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Reference Texts:
Pathologic Basis of Veterinary Disease, Zachary & McGavin (eds), 5th ed, 2012, Chapters 3.
Pathologic Basis of Disease, 8th ed, Kumar et al (eds), Chapters 2 & 3.
Goals:
1. Recognize, describe and interpret morphologic changes associated with inflammation (gross &
microscopic).
2. Associate the appearance of given lesion with its probable etiology (cause).
3. Learn the pathogenesis (mechanisms) of inflammatory disease processes.
INFLAMMATION
"Inflammation is one of the most important and most useful of our host defense mechanisms, and without an
adequate inflammatory response none of us or our patients would be living. Ironically it is also one of the most
common means whereby our own tissues are injured." (Slauson & Cooper, 2002)
Inflammation (literally, “burning”): is the reaction of vascularized living tissues to local injury, it comprises a
series of changes in the terminal vascular bed, in blood and in connective tissues to eliminate the offending
irritant and repair the damaged tissue.
It involves cellular / tissue, humoral and chemical (eg cytokines) participants.
The role of inflammation is to protect the body, contain and isolate injurious agents (destroy invading
organisms, inactivate toxins, etc.) and finally, achieve healing and repair.
Note: - It is very common to have some degree of necrosis in areas of inflammation and vice versa.
- Necrosis can not only occur within primary inflammation (with certain severe injurious agents); but
necrosis that is not initially the result of inflammation (eg infarction) will invariably incite a secondary
inflammatory response.
10 Generalities Regarding the Inflammatory Response: (adapted from Slauson and Cooper)
1. Inflammation is a process involving multiple participants.
2. Inflammation occurs only in living tissue.
3. It is a series of events which overlap and form a continuum.
4. It is a response to an initiating event.
5. It can be harmful.
6. It is primarily a defensive reaction (survival oriented).
7. It is fairly stereotypical in its early stages, regardless of the nature of the irritant.
8. Many components are found in the blood stream.
9. There are multiple overlapping pathways (ie. redundancies)
10. It is a “surface phenomenon” - cell membrane changes or signals are important.
Classification of Inflammation
Clinical example: A client presents you with a dead pigeon the necropsy reveals the presence of a fibrinous exudate
on the serosal surfaces and the air sac walls - this would indicate an acute process furthermore, the type of exudate
and the extensive nature of it would suggest a bacterial infection, very likely a chlamydial infection once it is examined
microscopically, the exudate is better characterized as being composed of macrophages and heterophils, which supports
the etiology of chlamydiosis when intracytoplasmic inclusions are noted, the etiology is further supported the
inclusions stain positively with Macchiavello’s stain, adding support to their identification as Chlamydophila sp to
confirm the infectious agent, an immunohistochemical stain is employed, identifying the organism as Chlamydophila
psittaci.
Necrosuppurative Chronic-
Hepatitis (Segmental)
active
Encephalitis Granulomatous Diffuse
Dermatitis Hemorrhagic
[*note: pneumonia is the term typically used for inflammation of the lung, not "pneumonitis"]
2) Inflammatory Exudates
The inflammatory process can be classified according to the predominant type of inflammatory cells (eg
neutrophils) and/or the kind of fluid or material present (eg fibrin, hemorrhage, etc).
a) Fibrinous Exudate
Accumulation of fibrin, resulting from increased vascular permeability (inflammatory edema) due to injury to
the endothelium and basement membranes and subsequent leakage of plasma proteins, including
fibrinogen.
Fibrinogen polymerizes perivascularly to form fibrin and is thus found within inflamed tissue or in body
cavities.
Time: acute process, it can form in minutes (and can persist for days)
Gross: yellow-white, or pale tan, stringy / elastic, shaggy meshwork (or fibrillar material) that gives a rough
irregular appearance to the tissue surfaces.
Casts of this friable material may form in the lumen of tubular organs (diphtheric pseudomembranes).
Fibrin can easily be broken apart and pulled from the underlying tissue.
Histo: thread-like eosinophilic meshwork or masses of solid amorphous eosinophilic material.
Outcome: - Small amounts of fibrin can be dissolved by enzymatic fibrinolysis or phagocytosed by
macrophages.
- When in large amounts, fibrin provides the support for the eventual growth of fibroblasts & new
capillaries (ie granulation tissue).
Do not confuse fibrinous exudate with fibrosis. A fibrinous exudate is indicative of an acute
process while fibrosis is a chronic process & occurs when fibroblasts synthesize and secrete
collagen (scar formation).
b) Necrotizing Inflammation
Characterized primarily by necrosis, with usually only small amounts of vascular and leukocyte
contributions.
Histo: severe necrosis (tissue destruction) with only scant evidence of vascular or leukocytic contributions.
Etiology: often associated with ischemia or in association with toxin-producing bacterial infection.
Example: Dry gangrene, Blackleg (Clostridium chauvoei), bacterial or viral hepatitis, etc.
f) Serous Exudation
Accumulation of fluid relatively rich in protein on body surfaces with little cellular infiltrate.
It can be the dominant pattern of exudation for a wide variety of mild injuries.
Time: usually peracute (minutes) to acute (hrs / few days).
Gross: pale yellow to transparent fluid, somewhat viscous.
Examples: traumatic blisters or a “runny” nose because of the cold.
g) Mucoid Exudate
Consists of mucus as well as variable numbers of inflammatory cells.
Gross: “Snotty”
Catarrhal inflammation: inflammation of a mucous membrane with marked increased flow of mucous
and/or exudate (mucoid or mucopurulent exudate).
h) Eosinophilic Inflammation
Eosinophils are the primary inflammatory cell type present.
In some cases it is possible to diagnose grossly because eosinophils possess granules which give the
affected tissue a green tinge.
Etiology: usually parasites or hypersensitivity reactions.
Examples: eosinophilic dermatitis due to flea bites.
INFLAMMATION AND REPAIR 7 Winter 2013
i) Non-Suppurative Inflammation
This is a microscopic diagnosis; mononuclear cells predominate, esp. lymphocytes and plasma cells.
Used mostly for reactions to viral infections in the brain (ie non-suppurative encephalitis).
Lymphocytic inflammation: lymphocytes are the predominant inflammatory cell type (eg lymphocytic
thyroiditis).
3) Distribution
Indicates the location of the lesion within an organ and, indirectly,
how much of it is affected.
Used both macro- and microscopically.
Focal: single abnormality or inflamed area within a tissue.
Multifocal: each focus of inflammation is separated from other
inflamed foci by an intervening zone of relatively normal tissue.
(when many crowded together can say “multifocal to coalescing”)
Locally (focally) extensive: a significant portion of an organ.
(“Segmental” often used for tubular organs, eg. segmental enteritis)
Diffuse: the entire organ (usually a viral or toxic cause).
4) Duration
Indicates how long the process has been underway.
Determination of duration can be very subjective; the morphologic
changes associated with an inflammatory process may not correlate with the onset of clinical signs; for
example, the high functional reserve of the liver and kidney allow for severe chronic lesions in these organs
in animals that die suddenly.
In general, duration is classified as peracute, acute, subacute and chronic.
Chronic-active is a debatable term, not liked by all pathologists.
a) Peracute inflammation
Very acute (very recent); usually caused by a potent stimulus.
Often there are few morphologic changes, as there is insufficient time to respond to the insult.
It is less common than acute inflammatory disease processes.
Time: 0-4 hours
Vascular involvement: hyperemia, slight edema, often hemorrhage.
Lymphatics: may be filled with edema fluid &/or fibrin.
Inflammatory cells: not usually numerous (ie few leukocytes).
Clinical signs: if highly pathogenic agent can see shock / sudden death, with few other signs.
Examples: bee sting, highly pathogenic virus.
b) Acute Inflammation
Has a short and often severe course.
Time: begins within 4-6 hours can last for 3-5 days.
Vascular Involvement: active hyperemia, edema (due to endothelial changes/damage of lymphatics and
small blood vessels), occasional fibrin thrombi within vessels.
Lymphatics: often filled with exudate and edema (they have an important role in exudate removal)
Inflammatory cells: neutrophils (suppurative exudate) usually predominate, but some mononuclear cells
(macrophages / lymphocytes / plasma cells) can be present.
Clinical signs: most associated with classical signs of inflammation: warm, red, swollen, painful, loss of
function.
c) Subacute inflammation
Transition period separating acute and chronic inflammation.
Time: from a few days to ~1 week.
Vascular Involvement:
Decline in the magnitude of vascular changes compared to acute inflammation (less hemorrhage,
hyperemia and edema) since endothelial damage repaired.
INFLAMMATION AND REPAIR 8 Winter 2013
No evidence of repair (ie fibrosis and angiogenesis) which is typical found in chronic inflammation.
Lymphatics: increased lymphatic drainage.
Inflammatory cells: characterised by a "mixed" or "pleocellular" inflammatory infiltrate; often predominately
lymphocytes & plasma cells with variable numbers of macrophages and fewer neutrophils.
d) Chronic Inflammation
Persists over a period of time (wks to months).
May follow an acute inflammatory response (if host fails to completely remove inciting stimulus) or develops
as an insidious, low-grade, subclinical process without history of a prior acute episode.
Time: variable, usually over 1 to 2 weeks to months/years.
Vascular involvement: proliferations of capillaries and small blood vessels (ie angiogenesis/
neovascularization).
Lymphatics: variable involvement; +/- proliferation and activation.
Histo: primarily see mononuclear inflammatory cells (macrophages, lymphocytes and plasma cells)
Macrophages: for phagocytosis and tissue debridement; can be in the form of “epithelioid” macrophages or
multinucleated giant cells.
Usually see evidence of repair; esp fibrosis and angiogenesis (granulation tissue / scar formation).
Clinical signs: primary dependent upon duration of illness and the location / severity of the inflammatory
lesions.
Note: many changes represented in chronic inflammation are also seen in areas of REPAIR.
e) Chronic-active Inflammation
Tissues exhibit all of the usual characteristics of chronicity, with superimposed features of acute
inflammation.
Can be due to repeated overlapping episodes of inflammation; usually because the host has failed to
adequately contain the inciting agent.
Time: chronic time frame (weeks to months) with exacerbations (acute episodes).
Vascular Involvement: can have vascular changes of both acute and chronic (neovascularization)
Lymphatics: may be inflamed.
Histo: both neutrophils and cells of chronic inflammation (ie mononuclear inflammatory cells) along with
fibrosis and angiogenesis.
Clinical signs: variable.
5) SEVERITY
The severity (extent) of the process must be evaluated; but recognize it is somewhat subjective.
Moderate Some Present Readily observed Moderate edema & evidence of hemorrhage
NOTE: other modifiers of severity are sometimes used, eg, extensive or marked (for severe); minimal (less
than mild), etc.
INFLAMMATION AND REPAIR 9 Winter 2013
ACUTE INFLAMMATION
3) Blood flow slows (stasis) and erythrocytes concentrated in capillaries and veins because of fluid loss
►RUBOR
4) Cellular events:
a) margination / rolling / adhesion of WBC’s (white blood cells) in capillaries and venules
b) emigration of WBC from post-capillary venules into tissue (exudation)
c) accumulation of WBC at sites of injury ►TUMOR
d) activation of inflammatory cells and production of chemical mediators ►DOLOR
e) removal of stimulus
Transudates are the result of increased hydrostatic pressure or decreased colloid osmotic pressure in the vasculature;
the fluid has low protein and cellular contents.
Exudates are fluids with more protein and more white cells that escape to the extravascular space when endothelial
gaps are opened or endothelial cells are damaged.
Modified transudate is an in between fluid (somewhat controversial); in Feline Infectious Peritonitis the fluid is protein
rich but cell poor.
INFLAMMATION AND REPAIR 12 Winter 2013
Cellular events are required to deliver leukocytes to the site of inflammation (contributing to the exudate) so
they can internalize pathogens through phagocytosis and kill or digest them by releasing proteolytic
enzymes, chemical mediators and reactive O2 species.
The main inflammatory cells are polymorphonuclear leukocytes (neutrophils/heterophils, eosinophils,
basophils), mast cells, mononuclear cells (monocytes/macrophages, lymphocytes, plasma cells), and
platelets.
Most cells, except for plasma cells, macrophages & mast cells, are normal inhabitants of the circulating
blood.
The total leukocyte (WBC) count in peripheral blood and the relative proportions of different white blood cells
may be greatly modified in the systemic response to inflammation and can, therefore, be used as a
diagnostic tool.
They enter into the inflammatory response in a definite sequence and each cell type plays a fairly distinctive
role, but some cells can have redundant functions.
Neutrophil granules
i) Azurophil Granules (primary granules): myeloperoxidase, lysozyme, elastase, etc.
ii) Specific Granules (secondary granules): leukocyte adhesion molecules, lysozyme, histaminase, etc.
iii) Tertiary granules (gelatinase granules): gelatinase, lysozyme, leukocyte adhesion molecules, etc.
Functions of neutrophils
i) Phagocytosis:
Ingest material (opsonized by C3b and Ig), neutralize & destroy it through the following mechanisms:
o production of oxygen free radicals
o hydrogen peroxide
o lysosomal enzymes
Non-opsonized material can also be ingested, but in a less efficient manner.
ii) Mediate tissue injury: via release of O2 free radicals and lysosomal enzymes into the tissue.
iii) Regulate inflammatory response: via releasing mediators (eg leukotrienes, platelet activating factor).
INFLAMMATION AND REPAIR 13 Winter 2013
*Heterophils
Heterophils are the equivalent of neutrophils in some species (eg rabbits, guinea pigs, rats, reptiles, fish
and birds), but they do not contain myeloperoxidase in their granules.
Differentiating heterophils from eosinophils is difficult because they have prominent eosinophilic
granules and thus resemble eosinophils.
Eosinophil granules
Vary in size depending on the species but all stain with acid dye eosin - hence their name.
The main components of the granules are:
Major basic protein
o Parasite (helminth) killing
o Induce histamine release from mast cells Pro-inflammatory
Eosinophil cationic protein
o Parasite (helminth) killing
o Shortens coagulation time and alters fibrinolysis
Histaminase: inactivates histamine Anti-inflammatory
Functions of eosinophils
i. Kill or damage helminths and other pathogens by antibody-dependent cell-mediated cytotoxicity.
ii. Cause and assist in hypersensitivity reactions, especially Type I hypersensitivities.
iii. Regulate inflammation, particularly to mast cell products.
iv. Phagocytosis, but much less than neutrophils.
Functions of macrophages:
i. Phagocytic and antimicrobial (oxygen radicals, lysozyme, etc).
ii. Recruit other leukocytes (chemokines / cytokines).
iii. Stimulate or modulate other cell activity (vascular effects).
iv. Clean up debris (host and foreign).
v. Source of epithelioid macrophages and multinucleated giant cells.
2) Endothelial Cells
Endothelial cells are the source of many pro-inflammatory substances; most important are Prostacyclin,
Prostaglandins (PGE2 and PGF2), PAF (platelet activating factor), IL-1, IL-8 and nitric oxide.
They also express adhesion molecules which allow leukocyte adhesion and migration to occur (see
next section).
They can also down-regulate the inflammatory response through production of TGF-β (anti-
inflammatory, pro-repair).
3) Fibroblasts
Fibroblasts produce IL-6 (an inflammatory interleukin that stimulates B and T cell proliferation) and
TGF-β (a mediator that down-regulates inflammation and promotes repair).
INFLAMMATION AND REPAIR 15 Winter 2013
1) Margination
2) Rolling & Adhesion Leukocyte adhesion cascade
3) Emigration
4) Chemotaxis
5) Phagocytosis and intracellular killing / degradation
6) Extracellular release of leukocyte products
7) Synthesis of chemical mediators of inflammation
Integrins
Selectins
1) Margination
Slowing and stagnation of the flow occurs due to vasodilation and increased vascular permeability.
Leukocytes fall out of the central column and tumble slowly to the periphery of the vascular lumen, until
they come in contact with the surface of endothelial cells of capillaries and post-capillary venules.
Adhesion occurs through adhesion molecules of which there are 4 main groups:
i. Selectins (P-selectin and E-selectin on endothelium and L-selectin on leukocytes)
ii. Mucin-like ligands (Sialyl-Lewis X, etc. on leukocytes)
iii. Integrins (CD11/CD18, etc. on leukocytes)
iv. Immunoglobulin superfamily adhesion molecules – IgSAM’s (ICAM, VCAM, MadCAM,
etc on endothelium, and PECAM on endothelium and leukocytes)
Rolling
o P-selectin is first to become activated due to release of histamine, thrombin & Platelet Activating Factor
(PAF).
o E-selectin follows in 1-2 hours, stimulated by the secretion of TNF-alpha and IL-1 by macrophages,
mast cells and/or damaged endothelial cells.
Arrest and adhesion
o L-selectin on leukocytes binds MadCAM (Mucosal addressin Cell Adhesion Molecule) on endothelial
cells.
Firm adhesion
o Leukocytes become activated and express integrins (eg CD11/CD18) which bind to endothelial
IgSAM’s, ie ICAM (InterCellular Adhesion Molecule) and VCAM (Vascular Cell Adhesion Molecule).
Note:
o In addition to E-selectin, other adhesion molecules (L-selectin, IgSAM’s and Integrins) are activated by
TNF-alpha & IL-1 secreted mainly by macrophages &/or damaged endothelial cells, as well as by IL-6,
C5a, PAF, etc.
o Adhesion molecules tend to have several names and, to make things more confusing, they are
classified in a slightly different manner depending on the textbook consulted, not to mention the internet
sources; the ones mentioned above are not the only ones, but they are the most important ones.
Leukocyte adhesion deficiency in Irish setter dogs (CLAD): First reported in 1979 as “canine
granulocytopathy syndrome.” It is now known to be due to CD11b/CD18 (integrin) deficiency. The
condition is characterized by delayed umbilical cord separation at birth, impaired wound healing, and
recurrent bacterial infections without pus formation.
INFLAMMATION AND REPAIR 17 Winter 2013
3) Emigration
The process by which leukocytes escape from the blood to perivascular tissues; moving to the site of
inflammation.
After firm adhesion (using integrins bound to IgSAM’s like ICAM or VCAM), the leukocytes insert large
cytoplasmic extensions (pseudopodia) into endothelial gaps.
Gaps have been created by actions of histamine and other chemical mediators (see vascular
changes) as well as by the leukocytes themselves.
PECAM (Platelet Endothelial Cell Adhesion Molecule), an IgSAM expressed on both endothelial
and leukocyte surfaces, is the adhesion molecule most directly responsible for this process.
To pass through the basement membrane of the vessel, the leukocyte must also secrete
collagenases (this produces gaps of less than 1 micron in diameter).
As the leukocyte leaves the vessel, it expresses β1 integrins that help it bind to extracellular
matrix (ECM) proteins in the perivascular tissue.
Emigration occurs in the postcapillary venule because it is there that adequate numbers of inter-
endothelial gaps and receptors are found (particularly histamine receptors).
Neutrophils are usually the first to emigrate; they predominate for the first 6-24 hrs, peaking at 4-6 hrs.
Monocytes usually follow neutrophils, peaking at 18-24 hrs and becoming predominant in 24-48 hrs.
Neutrophils typically do not last for more than 24-48 hrs, while monocytes are longer lived.
In viral infections, lymphocytes are the first to arrive and in some hypersensitivity reactions eosinophils
arrive first.
Blocking adhesion molecules as treatment for Multiple Sclerosis (MS) NOT ON THE EXAM
MS is the most common demyelinating disorder, with both genetic and environmental components; about 1/1000 people
in the US and Europe will be affected. It is an autoimmune attack on myelin sheaths in the brain that begins with CD4 T
cells migrating into the neuropil and recruiting other T cells and macrophages to the site . It results in relapsing and
remitting episodes of neurologic deficit due to white matter lesions, with steady neurologic deterioration. Therapy drugs
have been developed to stop the migration of lymphocytes into the neuropil, one of them is Natalizumab.
Natalizumab is an antibody that binds the α4β1 integrin (VLA4), the key molecule for homing and adhesion of
lymphocytes to brain capillaries. Because Natalizumab binds the α4β1 integrin it prevents its binding to the endothelial
immunoglobulin VCAM-1, blocking the adhesion cascade and preventing the lymphocyte’s migration into the neuropil.
Three months after its approval by the FDA, it was pulled from the market after causing progressive multifocal
leukoencephalopathy (PML) in 2 patients enrolled in clinical trials, one of which was fatal. Natalizumab may have
allowed for an opportunistic infection with a polyoma virus that caused the PML.
4) Chemotaxis
The directional migration in response to a chemical gradient of chemoattractant (aka chemotaxin).
The process is receptor-mediated and allows leukocytes to travel from the perivascular space to the
site of injury / infection.
All leukocytes respond to chemotactic stimuli; neutrophils are the fastest (within 90 minutes), followed
by monocytes / macrophages (several hours) and then lymphocytes (last ones there).
Chemoattractants (chemotaxins) can be exogenous or endogenous.
o Exogenous chemoattractants
LPS in the wall of Gram-negative bacteria; attract neutrophils, eosinophils, monocytes /
macrophages.
Foreign material (eg wood splinter).
o Endogenous chemoattractants are from plasma and / or necrotic tissues
Histamine - attracts eo’s
Complement (particularly C5a) - attracts neutrophils, eosinophils, monocytes, basophils.
Fibrin-degradation products (FDPs)- attracts neutrophils
Leukotrienes (e.g. LTB4) from arachidonic acid metabolism - attract neutrophils and
eosinophils.
Chemokines - a type of cytokine (signal molecule produced by leukocytes) which main
function is to attract leukocytes; ie make them migrate across capillaries and post-
INFLAMMATION AND REPAIR 18 Winter 2013
capillary venules.
IL-8 attracts mostly neutrophils, but may also attract macrophages and
eosinophils.
Chemokines not only stimulate locomotion (chemotaxis) but also activate
leukocytes to: produce inflammatory mediators, engage in phagocytosis, initiate
the oxidative burst, etc.
Mechanisms of Chemotaxis
Leukocytes have receptors on their membrane that bind the chemoattractant initiates chain of
biochemical reactions that cause increased intracellular calcium leads to assembly of contractile elements
responsible for cell movement towards the highest concentration of chemoattractant.
i. Microtubules allow the cell to orient toward the chemotactic gradient while microfilaments (actin and
myosin) are actually responsible for the movement (cytoskeletal movement or re-organization).
ii. Movement is achieved by formation of a pseudopod that pulls the remainder of the cell in its direction.
a) Phagocytosis
To engulf, kill and degrade foreign material; most commonly bacteria.
Cellular mechanisms are similar to those of chemotaxis (cytoskeletal re-organization) but aimed at
engulfing an injurious agent; steps include:
o Recognition and attachment of agent (in this case bacteria):
(Numbers correspond to figure) Mannose on bacterial wall is recognized directly by the
leukocyte’s mannose receptor or bacteria are opsonized by antibodies and complement
(C3b) fragments that are then
recognized by specific receptors
on leukocytes. (1)
o Engulfment:
Small cytoplasmic extensions
(pseudopods) project from the
leukocyte. (2)
Pseudopods wrap around the
attached particle until it is
engulfed.
Pseudopods meet and fuse,
forming a phagosome.(3)
o Phagolysosome formation
Fusion of lysosomal granules with
phagosome (4) forms the
phagolysosome (5) in which the bacteria is killed and digested.
Metchnikoff, the star fish, the water flea and their wandering cells NOT ON THE EXAM
Elie Metchnikof (1845-1915) was the co-winner of the 1908 Nobel Prize in Physiology or Medicine for discovering phagocytosis.
Metchnikoff, a naturalist by trade, began studying the “wandering cells” in star fish larvae. Larvae are transparent and so it is
possible to observe these cells engulfing pigment particles under a microscope. Metchnikoff suspected that the leukocytes in our
blood stream would similarly engulf microbes, and thus protect us from infection. “I suddenly became a pathologist”, he wrote in his
diary, and gave the wandering cells the name of phagocytes (greek for devouring cells). Watching phagocytes of the water flea,
another conveniently transparent animal, he noted that they were able to engulf and digest spores from a yeast that otherwise overrun
the water flea and kill it.
b) Intracellular Killing
Oxygen-dependent and independent mechanisms of bactericidal activity occur in the phagolysosome:
1. Oxygen-dependent mechanisms
These are the most common, and are based on the production of reactive oxygen
INFLAMMATION AND REPAIR 19 Winter 2013
iron
•
O2- + H2O2 OH- + •OH + O2
2. Oxygen-independent mechanisms
Mostly due to substances within leukocyte granules such as:
Lysozyme
o Produced and stored in lysosomes, mostly neutrophils and macrophages.
o Attacks bacterial cell walls (esp gram +ve bacteria).
Lactoferrin
o Iron-binding glycoprotein, sequesters iron so that it is unavailable for use
by bacteria.
Trypsin and chymase
o Neutral proteases in mast cell granules, which degrade ECM.
Major Basic Protein
o Cationic protein of eosinophils that has limited bactericidal activity but it is
cytotoxic to many parasites.
Cathepsin G
o Protease within azurophilic granules of PMN’s which has antimicrobial
properties for both Gram +ve and Gram –ve bacteria and some fungi.
c) Degradation
After the microorganism has been phagocytised, the pH in the phagolysosome drops to 4-5.
This acid pH is optimal for the action of degradative enzymes within lysosomes.
INFLAMMATION AND REPAIR 20 Winter 2013
There may be defects in the phagocytic cells that interfere with the destruction of microorganisms
• eg, chronic granulomatous disease of childhood where children have neutrophils with defective oxidases
incapable of producing superoxide anion (O2•) and therefore develop recurrent infections.
o One mediator can stimulate the release of other mediators by target cells (ie provide
amplification).
o Chemical mediators may have different effects on different cells.
o Chemical mediators are interactive and redundant, guaranteeing amplification and preservation
of the response even if one or more components of the response are deficient.
o Most are short-lived and have the potential to be harmful.
Effect Mediator
Histamine
Vasodilation Nitric Oxide
Prostaglandins: PGI2, PGE2, PGD2
Histamine
Complement: C3a & C5a (anaphylatoxins)
Increased Vascular Bradykinin
Permeability Oxygen metabolites (ROS)
Leukotrienes: LTC4, LTD4, LTE4
Platelet-activating factor (PAF)
Complement: C5a
Leukotrienes: LTB4 & LTC4
Chemotaxis
Chemokines such as TNF, IL-1, IL-8
Bacterial products such as LPS
IL-1, TNF, IL-6
Fever
Prostaglandins
Bradykinin
Pain Substance P
Prostaglandin (PGF2)
Oxygen metabolites (ROS)
Tissue Damage Nitric Oxide
Lysosomal Enzymes
a) Vasoactive Amines
Histamine and serotonin are believed to be the primary mediators in the immediate active phase of
increased permeability.
Vasoactive amines cause vasodilation and increased vascular permeability by causing endothelial
cells to round up, increasing intercellular gaps, and also increasing vesiculovacuolar transfer of fluids.
Vasoactive amines are stored within cells for immediate release.
Histamine
Extensively distributed in tissues, the main source being the mast cells that are normally present in
the perivascular connective tissue; it is preformed and stored in granules with heparin.
Present in granules of basophils and in platelets (some species).
Histamine is important mainly in early inflammatory responses and in type 1 hypersensitivity
reactions.
Histamine is important in the immediate active phase of increased vascular permeability.
INFLAMMATION AND REPAIR 22 Winter 2013
Serotonin
Present in platelets and some mast cells (not in humans).
Acts primarily on venules during the early phase of acute inflammation, when it is released from
mast cells, basophils and platelets.
Release of histamine and serotonin from platelets (the platelet release reaction) is stimulated when
platelets aggregate after contact with collagen, thrombin, ADP, and antigen-antibody complexes.
b) Plasma Proteases
Three interrelated systems important in the inflammatory response are found within plasma:
o Complement, kinin and clotting systems.
o All are capable of being activated by activated Hageman’s factor (factor XIIa of the
coagulation cascade).
Complement system
Set of plasma proteins that act together to attack extracellular forms of microbial pathogens.
It can be activated directly by certain pathogens or by antibodies binding to a pathogen.
When pathogens (microorganism) are coated with complement proteins their removal by leukocytes
is facilitated (opsonized) &/or they are directly killed by the membrane attack complex (MAC).
Besides facilitated removal & killing of targeted microorganisms, activated complement is also
involved in:
o Vascular permeability (esp C3a & C5a) – via histamine release from mast cells.
o Chemotaxis - C5a chemoattractant for neutrophils, monocytes, eosinopils & basophils.
Kinin System
The kinin system generates vasoactive peptides from plasma proteins called kininogens by the
action of specific proteases called kallikreins which ultimately leads to activation of bradykinin.
o Bradykinin has the following actions:
Vasodilation and stimulation of histamine release by mast cells increased
vascular permeability
Contraction of non-vascular smooth muscle
Produce pain
Activate the arachidonic acid cascade
INFLAMMATION AND REPAIR 23 Winter 2013
Clotting system
The clotting system and inflammation are intimately connected.
Intrinsic clotting system is a sequence of plasma proteins that can be activated by Hageman factor
(factor XII – produced in liver and circulating in inactive form).
The final phase of the cascade is the conversion of fibrinogen to fibrin by the action of thrombin.
Thrombin binds to a receptor on platelets, endothelium, smooth muscle cells and causes them to:
o Mobilize P-selectin to the cell membrane and express adhesion molecules for integrins.
o Produce chemokines.
o Induce cyclooxygenase-2 – production of prostaglandins.
o Produce platelet activating factor (PAF) & nitric oxide (NO).
o Change endothelial shape.
Note:
Factor XIIa (activated Hageman factor) initiates the clotting, kinin, complement and fibrinolytic systems;
which all have effects on inflammation (eg fibrin-split products can increase permeability).
Also note, the products of this initiation (kallikrein, factor XIIA, and plasmin) can, by feedback, activate
Hageman factor, resulting in significant amplification of the effects of the initial stimulus.
Cyclooxygenase Pathway
Two enzymes are able to produce these products: COX-1 and COX-2.
COX-1 is normally present (constitutively expressed) and necessary for everyday activities; also
synthesized at sites of inflammation.
COX-2 is transcriptionally regulated - present in various circumstances (eg inflammation).
the main 3 products resulting from this pathway are:
Thromboxane A2 is found in platelets and other cells is a potent platelet aggregator and
vasoconstrictor
Prostacyclin (PG I2) is found predominantly in endothelial cells; a potent inhibitor of platelet
aggregation and vasodilator.
Prostaglandins (PG’s E2, D2, F2α) cause vasodilation, increased vascular permeability & pain.
Lipoxygenase Pathway
Results in the production of leukotrienes (they have a conjugated triene chain and were first
isolated from leukocytes), and lipoxins (produced mainly as intermediates by neutrophils, they are
activated by platelet-leukocyte interaction), which have opposing effects.
o Leukotrienes
Exacerbate acute inflammatory response:
1. Increased vascular permeability (up to 1000X as potent as histamine)
2. Chemotaxis for leukocytes
3. Vasoconstriction
4. Also causes bronchoconstriction
INFLAMMATION AND REPAIR 25 Winter 2013
Leukotriene B4
1. Primary leukotriene synthesized by neutrophils, also made by macrophages
2. One of the most potent chemotactic agents for neutrophils and macrophages.
d) Lysosomal Constituents
Found mainly in neutrophils, macrophages and cytotoxic lymphocytes, but also in eosinophils and
mast cells (see previous “oxygen-independent mechanisms intracellular killing” and note granzyme
& perforin below).
Granzyme and perforin are present in granules of cytotoxic T lymphocytes and Natural Killer cells.
Perforin punches holes in the membrane of a target cell (usually one infected with a virus) and
allows granzyme to get into the cytoplasm and activate the caspase cascade that results in
apoptosis.
IL-1
TNF-α
Hypothalamus PGE2 /cAMP Vasomotor Centre Sympathetic nerves
(raised thermostatic set point)
Skin vasoconstriction
(IL-6, prostaglandins)
Reduce heat dissipation
FEVER
IL-5
Produced by helper T lymphocytes (CD4 Th2) and mast cells.
Affects proliferation, chemotaxis, and activation of eosinophils (important in parasitic
infections, allergies, etc.).
IL-6
Produced by T lymphocytes & macrophages; its major activities include B and T cell
proliferation.
Sometimes considered a “Master cytokine”, like IL-1 and TNF.
IL-8
Produced by leukocytes and endothelial cells.
It is a powerful chemoattractant and activator of neutrophils and to lesser degree monocytes
and eosinophils
IFN-γ
Produced by T lymphocytes and NK cells.
Activates macrophages and T lymphocytes, particularly against viral infections.
PDGF (Platelet Derived Growth Factor)
Produced by leukocytes, endothelial cells and fibroblasts.
It is most important in chronic inflammation but is present from the beginning.
Acts as a chemoattractant to leukocytes and mesenchymal cells (fibroblasts); one of its main
functions is stimulating the proliferation of fibroblasts.
Other growth factors, like Vascular Endothelial Growth Factor (VEGF), Transforming Growth
Factor beta (TGF-β) are particularly important in tissue regeneration and repair (see later
section).
Mediators typically have short half lives and are produced in short bursts while stimulus is present; they
are degraded soon after release.
Switch to anti-inflammatory lipoxins (from arachidonic acid) & production of anti-inflammatory cytokines
(eg TGF-β).
Inhibition of the production of TNF in macrophages.
b) Abscess Formation
Seen with pyogenic organisms (pus-forming bacteria) or foreign bodies.
c) Fibrosis
Repair by Connective Tissue Replacement.
Usually associated with substantial tissue destruction.
Occurs when affected tissue cannot regenerate.
Occurs when fibrin exudation is abundant and cannot be completely cleared.
d) Chronic Inflammation
Persistent stimuli (often specific agents) → progression to a chronic inflammatory process.
Its primary purpose is to contain and degrade pathologic agents that are difficult to eliminate.
INFLAMMATION AND REPAIR 28 Winter 2013
CHRONIC INFLAMMATION
Definition = inflammation of prolonged duration (weeks to months or years) in which inflammation, tissue injury
and attempts at repair occur at the same time (to varying degrees).
b) Histology - Mononuclear infiltration (macrophages, lymphocytes, plasma cells) and tissue destruction.
Attempts of healing via granulation tissue (fibrosis) & angiogenesis (neovascularisation).
*Note: Morphologic changes may not correlate with the onset of clinical signs. A chronic lesion may
develop as an insidious, low-grade, subclinical process without history of a prior acute episode. For
example, due to the high functional reserve of the liver and kidney it is common to find severe
chronic lesions in these organs in animals that die suddenly (with no prior clinical history disease).
5) Chronic-Active Inflammation
All chronic inflammatory lesions are “technically” active, if not, they would be healed &/or scar tissue.
The term “chronic-active” has been extrapolated from human pathology; use it only if there are large
numbers of neutrophils or fibrin in a chronic lesion.
1) Macrophages
“The prima donna of chronic inflammation”
This designation includes related cells of bone marrow origin, namely monocytes and macrophages,
Kupffer cells, sinus histiocytes, alveolar macrophages, microglial cells, etc.
a) Macrophage Functions
Phagocytosis of particulate matter, microbes and senescent cells.
Recruitment of T and B cells, at the same time as they themselves are recruited to inflammatory
sites by lymphocyte products (self-perpetuating stimulation, as long as the inciting stimulus
INFLAMMATION AND REPAIR 29 Winter 2013
remains).
Emigrate relatively early in acute inflammation, within 48 hrs they are the predominant cell type
and are "activated".
b) Epithelioid Macrophages
Specialized macrophages with more abundant eosinophilic cytoplasm and eccentrically located,
round to oval nucleus, thus resembling epithelial cells.
Possess numerous lysosomes and vacuolated cytoplasm.
Have fewer receptors and less phagocytic activity; they specialize in secretion of cytokines.
Can fuse together to form multinucleate giant cells.
Touton
Yale Rosen, MD “Atlas of Granulomatous Diseases”
d) Participation of macrophages in chronic inflammation
Continued recruitment of monocytes from circulation (if persistant agent) thanks to steady
expression of chemotactic factors, eg C5a, IL-8, PDGF, TGF- .
Macrophage numbers can also increase due to local proliferation (replication).
Macrophages can be activated by microbial products (eg LPS), cytokines (eg IFN-γ, IL-4) &
other mediators.
Activated macrophages become immobilized and long-
lived at the sites of chronic inflammation.
Macrophages cause tissue destruction, even when
properly activated.
* Tissue destruction is one of the hallmarks of
chronic inflammation.
GRANULOMATOUS INFLAMMATION
Gross appearance: Usually firm (due to fibrosis) but with variable distribution and demarcation.
Diffuse (or locally extensive) thickening of tissue (eg. Johne’s disease due to M. paratuberculosis).
Nodular lesions (granulomas), often with central caseous necrosis or suppuration (pyogranuloma).
Granuloma
Focal type of granulomatous inflammation, consisting of a central aggregate of macrophages (many
being epithelioid macrophages &/or multinucleated giant cells); which is surrounded by variable
numbers of primarily lymphocytes and plasma cells and often circumferential fibrous connective tissue.
Types:
o Simple granuloma: organized accumulation of macrophages and epithelioid cells, often
rimmed by lymphocytes.
o Complex granuloma: granuloma with a central area of necrosis (which may show dystrophic
calcification / mineralization). Necrosis may be due to release of oxygen free radicals &/or
lysosomal enzymes or ischemia.
o Pyogranuloma: core is rich in neutrophils; often these neutrophils have undergone
degeneration.
o Foreign body granulomas: often characterized by the abundance of foreign body giant cells.
Examples of foreign material: inert particles (eg silica, asbestos, etc), lipids
resistant to metabolism (eg mineral oil), plant material (eg wood splinters, grass
awns) suture material, hair, keratin, sperm, etc.
Pathogenesis
o Certain pathogens (antigens) stimulate macrophages or dendritic cells to activate T
lymphocytes (esp by IL-12).
o T cells secrete IFN-γ which promotes transformation of macrophages into epithelioid
macrophages, +/- multinucleated giant cells.
o If pathogen persists inflammation continues (esp maintained by TNF) and will organize into a
granuloma.
o Can also have recruitment of neutrophils (esp IL-8 & IL-17) or eosinophils (IL-5).
INFLAMMATION AND REPAIR 31 Winter 2013
Repair
The process by which lost or necrotic cells are replaced by vital cells; either by regeneration or fibrosis.
Even as cells and tissues are being injured, events that contain the damage and prepare for the
surviving cells to replicate are set into motion.
Stimuli that induce death in some cells can trigger the activation of proliferative pathways in others; ie
recruited inflammatory cells not only clean up the necrotic debris but also elaborate mediators that drive
the synthesis of new extracellular matrix.
* Because there are so many requirements for a return to normality, some degree of scarring is more common
than complete resolution for most significant lesions.
Tissues made up of stable cells include: Epithelial cells of the liver, kidney and lung
Endocrine organs
Smooth muscle cells
Fibroblasts
Endothelial cells
Wound Healing
May involve regeneration &/or fibrosis and results in restoration of tissue continuity, with or without
function.
Sequence of events, regardless of etiology:
o Induction of acute inflammatory response by initial injury.
o Parenchyma cells regenerate (if possible).
o Migration and proliferation of both parenchymal and connective tissue cells.
o Synthesis of ECM proteins.
o Remodelling of parenchymal elements to restore tissue function.
o Remodelling of connective tissue to achieve wound strength.
Factors that can Impair Wound Healing:
o Infections, nutritional factors, glucocorticoids, mechanical factors, poor perfusion, foreign
bodies, etc.
o Type and volume of tissue injured.
o Location of the injury.
Wound strength:
o At 1 week, ~10% wound strength and increases to ~70-80% at 3 months; then doesn’t get much
better.
o If sutured, immediate wound strength is ~70% (due to sutures); note sutures are usually
removed on day 10.
INFLAMMATION AND REPAIR 34 Winter 2013
Timeline
24 hours: - Neutrophils at the incision margin migrate toward the fibrin clot.
- Basal epidermal cells at edges of incision increase mitotic activity.
24-48 hours: - Basal epidermal cells start to migrate and proliferate with deposition of basement
membrane.
Day 3: - Neutrophils are replaced by macrophages that invade the incision space.
- Fibroblasts & collagen fibres are evident at incision margins (at first are vertically
oriented and do not bridge the incision).
- Epithelial cells continue to proliferate.
Week 4: - Scar is composed of fibrous connective tissue with few inflammatory cells.
- Tensile strength continues to increase with time.
Granulation Tissue
Terminology is used both histologically and grossly, but the term is derived from its gross appearance.
Located on the surface of wounds & is pink, soft, sometimes granular appearing tissue that bleeds easily.
Histologically, see proliferation of new small blood vessels and fibroblasts (characteristic features).
1. Liver
Varies from complete parenchymal regeneration to extensive scar formation.
Outcome of hepatic healing is dependent upon the insult, the location, the extent and the chronicity.
If the insult is mild, regeneration with minimal fibrosis or alteration of the morphology will be
accomplished.
If the injury is severe, fibrosis will be abundant and will result in morphologic and functional alterations.
2. Kidney
Variable regenerative capacity
o Maximal regenerative capacity in renal cortical tubules
o Minimal regenerative capacity in medullary tubules
o No regenerative capacity in glomeruli
May achieve excellent tubular epithelial regeneration in cases of mild injury (ie no destruction basement
INFLAMMATION AND REPAIR 36 Winter 2013
membranes).
If there is destruction of the extracellular matrix framework (along with the tubular epithelial necrosis), it
will result in repair with scar formation with incomplete regeneration.
Example:
3. Lung
Alveolar injury with intact basement membranes
o Regeneration if alveolar exudate (eg fibrin, cell debris) is cleared by neutrophils &
macrophages.
o Granulation tissue and intra-alveolar fibrosis results if the inflammatory cells fail to lyse the
alveolar exudate.
o Alveolar type 2 pneumocytes are the cells responsible for regeneration (they are the alveolar
stem cells).
After injury type 2 pneumocytes migrate to denuded areas and undergo mitosis to
generate cells with features intermediate between those of type 1 and type 2
pneumocytes; as they establish contact with other epithelial cells, mitosis stops and the
cells differentiate into type 1 pneumonocytes.
Alveolar injury with damaged basement membranes
o Results in fibrosis / scarring.
o Mesenchymal cells from the alveolar septa proliferate and differentiate into fibroblasts and
myofibroblasts.
4. Heart
Healing occurs by granulation tissue & scarring, since myocardial cells are permanent cells with very
limited proliferative ability.
5. Brain
Neurons are permanent cells with very limited proliferative ability; but the supporting stromal cells (glial
cells and perivascular / meningeal fibroblasts) are capable of a robust proliferative response.
When the neuropil (neuroparenchyma) is punctured by a sterile instrument, the lesion heals by
astrocytic gliosis & fibrosis; the lesion fills with a fibrous core derived from the meninges and
perivascular adventitia.
Astrocytes are stimulated by edema and ischemia, they are less vulnerable to injury than nerve cells;
so if astrocytes are not destroyed during injury, they form a branching network around the wounded
neuropil.
Microglial cells are migratory, actively phagocytic cells of the neuropil; (ie clean up the debris).
INFLAMMATION AND REPAIR 37 Winter 2013
6. Bone
Stages in fracture healing:
Soft callus = the organizing, predominantly uncalcified, connective tissue that provides some
anchorage between the ends of the fractured bones but offers no structural rigidity for weight bearing.
Hard callus = the hard bony tissue that develops around the ends of a fractured bone during healing.
o As it mineralizes the stiffness and strength of the callus increases to the point that controlled
weight bearing can be tolerated.