Pathology week 2 – Inflammation ver.

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Inflammation
‘a protective response of vascular tissue to local injury, the role of which is to contain and isolate injury, destroy
infection and inactivate toxins in order to achieve healing and repair’
- can also be harmful (hypersensitivity, chronic inflammation → fibrosis)
- 2 components - vascular wall response and inflammatory cell response
- mediated by circulating plasma proteins and factors in vessel wall or from inflam cells
- terminated when agent eliminated and mediators removed

Acute Inflammation
‘Short duration response to injurous agent lasting mins/hrs/ days, resulting in vasodilation, exudation of protein rich
fluid and migration of leucocytes - characterised by heat, redness, oedema, pain and loss of function’
3 components:
∆ in vessel caliber → ↑ blood flow (heat and redness)
∆ in microvasculature → plasma proteins/leukocytes leak out, produce exudate (oedema)
Leukocyte emigration from vessels → accum at site of injury (pain/oedema)

Vascular changes
- normal fluid exchange dependent on Starlings Law and intact endothelium
- usually hydrostatic P (fluid out) = osmotic P (fluid in) – Starlings Law
- inflammation → vasodilation → ↑ hydrostatic P
- ↑ vascular permeability → exudation protein → ↓ osmotic P
- total net effect outflow/extravasation
- stasis secondary to ↑ [RBC] and ↑ viscosity → WBC accumulation along endothelium (margination) →
emigration through vessel wall

Exudate = escape fluid, protein, blood cells from vessel into interstitium or body cavity - high prot, sp. gravity >1.020
Transudate - low prot, sp. gravity < 1.020
Purulent (pus) – inflammatory exudate rich in leucos and cell debris

Changes in vascular flow

1. transient vasoconstriction (secs)
2. vasodilation arterioles and capillary beds (histamine, NO) → ↑ flow
3. stasis (increased vascular permeability and outpouring of protein rich fluid)
4. leucocyte margination

Migration/extravasation of leukocytes
1. endothelial activation
a. mediators @ inflammatory sites cause ↑ expression E-selectins and P-selectins
i. histamine, thrombin, PAF, IL1, TNF
2. margination
3. rolling
a. tumble along, adhere transiently to endothelium
b. initial loose adhesion due to interaction between:
i. E-selectins and Sialyl Lewis X; P-selectins and modified glycoprotein
4. leukocyte activation and firm adhesion
a. leucos activated by chemokines (histamine, thrombin, PAF, IL1, TNF) → ↑ avidity of integrins
i. eg β2 integrin ↑ avidity from ICAM-1
ii. pavementing = endothelium lined by WBCs
5. transmigration across endothelium = diapedesis
a. mostly in venules and in lungs, through intercellular junctions
b. mediated by:
i. CD32 (PECAM-1) on WBC and endothelium
1. neutrophils predominate in 1st 5-24 hrs, monocytes in 24-48 hrs
2. neutrophils remain 2-4 days in pseudomonas
3. lymphocytes first to arrive in viral infections
4. eosinophils first to arrive in hypersensitivity reactions
6. chemotaxis
- locomotion along chemical gradient which allows accum neutrophils at site of injury
i. exogenous chemoattractants – bacterial products
ii. endogenous chemoattractants – complement esp C5a, lipoxygenase pathway esp leukotriene
B4, cytokines esp chemokines eg IL8

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Mechanisms of increased vascular permeability
1. Gaps due to endothelial cell contraction
Venules
Mediated by histamine, leukotrienes, bradykinin, substance P, LTC4, D4, E4
Immediate-transient response (<30mins)
2. Cytoskeletal reorganization (endothelial cell retraction)
Endothelial cells undergo structural reorganization of cytoskeleton
Mediated by IL1, TNF, hypoxia, direct injury
Delayed by 4-6 hrs, lasts >24hrs
2. Direct injury and endothelial necrosis
Arterioles, capillaries, venules
Results in endothelial cell necrosis/detachment, neutrophil recruitment, often plt attachment
Toxins, burns, chemicals
Immediate-sustained (hrs-days)
3. Leukocyte dependent injury
Venules, glomerular and pulmonary capillaries
Activated leucocytes release toxic O2 metabolites/enzymes that cause endothelial injury
Delayed, prolonged response
4. Increased transcytosis
Venules
Mediated by VEGF (vascular endothelial growth factor)
Occurs across channels consisting of interconnected clustered vesicles that form a vesiculovacuolar organelle
5. New blood vessel formation and leakage
Sites of angiogenesis
Persist until intercellular junctions form

Leukocyte adhesion
Determined by:
- binding of complementary adhesion molecules on leuco and endothelial surfaces
- chemical mediators (chemoattractants and cytokines) which modulate the surface expression or avidity of such
adhesion molecules
4 molecular families of adhesion receptors:
1. Selectins (E, P, L) bind via lectin to oligosaccharides on cell surface glycoproteins
2. Immunoglobulins – endothelial adhesion molecules which interact with integrins on WBCs
ICAM 1 = intracellular
VCAM 1 = vascular cell
3. Integrins – transmembrane adhesive heterodimeric glycoproteins made up of α and β chains
4. Mucin-like glycoproteins eg heparan sulphate

Mechanisms by which adhesion receptors are modulated to induce adhesion

1. redistribution of adhesion molecules to cell surface
a. P selectin on membrane Weibel-Palade bodies (intracytoplasmic endothel granules)
2. cytokine induction of endothelial adhesion molecules (IL1, TNF) – 1-2 hr delay
3. ↑ avidity of binding of integrins

Leukocyte activation
Activated by:
- bacterial products
- complement (C5a)
- leukotrienes (LTB4)
- cytokines (IL8)
TNF acts as primer by allowing increased stimulation of leucocyte by other agents
Biochemical events in leukocyte activation:
- receptor-ligand binding
- phospholipase C activation
- increased intracellular calcium
- activation of protein kinase C
Activated leucocytes respond by:
- chemotaxis (raised intracel Ca stimulates assembly of intracellular actin and myosin filaments)
- increased number and affinity of adhesion molecules
- production of arachidonic acid metabolites
- degranulation
- activation of oxidative burst

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Phagocytosis
3 steps:
1. Recognition and attachment:
- opsonisation of particles such as bacteria enhances efficiency of phagocytosis
- major opsonins: Fc fragment of IgG, C3b
- collectins – CHO binding proteins in plasma that bind to microbial walls

2. Engulfment:
- binding of opsonised particle triggers engulfment
- pseudopods flow around particle resulting in complete enclosure to form a phagosome
- membrane of phagosome fuses with lysosomal granule → discharge of contents into phagosome

3. Killing or degradation:
- oxygen dependent:
- phagocytosis stimulates O2 burst, glycogenolysis, production of reactive O2 metabolites
- generation oxygen metabolites due to rapid activation NADPH oxidase reduces oxygen to superoxide
- superoxide dissociates to hydrogen peroxide
- neutrophils also contain myeloperoxidase, which converts hydrogen peroxide to HOCl, which destroys
bacteria by halogenation – more effective than hydrogen peroxide
- dead microbes degraded by lysosomal hydrolases
- oxygen independent:
- via actions of substances in leukocyte granules
- bacteriacidal permeability incr protein (BPIP), lysozyme, lactoferrin, major basic protein, defensins

Neutrophilic substances that are mediators of endothelial injury

- mediators of endothelial injury, tissue damage, amplify effects of initial stimulus
o lysosomal enzymes (in granules)
o O2-derived active metabolites
o products of arachidonic acid metabolism incl PGs and leukotrienes
- examples of leukocyte-induced injury:
o acute: ARDS, acute rejection, asthma, GN, reperfusion, septic shock, vasculitis
o chronic: arthritis, asthma, atherosclerosis, chronic lung disease, chronic rejection

Termination of acute inflammation

- mediators produced in short burst, short half life
- anti-inflammatory arachidonic metabolites
- anti-inflammatory cytokines (transforming growth factor β – TGF-β)

Chemical mediators of inflammation

Arise from plasma or cells.
Cell derived mediators pre-formed or newly synthesized.
Can stimulate release of further mediators and therefore amplify a response.
Most short-lived.

Cell preformed mediators

Vasoactive amines – first mediators to be released in inflammation
Histamine, serotonin

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 Histamine
formed by decarboxylation L-histidine by histidine decarboxylase
once formed histamine either stored or rapidly deactivated.
found in mast cells, basophils, platelets
Serotonin
formed by decarboxylation of aa L tryptophan.
once formed serotonin either stored or rapidly deactivated
90% found in enterochromaffin cells of GIT
also in platelets and mast cells.
Neurotransmitter actions in brain control mood, sleep, appetite,
temperature regulation.
Also found in enteric nervous system.
Metabolised by monoamine oxidases.

Release from mast cells (degranulation) caused by

Physical injury, IgE reactions, C5a, C3a (anaphylotoxins), Substance P, IL1, IL8
Release from platelets caused by
Collagen, thrombin, ADP, PAF, Antigen-antibody complexes
Actions
Histamine
Combines with specific H1 H2, H3 receptors
Vasodilation (but constricts large arteries).
Vascular leakage (causes venular gaps).
Smooth muscle contraction (GIT, bronchi)
Stimulates gastric acid secretion (plus small and large intestine).
Stimulates nerve endings – pain and itch.

Serotonin
Combines with specific 5HT receptors (7 major subtypes).
Smooth muscle contraction (GIT, bronchi).
Vasodilation - especially skeletal muscle and heart (constricts large arteries).
Direct chronotropic and inotropic effect (clinically insignificant).
Prokinetic action on GIT 5HT3 receptors control vomiting reflex.
Stimulates nerve endings – pain and itch.
Hypotension and bradycardia via chemoreceptor (Bezold Jarish) reflex.
Lysosomal contents
Source
Neutrophils, macrophages
2 major types of granules:
Specific (secondary)
Primarily release contents extracellularly
Lactoferrin, lysozyme, alk phos, collagenase, leukocyte adhesion
molecules, plasminogen activator, phospholipase A2

Azurophilic (primary)
Primarily release their contents into phagosomes
Myeloperoxidase, acid hydrolases, neutral proteases, elastase
Cationic proteins, phospholipase A2

Note that proteases are kept in check by serum antiproteases

Cell newly-formed mediators

Arachidonic acid metabolites (eicosanoids)

Arachidonic acid released from membrane phospholipids by action of phospholipase A2, mechanical
or chemical stimuli or other mediators (C5a).
Arachidonic acid metabolized by 2 classes of enzymes.

Cyclooxygenase pathway (mediated by COX1 and COX2)

PGD2, PGE2 PGF2alpha
Vasodilation and oedema
PGI2 (prostacyclin)

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 Decreased platelet aggregation
Vasodilation
Primarily made by endothelial cells.
PGG2
PGH2
Thromboxane A2
Platelet aggregation
Vasoconstriction.

5-Lipoxygenase Pathway
Leukotriene A4
Leukotriene B4
Potent chemotaxic agent and neutrophil activator
Leukotriene C4, D4, E4
Vascular leakage, vasoconstriction, Bronchoconstriction (1000 times more potent
than histamine)

Inflammatory Actions of Eicosanoids

Action Metabolite
Vasoconstriction Thromboxane A2, leukotrienes C4, D4, E4
Vasodilation PGI2, PGE1, PGE2, PGD2
Increased vascular permeability Leukotrienes C4, D4, E4
Chemotaxis, leukocyte adhesion Leukotriene B4, HETE, lipoxins

Platelet activating factor

Formed from phospholipid by action of phospholipase A2
Source
Mast cells, platelets, basophils, leucocytes, endothelium
Actions
Stimulates plt to produce TxA2 from arachidonic acid resulting in plt aggregation
Bronchoconstriction
Vasoconstriction
Low dose vasodilation and incr vasc perm - 100-10000x more potent than histamine
Leucocyte activation

Cytokines

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 Polypeptide short acting mediators of acute/chronic inflamm and immune response.
Autocrine, paracrine, endocrine effects and exert action by binding specific receptors
The term ‘cytokine’ inclusive of monokines, lymphokines, colony stimulating factors, interleukins,
chemokines and growth factors
Source
Macrophages, lymphocytes, endothelium, connective tissue.
Actions
Mediation of natural immunity
IL1, TNF
Regulation of lymphocyte function
IL2, IL4, IL10, TGF
Activatation of inflammatory cells
IL5, IL10, IL12, TNF, IFN
Chemotaxis
Stimulation of haematopoesis
IL3, IL7, GM-CSF, M-CSF, G-CSF

IL1 and TNF most important in regulating acute inflammation.

Bacterial products/immune complexes/toxins/injury/cytokines activate macrophages – production
IL1/TNF
Actions include
Endothelial activation
Leuko adherence, PGI synthesis, ↑procoagulant activity, ↓anticoagulant
activity, ↑IL1/IL8/IL6/PDGF
Fibroblast activation
↑ proliferation, ↑ collagen synthesis, ↑ collagenase, ↑ protease, ↑ PGE
synthesis
Leucocyte activation
↑ IL1/IL6
Acute phase response
Fever, neutrophilia, anorexia, ↑slow-wave sleep, haemodynamic effects of
septic shock

Nitric Oxide
Gas produced by endothelial cells, macrophages and neurons.
Synthesised from L-arginine, oxygen and NADPH
Acts via increase in cGMP which in turn causes smooth muscle relaxation
Plays an important role in vascular function during inflammation
Actions
Potent vasodilator
Decreases platelet adhesion and aggregation.
Inhibits mast cell induced inflammatory effect
Reduces leucocyte recruitment by inhibiting rolling and adhesion. Antimicrobial
action due to interaction between NO and reactive oxygen metabolites

Oxygen derived free radicals

Oxygen derived free radicals released from leukocytes after exposure to chemotactic agents, immune
complex or as result of phagocytosis; dependent on NADPH system

Oxygen derived free radicals include

Superoxide, hydrogen peroxide, hydroxyl radicals
Actions
Increased release of chemokines
Endothelial activation
Endothelial cell damage and increased vascular permeability.
Inactivation of antiproteases
Injury to specific cell types
Red cells, tumour cells, parenchymal cells

Free radicals detoxified by antioxidants:

Ceruloplasmin, transferring, superoxide dismutase, catalase, glutathione peroxidase

Neuropeptides

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 Role in initiation of inflammatory response
Substance P
Transmission pain signals, regulate BP, stimulate immune cells, mediator
of increasing vascular permeability, plasma influx, amplification of inflam
response

Plasma derived mediators (mainly synthesized by the liver)

Complement system
Complement system consists circulating proteins that when activated result in formation of products important
for innate and adaptive immunity
Activation of complement system occurs via classic or alternative pathway
Most critical step is cleavage of C3
Culminates in lysing of microorganisms by membrane-attack complex (MAC)
Also causes:
Increased vascular permeability
Chemotaxis
Opsonization
Components present in plasma as inactive forms C1-C9

Classic pathway
Triggered by Ag-Ab complexes that activate C1
produce C4a and C4b2a from C4 and C2
C4b2a (C3 convertase) and C3b combine to form C4b2a3b (C5 convertase)
Alternative pathway
Triggered by endotoxin from microbial surfaces, complex polysaccharides, aggregated
globulins combine with properdin system (factors B and D) plus C3b to form C3bBb
(alternative C3 convertase)
Combined pathway
C4b2a (C3 convertase) and C3bBb (alternative C3 convertase) both cleave C3 to form C3a
and C3b
C3b binds to classical and alternative C3 convertase to form C5 convertase, which cleaves
C5 to C5a and C5b – initiates assembly of MAC

Important products:
C3a, C5a (anaphylatoxins)

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 Increases vascular permeability
C5a activates lipoxygenase pathway, is a powerful chemotactic agent and leucocyte
activator
Vasodilation due to histamine release from mast cells
C3b (opsonin)
Enchances phagocytosis
C5b-9 (membrane attack complex)
Lyses cells
Stimulates arachidonic acid metabolism and production of toxic oxygen metabolites
in leucocytes
Complement inhibitors
Complement assembly is closely controlled by protein inhibitors
Protects cells against inappropriate lysis
Decay accelerating factor - regulates C3 and C5 convertases
Factor I – proteolytically cleave C3b
C1 inhibitor – inhibits C1 binding to immune complex
Deficiency leads to angioneurotic oedema

Kinin system
Trigger – exposure to collagen fibres (or any electronegative surface), or trauma to blood
Activation of factor XII catalysed by Kallikrein and HMW kininogen
XIIa converts prekallikrein to kallikrein.
Kallikrein cleaves HMW kininogen to produce bradykinin.
Kallikrein activates Hageman factor – autocatalytic loop
Bradykinin causes vasodilation, ↑vascular perm, contraction smooth muscle, pain

Clotting system
Intrinsic pathway activated by Hageman factor (XII), circulates in inactive form
Contact between Hageman factor and exposed collagen results in activation of factor XII and
conversion of prothrombin to thrombin (IIa) and fibrinogen to fibrin (insol)
Fibrinopeptides also produced - increase vascular permeability and are chemotactic.
Factor Xa also acts by increasing vascular permeability.

Activated Hageman Factor (XIIa) initiates 4 systems involved in inflammation:

1. kinin system (vasoactive kinins)
2. clotting system (thrombin, fibrinopeptides, factor X)
3. fibrinolytic system (plasmin and degrades fibrin)
4. complement system (anaphylatoxins)

Mediator Summary
Vasodilation
Prostaglandins, NO, Histamine
Vascular leakage

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 Vasoactive amines, C3a/C5a, Bradykinin, Leukotriene C4/D4/E4, PAF, Substance P
Chemotaxis, leucocyte activation
C5a, Leukotriene B4, Chemokines, IL1, TNF, Bacterial products
Fever
IL1, TNF, Prostaglandins
Pain and itch
Histamine, Serotonin, Prostaglandins, Bradykinin

Outcomes of inflammation
Depends on:
Nature/intensity of injury
Site/tissue affected
Responsiveness of host
1. Resolution
Usual outcome if injury limited, short-lived, little tissue damage
Involves
Neutralization/decay of chemical mediators
Return of normal vascular permeability
Drainage of oedema fluid into lymphatics or by pinocytosis
Phagocytosis of apoptotic macrophages
Necrotic debris cleared by macrophages
Disposal of macrophages
2. Abscess Formation
Esp in infections with pyogenic macrophages
3. Fibrosis/healing
Involves regeneration and fibrosis
Occurs after:
Substantial tissue damage
If tissues can’t regenerate
Abundant fibrin exudate
4. Chronic Inflammation
If acute inflammatory response not resolved due to:
Persistent injurious agent
Interference with normal healing

Chronic inflammation
‘Inflammation of prolonged duration (weeks or months) in which active inflammation, tissue destruction and
attempts at healing and repair are proceeding simultaneously’

Causes
Persistent infection - TB, Treponema pallidum
Prolonged exposure to endogenous or exogenous toxic agents - Silica
Autoimmunity - Rheumatoid arthritis

Features
Infiltration by macrophages, lymphocytes, plasma cells, eosinophils.
Tissue destruction
Connective tissue replacement, angiogenesis and fibrosis

Monocytes/macrophages
Macrophages predominant cell type in acute inflammation after they replace neutrophils (first 48 hours)
Also most important cell type in chronic inflammation
Migration of monocytes analogous to migration of neutrophils in acute inflammation
Monocytes enter blood from bone marrow
Circulate for 24 hours then become tissue macrophages, persist for about 3 months.
Macrophages differentiate into:
Kuppfer cells
Pulmonary alveolar macrophages
Microglia
Osteoclasts
Functions:

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 Antigen presentation to T cells
Phagocytosis
Activated macrophages migrate in response to chemotactic stimuli to engulf and kill
bacteria
Also phagocytose bacteria opsonised by IgG or C3b

As well as antigen presentation and phagocytosis, macrophages can be activated by

Cytokines (especially gamma interferon) from T lymphocytes
Bacterial endotoxin
Extracellular matrix proteins
Other chemical mediators

Activated macrophages secrete a wide variety of products that result in tissue injury and fibrosis
Tissue injury
Reactive oxygen metabolites
Proteases
Neutrophil chemotactic factors
Coagulation factors
Arachidonic acid metabolites
NO
Fibrosis
Growth factors
Cytokines
Angiogenesis factors
Collagenases (remodeling function)

If inflammation persists, macrophage numbers are maintained by continuing recruitment, local

proliferation and immobilization of macrophages.

Granulomatous inflammation
‘a distinct form of chronic inflammation in which the predominant cell type is an activated macrophage with a
modified epithelioid appearance’

Granuloma
‘a focal area of granulomatous inflammation consisting of a microscopic aggregation of macrophages,
langhans cells and surrounding lymphocytes and plasma cells.
There may also be an enclosing rim of fibroblasts’

2 types
Foreign body (silica)
Immune
Tuberculosis
TB is unique in that the granuloma (tubercle) has a central area of
caseating necrosis
Leprosy, Syphilis, Sarcoid, Lymphogranuloma inguinale, Brucellosis, Berylliosis

Other cells in chronic inflammation

4 other types: lymphocytes, mast cells, eosinophils, neutrophils

Macrophage-lymphocyte interactions in chronic inflammation

Morphological patterns in acute and chronic inflammation

Severity, cause and site of inflammation will decide morphology.

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Serous inflammation
Tissue fluid accumulation, indicates modest increased vascular permeability
In pericardial/peritoneal/pleural cavities called effusion; burn blisters
Fibrinous inflammation
More marked vascular permeability, exudate with large amounts of fibrinogen
Converted to fibrin thru coag system
Involves serosal surfaces – fibrinous pericarditis or pleuritis
Suppurative/Purulent inflammation
Purulent exudate: pus – neutrophils, necrotic cells, oedema.
Ulcers
Local erosions epithelium by sloughing of inflamed necrotic tissue eg gastric, venous

Systemic effects of inflammation

Acute phase response - IL1 and TNF play a key role
Endocrine and metabolic - Secretion of acute phase proteins by liver:
CRP, Serum amyloid A and P, Complement, Clotting factors.
CRP, amyloid A and P act as opsonins
Autonomic - Redirection of blood flow from skin to deep vascular beds
Behavioural - Rigors, anorexia, somnolence, malaise
Fever
Results in increased immune efficiency, may reduce replication rate of infecting agent
Peripheral cytokines signal hypothalamus and vasomotor centre to cause peripheral vasoconstriction
Leukocytosis
Bacteria – neutrophilia
EBV, mumps, german measles – lymphocytosis
Asthma, parasites – eosinophilia
Typhoid, viruses, rickettsiae, protozoa – leukopenia

Consequences of defective or excessive inflammation

- defective – increased risk infection, delayed wound healing
- excessive – allergies, autoimmune diseases, atherosclerosis, Alzheimers

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