes Endotoxin
(collagenase, elastase) Silica 3)
Lipids Hyperoxia (leukotrienes,
prostaglandins, thromboxanes)
4) IL-1 Cigarette smoke 5)
TNF-a (mitochondrial toxin, Cadmium chloride
Alcohol reactive oxygen
species)
Toxicology 160, 111 ? 8 (2001). Ann. Rev.
Pharmacol. Toxicol. 35, 655 (1995).
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Table 1 Toxicants whose pathophysiology is
thought to be associated with macrophages and
inflammatory mediators Pulmonary
toxicants Hepatotoxicants Ozone Acetaminoph
en Sulfuric acid Carbon tetrachloride Nitrogen
dioxide Lipopolysaccharide Cigarette
smoke Galactosamine Hyperoxia 1,2-dichloroben
zene Bleomycin Cadmium Amiodarone Allyl
alcohol Titanium dioxide Endotoxin Silica Asbest
os Cadmium chloride
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• Autoimmune disease drug therapy
• 1. Alkylating Agents (anticancer cytostatic
  drugs)
• Inhibit cell division of both T cells and B
  cell (lymphocytes)
• Cyclophosphamide, nitrosoureas, platinum
  compounds
• Efficient therapy for systemic lupus,
  autoimmune hemolytic anemias, Wegeners
• 2. Antimetabolites Interfere with synthesis of
  nucleic acids
• Folic acid analogues (METHOTREXATE) binds
  dihydrofolate reductase and prevents synthesis of
  tetrahydrofolate. Used in treatment of autoimmune
  diseases (rheumatoid arthritis) and in
  transplantations
• Purine analogues (AZATHIOPRINE, or MYCOPHENOLATE
  (CellCept) inhib. IMP dehydrogenase and GMP
  synth. Treatment of autoimmune diseases
  (Wegeners,psoriasis ) also prevents transplant
  rejection. Mercaptopurine (leukemia,Crohns).
• Pyrimidine analogues LEFLUNOMIDE
• 3. Cytotoxic anticancer e.g. mithramycin,
  dactinomycin inhibits RNA transcription and DNA
  replication
• 4. Biologics anti-cytokine antibodies
• Etanercept for RA blocks TNFa, (not Wegeners)
• Infliximab for Crohns disease, an artificial
  antibody that blocks TNFa (not W).
• Rituximab kills CD20 B cells in lymphomas,
• Abatacept, a fusion protein inhib.T cell
  costimulation
• Stelera , NEW for psoriasis, antibody blocks TNFa
  5 shots p.a. (50,000)

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T lymphocytes formed in b.m. mature in thymus

• 1) T helper cells help B cells mature into plasma
  cells.
• CD4T cells express CD4 protein on their surface
• 2)19 Cytotoxic T cells destroy viral infective
  tumor cells
• 3)Memory Tcells CD4(lost in AIDs) or
  CD8(cytotoxic)
• subset of antigen T cells that persist long after
  infection.
• 4) Regulatory T cells (suppressor T cells)
• 5)Natural killer (NK) largest T cell.Interferons
  cause
• cytotoxic granule release
• 6) Antigen-presenting cell (APC)

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B cells bursa of fabricius(birds)

• Plasma cells are large B cells exposed to
  antigens and 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 IgMgtIgG in peritoneal pleural
  cavities
• B-2 cells

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Treatment

• Immunosuppressants and anti-inflammatories
• B cell depleting agents like rituximab
• Anti-cytokine therapies like tocilizumab
• No truly effective therapies exist