PHM142 Metabolic Biochemistry and Immunology WHITE CELLS CHEMICAL WARFARE lecture outline

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PHM142 Metabolic Biochemistry and Immunology WHITE CELLS

CHEMICAL WARFARE lecture outline1) 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

2White (Immune) Cells Type of cell

Red blood cells (erythrocytes)

White blood cells (leukocytes) polymorphonuclear/granular leukocytesNeutrophils (50 60%)Eosinophils (1 4%)

Basophils (0.5 2%)

Mononuclear leukocytes:

Lymphocytes (20 40%)

Monocytes (2 9%)( macrophages in extravascular tissue)Killer cells

Megakaryocytes ( platelets)

Main function

Transport O2 and CO2

Destroy invading bacteria

Phagocytose & kill invading antigensDestroy 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

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Thin sections of the major types of white blood cells (leukocytes) foundin the circulation, showing the variety of internal structures observed.

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Macrophage3

4Molecular mechanisms of neutrophil functionAmer J Med. 109, 33-44 (2000)

Neutrophil membrane electron transport

CYTOSOL

PHAGOSOME MEMBRANE

PHAGOSOME VACUOLE

H2O2

H2O

GSH

GSSG

NADP+

NADPH

Glucose

glucose-6-phosphate

glucose-6-phosphatedehydrogenase

6 P gluconic acid

H2O2 O2.-

O2

NADP+

NADPH

Glucose-6-P

Gluconic acid

NADPHoxidase

Intracellularly in white cells

H2O2 + Cl- HOCl Bactericidalmyeloperoxidase

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2

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Fe2+

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))

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Living with a killer: The effects of hypochlorite/hypobromite on mammalian cells Chem.Res.Toxicol.21,1011-6 (2008)

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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. 109:33 44.

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NADPH oxidase (Nox)Activation DON’T MEMORISE!

Proton Channel Function

Medicine 79, 170-200 (2000)

b558

+ H+

ProtonChannel

(phosphorylationby kinase)

gp91phox is b558 with proton channel (or K+?)

phox is “phagocyte oxidase”

membrane

Vacuolar space

b558

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bact.

• 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).

Chronic Granulomatous Disease (CGD)

NADPH OXIDASE DEFICIENCY

White Cell

0.1 M Chloridein blood

• 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

NADPH oxidase family - makes antibacterial H2O2

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leukocytes

Synovium (synovial fluid)

From Am J Med. (2000). 190:33-44

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).

Babior, 2000. American Journal of Medicine. 109:33 44.

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

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Side effects of COX-1 inhibitors• 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

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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

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14Rheumatoid Arthritis Therapy

Repetitive hypoxia in joints, and endothelial cells

ATP hydrolysis

Hypoxanthine from ATP

ROS

Reperfusion O2 +endothelial cell xanthine oxidase

ALLOPURINOLinhib.

endothelial cell nitric oxide

peroxynitriteDestruction of joint

Uric acidMonosodium urate crystals in joint

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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

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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) .

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Biochem J. 358, 233-239 (2001),

Journal of Biological Chemistry270 (7) 2905-2913(2000).

MPO

• 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

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MACROPHAGES - chemical warfare function

1) Endocytosis and exocytosis via specific receptors for IgG and C3 coated in bacteria2) H2O2 production by NADPH oxidase to kill mycobacteria3) Arachidonate oxidation to prostaglandin4) Cytokine production - upon activation by PDGF a) lipopolysaccharide (endotoxin) TNF- b) immune system activation BCG infection IL-1 c) inflammation or interferon (IFN-) PAF TGF and 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)

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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).

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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 specificregions 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.

22Macrophages and Tissue Injury

Toxicant

Target tissues

Activated macrophagese.g. Kupffer cells

Mediators

Cytotoxicity Amplification

Model for the role of macrophages in tissue injury by generating inflammatory mediators.

Gold thiomalateGadolinium chloride(macrophage inhibitor)

Tissue Injury

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.

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24 Inflammatory mediators implicated in toxicity Toxicant

Mediator Lung Liver

1) Reactive oxygen intermediates Ozone Endotoxin (H2O2, .OH) Asbestos Acetaminophen

Amiodarone Corynebacterium parvum Reactive nitrogen intermediates Bleomycin (peroxynitrite) Carbon tetrachloride

1,2-dichlorobenzenePhenobarbitalEndotoxin

2) Hydrolytic enzymes Endotoxin (collagenase, elastase) Silica 3) Lipids Hyperoxia (leukotrienes, prostaglandins, thromboxanes)

4) IL-1 Cigarette smoke

5) TNF- (mitochondrial toxin, Cadmium chloride Alcohol reactive oxygen species)

Toxicology 160, 111 8 (2001).Ann. Rev. Pharmacol. Toxicol. 35, 655 (1995).

4. Biologics: anti-cytokine antibodies • TNF 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.

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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

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Immunosuppressants and anti-inflammatories RA

• B cell depleting agents like rituximab

• human monoclonal antibody against IL-6R cytokine

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Biomarkers for inflammation diseasePro-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

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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

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