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Pathology: Free Radical Injury, Acute and Chronic Inflammation

August 15 | Dr. DeRisio (and Dr. Sattar)

Free Radical Injury

Free radical = chemical species w/ unpaired electron in outer orbit that can do damage within the cell

Physiologic Generation of Free Radicals Occurs during Oxidative Phosphorylation

Cytochrome c oxidase transfers electrons to O2 as final e- acceptor during ox phos

o Drives proton gradient which drives production of ATP

o O2 accepts 4 electrons, after which water is generated

If it doesn’t accept all 4, free radicals are generated:

If it accepts 1 superoxide (O2∙-)

If it accepts 2 peroxide (H2O2)

If it accepts 3 hydroxyl ion (∙OH) radicals

If it accepts 4 water (H2O)

Pathogenic Generation of Free Radicals

Ionizing radiation – radiation hits water in tissues causing formation of hydroxyl free radical (∙OH)

o Of all free radicals, hydroxyl free radical is the most damaging

Inflammation

o When neutrophils come in to battle an infection, two mechanisms can happen to kill microbe:

(1) Oxygen-dependent rxn starts w/ “oxidative burst”; free radicals are generated

O2 is acted on by NADPH oxidase to become superoxide

Superoxide is acted on by superoxide dismutase to hydrogen peroxide

Hydrogen peroxide is acted on by myloperoxidase to become bleach (HOCl)

(2) Oxygen-independent

Metals (ie: copper and iron)

o Copper and iron are usually tightly bound in the body

Iron is bound as soon as it enters the blood b/c when free, free radicals are generated

Fenton reaction allows Fe to generate hydroxyl free radical (most damaging)

o If copper or iron builds up, we get diseases

Fe buildup = hemochromatosis

Tissue damage (cirrhosis of liver); excess iron generates free radicals

Cu buildup = Wilson’s disease

Tissue damage bc of generation of free radicals

Sample question: What’s the underlying mechanism of damage in hemochromatosis or in

Wilson’s Disease? Pathologic generation of free radicals

Drugs and chemicals

o Acetaminophen – goes to liver, gets converted by P450 and free radicals are generated

High dose acetaminophen can be taken to commit suicide via liver necrosis, hepatocytes convert

it and free radicals are generated

o CCl4 (see below)

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Free Radical Damage

Peroxidation of lipids (lipid membrane)

Oxidation of DNA and proteins

o Results in damage to the cell

Particularly important in oncogenesis

o Antioxidants fight off free radicals to prevent cancer and fight off aging

Elimination of Free Radicals

Antioxidants

Vitamin A, C, E

Enzymes

(1) Superoxide dismutase (SOD): removes superoxide

(2) Catalase: removes peroxide

(3) Glutathione peroxidase: removes hydroxyl ions

Metal carrier proteins

Copper and iron can create free radicals (iron via Fenton reaction)

o Thus, we have carrier proteins to hide the metals to free radicals aren’t generated

Transferrin in blood tightly binds Fe

Ferratin in liver tightly binds Fe

Free Radical Injury

CCl4 (carbon tetrachloride)

Found in dry cleaning industry

When in blood, converted to CCl3∙ in the P450 system of the liver

o CCl3∙ is a free radical that causes reversible damage (cell swelling)

When cell swells, rough ER will also swell and ribosomes

will pop off to reduce protein synthesis

Decrease in protein synthesis results in lack of

apolipoproteins, which remove fat from liver

Fat comes in the liver but can’t leave classic presentation = FATTY LIVER

o Image above: red circle indicates hepatocyte, white bubbles are fat cells

Reperfusion Injury

When blood supply to an organ is cut off, tissue dies and cell membrane is damaged

o Ex: coronary artery occlusion heart tissue dies enzymes (troponins) leak into blood

If blood is returned to the organ, oxygen + inflammatory cells return with it

o Inflammatory cells reacting w/dead tissue + presence of oxygen = free radicals are generated

Free radicals further damage cardiac myocytes cardiac enzymes continue to rise after opening

artery/returning blood to injured organ (classic manifestation of reperfusion injury)

Inflammation

Definition = taking cells FROM within a blood vessel and bring them OUT to the tissue (usually in response to infection)

Acute inflammation: when neutrophils leave blood vessel into tissue space

Chronic inflammation: when lymphocytes leave blood vessel and into tissue space

REMEMBER THIS:

If O2 accepts 1 e- superoxide (O2∙

-)

If O2 accepts 2 e- peroxide (H2O2

)

If O2 accepts 3 e- hydroxyl ion (∙OH)

If O2 accepts 4 e- water (H2O)

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BV BV N N

N N

E

E

Acute Inflammation

Characterized by Presence of (1) Edema and (2) Neutrophils in Tissue

Edema (E): fluid from blood vessels (BV) that fills the interstitial space

Neutrophils (N): key inflammatory cells that leave blood vessels (BV)

and get into tissue space (hallmark of acute inflammation)

Arises in Response to (1) Infection or (2) Tissue Necrosis

Goal is to eliminate pathogen or clear necrotic debris

o Neutrophils destroy by eating/consuming the infectious pathogen

Difference between necrosis v. apoptosis: necrosis is followed by acute inflammation

Ex: myocardial infarction causes increase in patients white count (↑ neutrophils)

o 12 hours after

o 24 hours after: neutrophils and acute inflammation

Immediate Response with Limited Specificity

Response is immediate, caveat is that it has limited specificity

o Generalized response to stimulus, not a specific attack to a particular antigen

Innate immunity = includes: epithelium covering body surfaces, mucous secreted by cells, complement system

(series of protein in inactive state in serum that can be activated), mast cells, macrophages (consume pathogens

and present them to further immune response), neutrophils, basophils, eosinophils; broad, nonspecific system to

defend host against microbes

Adaptive immunity = longer but more specific response; includes: lymphocytes (produce Abs and have T-cell

receptors for a specific target)

Mediated by Several Factors

Toll-like Receptors (TLRs)

Present on cells of innate immune system (macrophages and dendritic cells)

o Recognize PAMPS (pathogen associated molecular patterns) – patterns of molecules on pathogens

recognized by TLRs and lets the body know there is an invader

CD14 on macrophages recognizes PAMP called LPS on outer membrane of gram neg bacteria

TLR activation results in upregulation of NF-κB (on switch to turn on acute inflammatory response)

o Leads to production of multiple immune mediators

Present on cells of adaptive immune system

o Mediate chronic inflammation

Arachidonic Acid

Released from the phospholipid cell membrane by phospholipase A2

Acted on by (1) cyclooxygenase or (2) 5-lipooxygenase

o Cyclooxygenase produces prostaglandins (PGs)

PGI2, PGD2, PGE2 mediate vasodilation (in arteriole) and increased vascular permeability (occurs

at post-capillary venule)

PGE2 also mediates fever and pain (fEEEEEEver for PGEEEEEE2)

o 5-lipooxygenase produces leukotrienes (LTs)

LTB4 attracts and activates neutrophils

REMINDER

There are 4 key mediators for

attracting neutrophils: LTB4,

C5a, IL8, bacterial products

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LTC4, LTD4, and LTE4 mediate vasoconstriction, bronchospasm, and increased vascular

permeability

Basically, these cause smooth muscle to contract

o Smooth muscle in arteriole contracts vasoconstriction

o Smooth muscle in bronchus bronchospasm

o Pericytes (layer of cells underneath epithelium) have smooth muscle contractile

function and when they contract, they pull apart the epithelial cells allowing for

fluid to leak from post-capillary venule into the interstitial space increased

vascular permeability

Mast Cells

Widely distributed throughout connective tissue in body

Activated by:

o (1) Tissue trauma – mast cells get activated to help initiate immune response

o (2) Complement proteins: C3a and C5a activate mast cells

o (3) Cross-linking of cell-surface IgE by antigen

Mast cells express IgE on their surface, if an antigen comes by and cross-links these IgE, the mast

cell is activated

Once activated, the mast cells undergo an immediate response:

o Release of preformed histamine granules

Mediates (1) vasodilation of arterioles; (2) increased vascular permeability at postcapillary venule

After releasing the histamine granules, the mast cell undergoes a delayed response:

o Production of arachidonic acid metabolites (particularly leukotrienes)

Complement Proteins

Proinflammatory serum proteins

“Complement” inflammation

Circulate as inactive precursors

Activation can occur via three different pathways:

o (1) Classical pathway C1 binds to IgG or IgM that is bound to antigen (“GM makes classic cars”)

o (2) Alternative pathway microbial products directly activate complement

o (3) Mannose-binding lectin pathway MBL binds mannose on microorganisms to activate complement

Result of activation = generation of C3 convertase

o C3 convertase converts C3 C3a & C3b

C3b helps C5 convertase

o C5 convertase converts C5 C5a & C5b

C5b complexes with C6 and C9 to form MAC

o Formation of membrane attack complex (MAC)

Produces a hole in the membrane for lysis of the microbe

Key products of complement pathway:

o C3a & C5a: trigger mast cell degranulation

o C5a: chemotactic for neutrophils

o C3b: opsonin for phagocytosis

When neutrophils get in tissue, they consume and then destroy whatever they eat

Phagocytosis is usually blind, but opsonins can assist and tell neutrophil what to consume

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o MAC: lyses microbes by creating a hole in the cell membrane

C5b joins with C6 and C9 to make MAC

Hageman Factor

Inactive proinflammatory protein produced in liver

Activated upon exposure to subendothelial or tissue collagen

Plays an important role in DIC

Activates:

o Coagulation and fibrinolytic systems*

o Complement

o Kinin system – cleaves HMWK to bradykinin increased vascular permeability, vasodilation, & pain

Remember: pain is mediated by BRADYKININ and PGE2

Symphony of Mediators Result in Cardinal Signs of Inflammation

Redness (rubor) and Warmth (calor)

Due to vasodilation, which results in increased blood flow

Occurs via relaxation of arteriolar smooth muscle

Key mediators: histamine*, PGs, and bradykinin

Swelling (tumor)

Due to leakage of fluid from postcapillary venules into interstitial space

Key mediators: histamine*, tissue damage

Pain (dolor)

Key mediators: bradykinin, PGE2

Sensitize sensory nerve endings

Fever

Pyrogens cause macrophages to release IL-1 and TNF

Increase COX activity in perivascular cells of hypothalamus

Increased PGE2 raises temperature set point creating fever

Neutrophil Arrival and Function

Step 1: Margination

Vasodilation slows blood flow in postcapillary venules

Cells marginate from center of flow to the periphery

Step 2: Rolling

Cells that have marginated need to slowdown

o So they hit “selectins” (speed bumps) which are upregulated on endothelial cells

P-selectin is released from Weibel-Palade bodies (mediated by histamine)

Weibel-Palade bodies contain 2 important proteins: van Willebrand factor & P-selectin

E-selectin is induced by TNF and IL-1

Selectins

o Bind sialyl Lewis X on leukocytes

Three phases of acute inflammation:

(1) F: fluid phase; (2) N: neutrophil phase (after

24hr); (3) M: macrophage phase

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o Interaction results in rolling of leukocytes along vessel wall

“Leukocytes” is generic WBC, but we’re talking about neutrophils when we discuss rolling

Step 3: Adhesion

Cellular adhesion molecules (CAMs: VCAM & ICAM) upregulate on endothelium by TNF and IL-1

Integrins upregulated on leukocytes by C5a and LTB4

Interaction results in firm adhesion to vessel wall

Clinical Correlation: Leukocyte Adhesion Deficiency

Autosomal recessive defect of integrins (CD18 subunit)

No integrin = no adhesion = never going to be drawn into tissue

o Findings:

Delayed separation of umbilical cord

Increased circulating neutrophils

50% of neutrophils hang out in blood vessels in the lung

o If needed, they can come out into the blood

o Without adhesion, they float around in the blood

Recurrent bacterial infections that lacks pus formation

What is pus? Dead neutrophils sitting in fluid

If no neutrophils can get in the tissue, no pus can be formed so they will have recurrent

bacterial infections that lacks pus

Step 4: Transmigration and Chemotaxis

Leukocytes transmigrate across endothelium of POSTCAPILLARY VENULES

Move toward chemical attractants (chemotaxis)

o Neutrophils are attracted by bacterial products (IL8, C5a, LTB4)

Step 5: Phagocytosis

Consumption of pathogens or necrotic tissue

Enhanced by opsonins (IgG and C3b)

o Molecules neutrophils recognize and say “oh, this is what I am supposed to eat”

Pseudopods from leukocytes extend to form phagosomes

o Internalized and merged with lysosomes to form phagolysosomes

Lysosomes contain highly degradative enzymes to break stuff down

Clinical Correlation: Chediak-Higashi Syndrome

Protein trafficking defect (autosomal recessive)

o Microtubule defect, phagosome cannot travel to the lysosome

Characterized by impaired phagolysosome formation

Clinical features:

o Increased risk of pyogenic infections b/c organisms can’t destroy what the neutrophil has consumed

since the phagosome cannot get to lysosome (train tracks are broken)

o Neutropenia – cells cannot divide properly so you don’t get the total number you should be getting

o Giant granules in leukocytes

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Granules within the leukocytes are produced by golgi apparatus and then move out to the

periphery; however, without a railroad system, they can’t move and bunch together as one big

granule

o Defective primary hemostasis – dependent on platelets

o Albinism

Pigment of the skin occurs by melanocytes which distributes pigment to the keratinocytes

Without being able to hand them off (due to railroad system defect), no pigment goes anywhere

o Peripheral neuropathy

Railroad/protein trafficking defect, so long nerves don’t get what they need distally from their

nucleus

Step 6: Destruction of Phagocytosed Material

Can occur in two ways:

(1) oxygen dependent killing

o Most effective method

o HOCl generated by oxidative burst in phagolysosomes destroys phagocytosed microbes

o Mechanism:

O2 converted to superoxide by NADPH oxidase [aka “oxidative burst”]

Superoxide converted to hydrogen peroxide by superoxide dismutase (SOD)

Hydrogen peroxide is converted to bleach (HOCl) by myeloperoxidase (MPO)

HOCl is key molecule that destroys phagocytosed materials

Clinical Correlation: Chronic Granuloma Disease (CGD)

o Poor O2-dependent killing

o Due to NADPH oxidase defect (X-linked or autosomal recessive)

Patients cannot produce HOCl from the normal pathway

However, they CAN take H2O2 from the microbe and convert that to HOCl

o Catalase destroys H2O2 though, so catalase + bacteria will destroy its H2O2, so

that cannot be used either and HOCl will NEVER get formed

o Leads to infection and granuloma formation w/ catalase positive organisms:

S. Aureus, P. cepacia*, S. marcescens, Nocardia, Aspergillus

o Nitroblue tetrazolium test (NBT test): can we convert oxygen to superoxide?

Used to screen for CGD

Turns blue if NADPH oxidase can convert O2 to O2-

Remains colorless if NADPH oxidase is defective (ie: patients with CGD)

Clinical Correlation: MPO deficiency

o Patients cannot form HOCl b/c of an MPO deficiency (defective conversion of H2O2 to HOCl∙)

o Remain relatively asymptomatic; but are prone to candida infections

o NBT test would be normal in these patients

(2) oxygen-independent killing

o Less effective method

o Occurs via enzymes present in leukocyte secondary granules

ex: lysozyme and major basic protein

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P

L

Step 7: Resolution

Neutrophils undergo apoptosis**

o Pus = dead neutrophils within fluid

Disappear within 24 hours after resolution of inflammatory stimulus

Macrophage Predominate After Neutrophils (third phase, see p. 5)

Peak 2-3 days after inflammation begins

Derived from monocytes in blood

o Arrive via margination, rolling, adhesion, and transmigration

Ingest via phagocytosis

Destroy phagocytosed material using enzymes in secondary granules

o Lysozyme: very important

Manage the next step of the acute inflammatory response

o Resolution and healing: IL-10 and TGF-β these both shutdown the inflammatory process

o Continued acute inflammation: IL-8 cytokine produced by macrophages to call in neutrophils*

“Acute” inflammation not defined by time, it’s defined by mediators

IL-8 calls on more neutrophils, this is still considered “acute inflammation” even though it could

still be going on 6 months later

o Abscess: area of fibrosis

Macrophages can form a wall of fibrosis around area of infection so area of inflammation gets

blocked in

o Chronic inflammation

Macrophages ingest antigens of microbe and present it on MHCII which results in activation by

helper T-cells in chronic inflammation

Chronic Inflammation

Characterized by Lymphocyte and Plasma Cells in Tissue

Delayed response, but more specific (adaptive immunity)

Inflammation = getting what’s on the inside of a blood vessel into the surrounding tissue

o Acute inflammation – neutrophils go from BVs tissue

o Chronic inflammation – lymphocytes (L) (can also become plasma cells) go from BVs tissue

Cells are mononuclear, don’t have multi-

lobulated nucleus like a neutrophil

Plasma cells (P): characterized by offset clock-

face nucleus w/glossy clearing next to it

Stimuli for Chronic Inflammation

Persistent infection *most common

Infection with viruses, mycobacteria, parasites, and fungi

Autoimmune disease

Foreign material

Some cancers

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WAYS TO ACTIVATE CASPASES

Intrinsic mitochondrial pathway:

cytochrome C leaks out of

mitochondria to activate caspases

Extrinsic receptor pathway:

Fas/Fas ligand

CD8+ cytotoxic T cell:

releases granzyme to activate

caspases

Lymphocytes can be divided into T cells and B cells:

T Lymphocytes

Produced in the bone marrow as progenitor T cells

Further develop in the thymus (where the T cells “go to college”)

o TCR undergoes rearrangement

o Cells can become CD4+ helper T cells or they can “graduate” to become CD8 cytotoxic T cells

Use TCR complex for antigen surveillance

o TCR complex (complex includes CD3) recognizes antigens presented on MHC

T cells can only recognize antigens presented on MHC

CD4+ T cells MHC Class II

CD8+ T cells MHC Class I

Activation of T cells requires: (1) binding of antigen/MHC complex and (2) additional 2nd

signal

CD4+ T cell activation

o Extracellular antigen is phagocytosed, processed, and presented via MHC class II (APCs)

APCs = macrophages, dendritic cells

o 2nd

activation signal = B7 on APC binds CD28 on CD4+ T cells

o What do activated CD4+ helper T cells do?

Secrete cytokines that “help” inflammation

Divided into two subsets: one that helps B cells & one that helps CD8+ cytotoxic T cells

TH1 subset [helps the CD8+ T cells]

o IL-2: T-cell growth factor and CD8+ T cell activator

o IFN-ϒ : macrophage activator

TH2 subset [helps the B cells]

o *IL-4: class switching to IgG and IgE

o *IL-5:

Eosinophil chemotaxis and activation

Maturation of B cells plasma cells

Class switching to IgA

o IL-10: inhibits TH1 subset

CD8+ cytotoxic T cell activation

o Intracellular antigen is processed and presented on MHCI

o 2nd

activation signal = IL-2 from CD4+ TH1 cell provides

o Cytotoxic T cells are activated for killing

o What do activated CD8+ cytotoxic T cells do? KILL THEIR TARGET!

o Secretion of (to make a hole) and perforins granzyme

induces apoptosis of the target cell

[ are the enzymes that mediate apoptosis] CASPASES

o Expression of FasL binds Fas on target cell to activate apoptosis

B Lymphocytes

Immature B cells are produced in bone marrow

Undergo Ig rearrangement to become naïve B cells that express surface IgM and IgD

Activation of B cells:

o (1) antigen binding by surface IgM (which causes B cell to become plasma cell that secretes IgM) OR

o (2) B-cell consumes and presents antigen to CD4+ helper T cells via MHCII

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G

EH EH

EH

EH

L

Noncaseating granuloma. Note: EH cells have nuclei and are not

necrotic.

Caseating granuloma. Note: Big clump of dead cells in the middle

surrounded by EH cells.

2nd

activation signal = CD40 receptor on B cells binds CD40L on helper T cell

o What do activated B cells do?

Helper T cell now secretes IL-4 and IL-5

These mediate B-cell isotype switching, hypermutation, and maturation to plasma cells

so that they can secrete IgM, IgD, IgG, IgE, etc

Chronic inflammation is divided into granulomatous and non-granulomatous inflammation:

Granulomatous Inflammation

Characterized by granuloma

Key cell: *epithelioid histiocytes (EH)* (macrophages with

abundant pink cytoplasm)

o Aggregation of these cells = “granuloma”

Surrounded by giant cells (G) and rim of lymphocytes (L)

Divided into noncaseating and caseating subtypes

Noncaseating granulomas lack central necrosis.

These types of granulomas can be caused by:

Reaction to foreign material

o Ex: pt has breast cancer removed followed by breast

implants. They leak and lead to a reaction leading to

enlarged axillary lymph nodes

Sarcoidosis

o Distinguishing feature of this is noncaseating granulomas in different areas of the body (particularly in the

lung)

Beryllium exposure

Crohn’s disease

o Distinguishing feature of this is noncaseating granulomas

o NOTE: distinguishing feature of ULCERATIVE COLITIS is crypt abscesses

Cat scratch disease

o Distinguishing feature of this is noncaseating stellate-shaped granulomas in the neck

Caseating granulomas exhibit central necrosis.

These types of granulomas can be caused by

TB

o AFB stain checks for TB

Fungal infections

o GMS stain checks for fungus

Steps Involved in Granuloma Formation [both caseating AND noncaseating are formed this way!]

Macrophages present antigen via MHCII to CD4+ helper T cells

Macrophages secrete IL-12, inducing CD4+ helper cells to

differentiate into TH1 subtype

TH1 cells secrete IFN-ϒ, which converts macrophages to

epithelioid histiocytes and giant cells