inflammation and repair - university of prince...

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.

Cardinal Signs of Inflammation:

1. Heat (calor) 2. Redness (rubor) 3. Swelling (tumor) 4. Pain (dolor) 5. Loss of Function (functio laesa)

Ex-libris of the International Inflammation Club, Willoughby & Spector

Inflammation & Repair General Pathology (VPM 152), Winter 2013

These signs are mainly due to the vascular events of vasodilation (increased blood flow) and increased vascular permeability (movement of plasma fluids, proteins and inflammatory cells from the lumen of the vascular system out into the tissues).

INFLAMMATION AND REPAIR Winter 2013

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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 What is different in an otherwise stereotypical reaction?

Depending on the host and initiating agent, the appearance of inflammation will vary. Differences reside in the nature of the exudate, the distribution, the time course and the severity of the reaction.

Why classify inflammation? Using specific criteria, it is possible to provide a brief descriptive evaluation, also known as morphologic diagnosis, for each type of inflammatory response.

The morphologic diagnosis helps point towards the cause of the lesion (etiology). Example: A morphologic diagnosis of “pneumonia, granulomatous, multifocal and

severe” in a chicken suggests “mycobacterial pneumonia” (the etiologic diagnosis). Why bother?

The ability to formulate and understand a morphologic diagnosis makes communication between clinicians and pathologists, immunologists, molecular biologists, etc. easier and more productive.

An understanding of histopathology, as well as other ancillary tests used by pathologists, will make for a better understanding of a disease process (and ability to interpret a pathology report).

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.

What is immunohistochemistry? NOT ON THE EXAM Immunohistochemistry (IHC) refers to the process of localizing antigens (eg. proteins) in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues. It takes its name from the roots "immuno," in reference to antibodies used in the procedure, and "histo," meaning tissue (compare to immunocytochemistry). Immunohistochemical staining is widely used in the identification of the lineage of abnormal cells such as those found in cancerous tumors, and for the detection of infectious agents, as in the case of the pigeon above. Although many infectious agents are very characteristic and can thus be identified through routine histology, there are cases when they are too small (virus) or too similar to others to be identified just by looking at them under the microscope - in those cases, we use IHC. The method is based on binding a colorant to an antibody that is specific to particular protein present only in the suspected infectious agent – ie the tissue will stain ONLY if the agent is present.


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Classification of Inflammatory Reactions:

[*note: pneumonia is the term typically used for inflammation of the lung, not "pneumonitis"] 1) Organ and Anatomic Modifiers terms used to indicate the organ, or a specific area within an organ, that is inflamed. Most common ones are listed in the table on the next page (Slauson and Cooper, 2002, p. 150).

ORGAN ANATOMIC MODIFIERS

EXUDATE DISTRIBUTION DURATION SEVERITY

Nephritis Interstitial, glomerulo-, etc Suppurative (purulent)

Focal Peracute MInimal

*Pneumonia Brocho-, interstitial, etc Fibrinous Multifocal Acute Mild

Enteritis Fibrinopurulent (Coalescing) Subacute Moderate

Myositis Necrotizing Locally Extensive Chronic Marked or Severe

Hepatitis Necrosuppurative (Segmental) Chronic-active

Encephalitis Granulomatous Diffuse

Dermatitis Hemorrhagic


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You must learn the following word roots!


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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.

* Fibrinous + Necrotizing = Fibrinonecrotizing inflammation: Typically occurs on a well-vascularized epithelial surface (eg: trachea, intestine, etc), characterized by necrosis of the surface epithelium and presence of fibrin.

Pseudomembranes / diphtheritic membranes are a form of fibrinonecrotic exudates; the fibrin and necrotic surface epithelium forms a structure which resembles the luminal surface of the tissue (looks like the affected tissue is covered by a membrane).

in the gut, a “cast” of yellow-white material (fibrin and necrotic mucosa) can fill the lumen.

Diphtheria, the strangling angel of children NOT ON THE EXAM Before vaccination, diphtheria was one of the main causes of death in children (those under 15 years of age). Caused by the Gram(+) bacterium Corynebacterium diphtheriae, it causes necrosis of the laryngeal and tracheal mucosa, forming a layer of fibrin and necrotic debris, the so-called diphtheric pseudomembrane, that can eventually fill the lumen and kill by suffocation. The damage is due to an exotoxin; the type of exotoxin produced is a major determinant of mortality (from 20-80%). In the 1890s Von Behring developed an antitoxin from the serum of guinea pigs that was able to treat the disease by neutralizing the exotoxin. He was awarded the Nobel Prize in Medicine in 1901 for his work. Later, a vaccine was developed, resulting in the disease becoming almost a thing of the past, except for the occasional outbreak in some regions such as the USSR in the mid-1990s.

c) Suppurative (Purulent) Exudate Grossly it is called pus and is composed of large numbers of neutrophils and dead tissue cells (cellular debris).

Neutrophils have proteolytic enzymes, including myeloperoxidase, that cause tissue necrosis. Birds / reptiles / amphibians / some mammals (eg. rabbits) have heterophils instead of

neutrophils. Heterophils lack myeloperoxidase, so they form caseous exudates, rather than purulent (liquefactive) exudates.


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Etiology: suppurative lesions are often bacterial in origin. Time: usually acute (hours). Gross: - May be fluid or viscous; may be within a cavity or within tissue.

- Over time, localized suppurative inflammation may be surrounded by a connective tissue capsule to form an abscess (chronic suppurative inflammation).

* Fibrinous + Suppurative = Fibrinosuppurative (fibrinopurulent) inflammation - Exudates have an abundance of both fibrin and neutrophils.

d) Granulomatous Exudate / Inflammation Inflammatory response where macrophages predominate, along with more or less abundant lymphocytes, plasma cells and often multinucleated giant cells.

Gross: single or multiple firm pale nodules in tissue; generally along with caseous necrosis / exudate. Histo: macrophages are clustered around the causative etiologic agent, or around a central necrotic area, or simply as organized nodules (see discussion of granulomatous inflammation in chronic inflammation section for more details)

Time: always chronic (weeks to months) Etiology: - Non-digestible organism or particle that serves as a chronic inflammatory stimulus.

Infectious agents like Mycobacterium sp, Actinomyces bovis, Blastomyces dermatitidis, Coccidioides immitis.

Non-infectious agents like mineral oil, foreign bodies, etc. * Granulomatous inflammation + neutrophils = Pyogranulomatous inflammation - Clusters of neutrophils are admixed with the macrophages, usually with the neutrophils at the core of a cluster of macrophages.

e) Hemorrhagic (sanguineous) Inflammation Hemorrhage is the predominant feature, ie it occurs due to severe injury to blood vessels (necrosis of the vessel wall) or marked diapedesis.

Time: peracute to acute (minutes to hours) Examples: rabbit hemorrhagic disease (caused by a calicivirus); “hemorrhagic fevers” of primates (eg. ebola)

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). * Mucus + purulent exudate = mucopurulent exudate - Contains abundant mucus and pus.

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.


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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.


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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.

EXTENT OF INJURY TISSUE DAMAGE INFLAMMATORY CELLS VASCULAR INVOLVEMENT

Mild Absent to Minimal Few Slight

Moderate Some Present Readily observed Moderate edema & evidence of hemorrhage

Severe Substantial Abundant Massive edema & hemorrhage may be seen

NOTE: other modifiers of severity are sometimes used, eg, extensive or marked (for severe); minimal (less

than mild), etc.


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ACUTE INFLAMMATION Inflammatory reactions can be triggered by a variety of stimuli, including:

Infectious agents (viruses, fungi, bacteria, etc)

Trauma

Physical & Chemical agents

Tissue Necrosis

Foreign Material

Immune Reactions

The main function of inflammation is to move defense mechanisms from the vascular system out to the tissues, as a response to an injurious (inflammatory) stimulus, and to initiate repair.

Inflammation can be somewhat subjectively divided into ACUTE (immediate and short term responses, necessary repair is minimal) and CHRONIC (long term response, usually rich in repair mechanisms).

Both acute and chronic inflammation have two major components: Vascular Changes and Cellular Events, that follow a predetermined sequence.

Vascular changes allow circulating blood cells to slow down and make their way into the adjacent tissues. Cellular events result from the activation of inflammatory cells and repair tissue. These occur at the same time, but are studied separately for clarity’s sake (that’s what we hope, anyways).

Sequence of Events in Acute (1-5) and Chronic (5) Inflammation

1) Vasodilation (increased blood flow) ►CALOR & RUBOR arteriolar dilation (sometimes after vasoconstriction for a few seconds) and opening of new capillaries increases the amount of blood to tissue (ie hyperemia / redness) result of histamine and nitric oxide (primarily) acting on vascular smooth muscle

2) Increase permeability of microvasculature (esp postcapillary venules)

outpouring of fluids into extravascular tissues ►TUMOR

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

5) Tissue Damage / Repair ►LOSS OF FUNCTION


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Vascular Changes in Acute Inflammation The hallmark of acute inflammation is increased vascular permeability leading to escape of protein-rich exudate.

Five mechanisms of increased vascular permeability are described: 1. Retraction of Endothelial Cells (in venules)

a) Endothelial cell contraction

Rapid and transitory (lasts 15-30 min); ie reversible. Inflammatory stimuli cause release of inflammatory mediators (histamine, bradykinin, leukotrienes, etc.) Binding of mediator to receptor contraction of endothelial cells widening of intercellular junctions Occurs mainly in venules (not capillaries and arterioles).

b) Delayed prolonged leakage In some forms of mild injury (eg mild burns, UV irradiation) vascular leakage begins after a delay of 2 to 12 hrs, but lasts for several hours to days.

Thought to be due to endothelial cell contraction &/or mild endothelial degeneration.

2. Direct endothelial injury

Arterioles, venules and capillaries affected. When severe injurious stimuli (eg severe burns, bacterial toxins, virus) cause endothelial necrosis & detachment.

An immediate sustained response; ie leakage starts immediately after injury and lasts several hours to days until damaged vascular structures are repaired (or thrombosed).

3. Leukocyte dependent endothelial injury

Neutrophils that adhere to the endothelium (capillaries and postcapillary venules) during inflammation may also injure the endothelial cells and thus amplify the reaction.

Associated with the later stages of inflammation


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4. Increased transcytosis (active transport mechanism) Normally there is some transport of fluid through endothelial cells by channels of interconnected, uncoated vesicles and vacuoles (vesiculovacuolar organelles).

Certain factors (eg vascular endothelial growth factor = VEGF, histamine) can increase the number and size of these channels.

5. Leakage from new capillaries (regenerating during neovascularization)

During the repair process, proliferating endothelial cells are leaky. Mediated by VEGF (vascular endothelial growth factor)

TRANSUDATE VERSUS EXUDATE

EXUDATE MODIFIED

TRANSUDATE TRANSUDATE

DEFINITION Inflammatory Non-inflammatory

ETIOLOGY Inflammation / infection ( vasc. perm.)

Long-term transudates or vasc. perm.

Non-inflammatory edema ( hydro. psi, colloid psi, blocked lymphatics)

SPECIFIC GRAVITY > 1.025 1.017-1.025 < 1.017

PROTEIN CONTENT > 30 g/L 25-75 g/L < 25g/L

CLOTTABLE Often Varies Rarely

INFLAMMATORY CELLS

Many

(>5,000-7,000 cell/μL)

Few (1,000-7,000 cell/μL)

Occasional (<1,500/μL)

BACTERIA Often Rare

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.


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Cellular Events in Acute Inflammation 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.

Virchow vs. Cohnheim, the origin of pus NOT ON THE EXAM Rudolph Virchow, the father of cellular pathology, was convinced that the inflammatory cells making up the suppurative

exudate (pus) in an inflammatory site came from the connective tissue, were in fact always there, just waiting to act. One

of his students, in a definitive experiment that may have some trouble getting approved by animal care committees these

days, proved they came from the blood: Julius Cohnheim dissected a live frog, put a drop of acetic acid on its mesentery

and observed through a magnifying glass how the blood flow slowed down and the white blood cells came out of the

vessels and into the tissue, to form an inflammatory focus.

Leukocytes involved in inflammation 1) Neutrophils (aka = polymorphs, Polys, PMN's, Neuts) Neutrophils have rapid amoeboid movement, respond to a wide variety of chemotaxic compounds and have good phagocytic and bactericidal activities – to kill microorganisms, tumor cells and eliminate foreign material.

Neutrophils are the major cellular defence system against bacteria and a major part of the innate immune system; they are the first line of defence (ie first to respond).

Neutrophils are an end cell (they do not divide); their average time in circulation is likely just a few days (until recently thought to be only 6 hrs in circulation); marrow produces 5-10 X 1010 per day.

Once out of circulation they don’t return & may live for 1-2 days in tissue. They die if granules are released. There are 2 distinct pools of neutrophils in the blood:

The Marginating Pool is composed of neutrophils within blood vessels but lying out of the flow, ie "marginated" against the walls; but they can be mobilized very quickly (note: the bone marrow also has a storage pool of neutrophils which can be released when needed).

The Circulating Pool is all the other neutrophils in circulation. 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).


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*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.

2) Eosinophils (aka eo’s)

Numerous at inflammatory sites which result from parasites and allergic / immune-mediate disease, but may be present in any exudate. Some live in tissues in contact with environment such as intestine, skin, lung and mucous membranes.

Corticosteroids cause a reduction in the release of eo’s from the bone marrow. The most important cytokines for production and recruitment of eo’s is IL-5. Histamine is very eotactic (attracts 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.

3) Basophils and Mast Cells

Basophils are rare circulating granulocytes while mast cells are relatively numerous and are found in perivascular sites, particularly in areas of contact with the environment (lung, gut, mucous membranes and skin).

Both are derived from bone marrow and have similar functions; but they come from separate stem cell lineages (ie basophils don’t become mast cells when they move into circulation).

Basophils and mast cells share many characteristics: o They contain abundant cytoplasmic metachromatic granules (stain magenta with toluidine blue)

that are rich in histamine, proteases, and potent inflammatory mediators (they don’t die after releasing their granules, unlike neutrophils).

o Membrane receptors bind the Fc portion of IgE antibody (these cells mediate Type 1 hypersensitivity reactions).

o Produce cytokines (eg TNF- , IL-3,-4,-5,-10,-13, IFN-γ) and arachidonic acid metabolites (eg leukotrienes).

o Mast cells are the major source of histamine in acute inflammation.

Functions of mast cells Intimately involved in acute inflammation, particularly hypersensitivity reactions. Activated by IgE-bound antigens (parasites, pollen, and other allergens), as well as Substance P from

nerves and macrophages. Cross-linking of IgE membrane receptors on mast cells causes the release of histamine (in granules)

o Histamine causes smooth muscle dilation in arterioles (vasodilation) and increased permeability in venules.

Recruitment of eosinophils via IL-5, leukotriene C4.


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4) Macrophages / Monocytes (“The most dynamic and gifted of the leukocytes.”)

Macrophages (MØ’s or histiocytes) are derived from circulating blood monocytes of bone marrow origin; (note, monocytes require activation by various chemical mediators to become macrophages).

Macrophages can also originate from division of resident macrophages (ie they can proliferate locally). Macrophages can be relatively long-lived (T½ 30-60 days) in tissues. Monocytes do not have a large reserve pool in bone marrow; remain in circulation for 24-72 hrs. Monocytes are motile (8-12 hrs to get to sites of inflammation), but sluggish compared to PMNs.

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.

5) Lymphocytes and Plasma Cells

Lymphocytes and plasma cells are the main cells of immune reactions (review immunology notes). They are needed for antibody response (B lymphocytes and plasma cells, activated by CD4 helper T

cells), delayed cellular hypersensitivity responses (CD4/TH1 & CD8 cytotoxic T lymphocytes) and down regulation of the immune system (regulatory or suppressor T cells).

They are less motile than neutrophils and monocytes.

Non-leukocytes involved in inflammation (platelets, endothelial cells and fibroblasts) 1) Platelets (as inflammatory cells)

In addition to their role in hemostasis and coagulation, platelets contain cytoplasmic granules and release their products through secretory degranulation.

The most important products for the inflammatory response are P- selectins & histamine. Functions of platelets in the inflammatory response:

i. Release histamine (& others mediators) to increase vascular permeability and provide local amplification.

ii. Produce adhesion molecules (P-selectin) to facilitate leukocyte migration from blood to tissues. iii. Release cationic inflammatory mediators that directly activate C5 (chemotactic for leukocytes).

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).


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SEQUENCE OF CELLULAR EVENTS & THE LEUKOCYTE ADHESION CASCADE Accumulation of leukocytes is the most important feature of the inflammatory reaction.

Leukocytes engulf and degrade bacteria, immune complexes, and the debris of necrotic cells.

Leukocytes get to sites of inflammation by adhesion to vascular walls and transmigration through them.

This process is regulated by the “Leukocyte Adhesion Cascade”, characterized by binding of complementary adhesion molecules on membranes of leukocytes and endothelial cells.

Chemical mediators such as chemoattractants and cytokines affect these processes.

SEQUENCE OF LEUKOCYTE EVENTS 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

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.

2) Rolling & Adhesion Marginated leukocytes line the endothelium.

Leukocytes start to become adhered to the surface of endothelial cells through various adhesion molecules (described below).

Adhesion to the endothelium is at first loose, allowing the leukocytes to roll along the endothelial surface.

As adhesion becomes firmer, the leukocytes become stationary and can then begin to migrate through the endothelium and into the site of inflammation.

Selectins

Integrins


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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.

Endothelial Molecule Binds to Leukocyte Molecule Effect

P-selectin and E-selectin Mucin-like ligands (Sialyl-Lewis X) Rolling

IgSAM’s (MadCAM, etc) L-selectin Arrest & Adhesion

IgSAM’s (ICAM, VCAM) Integrins (CD11/CD18, etc) Firm adhesion

IgSAM’s (PECAM) IgSAM’s (PECAM) Emigration

GENETIC DISEASES DUE TO FAULTY ADHESION Bovine Leukocyte Adhesion Deficiency (BLAD): An autosomal recessive disease of Holsteins, characterized by an increased susceptibility to infectious agents. Affected cattle are homozygous for a single point mutation at position 128 in the β subunit of the CD18 gene. The molecular basis of the

neutrophil dysfunction is impaired expression of the 2 integrin (CD18, and other) class of leukocyte adhesion molecules, resulting in inadequate passage of these cells into the perivascular tissue and overlying epithelium. Affected cattle (range 2 wks to 8 months of age) have inadequate mucosal immunity resulting in chronic, recurrent respiratory and gastrointestinal infections without pus formation, and persistent neutrophilia. 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.


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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-


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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.

5) Phagocytosis, Intracellular Killing / Degradation

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


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species in the “respiratory burst” part of phagocytosis.

Superoxide anion (•O2-)

Hydrogen peroxide (H 2O2)

Hydroxyl radical (•OH) Various enzymatic processes are involved in the production of reactive oxygen species:

(i) NADPH oxidase

Present in the lysosomal membrane that is now fused with the phagosome. Produces H2O2 and O2

•

(ii) Myeloperoxidase-dependent killing (= H202-Myeloperoxidase-Halide System):

The quantities of H2O2 produced by the phagolysosomes are insufficient to induce effective killing of bacteria.

Azurophilic granules of neutrophils contain the enzyme myeloperoxidase (MPO), which in the presence of a halide such as Cl - (or iodine or bromide), converts

hydrogen peroxide (H2O2) to hypochlorous acid (HOCl), a powerful oxidant and

antimicrobial agent. A similar mechanism is also effective against fungi, viruses, protozoa, and helminths.

H2O2 + Cl- Myeloperoxidase HOCl

(iii) Haber-Weiss Reaction: Requires iron to produce hydroxyl radicals (•OH), an extremely reactive oxidant.

•O2- + H2O2

iron OH- + •OH + O2

(iv) Nitric Oxide (NO): Production of peroxynitrite (ONOO-) via superoxide anion

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.


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SURVIVAL OF PHAGOCYTOZED MICROORGANISMS

A few microorganisms have developed strategies to survive and avoid killing after phagocytosis, eg:

a) Escape the phagolysosome and grow in the cytoplasm,eg Rickettsia sp, Listeria monocytogenes

b) Block the lysosome-phagosome fusion, eg Toxoplasma gondii

c) Survival within phagolysosome, eg Mycobacterium sp., Coxiella burnetii

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.

6) Extracellular Release of Leukocyte Products Leukocytes do not only release toxic metabolites or enzymes into phagolysosomes, they also release

them into the site of inflammation. This extracellular release helps kill microorganisms and enhances the inflammatory reaction, but can

also cause tissue necrosis. There are 4 mechanisms whereby phagocytic cells release these and other potent chemicals:

a) Lysosomal Suicide (cytotoxic release)

Pathogenic bacteria overwhelm the leukocyte o Phagolysosomes rupture into the cytoplasm and release potent hydrolytic lysosomal enzymes

leukocyte is killed release of lysosomal enzymes into local environment. Leukocyte dies for any other reason (eg due to leukotoxin from a bacteria)

o Release of lysosomal enzymes into local environment.

b) Regurgitation during feeding

Lysosome fuses with phagosome before the phagosome is closed (during so-called “feeding frenzies”) lysosomal contents are released to environment.

c) Reverse endocytosis (frustrated phagocytosis)

Phagocytic stimuli are too large to be internalized a phagosome cannot be formed the lysosomal contents are discharged (in frustration!). For example: bacteria on a fibrin meshwork, Ag-Ab complexes deposited in a basement membrane

(eg glomerular BM), immune complexes on joint surface, etc.

d) Neutrophil Extracellular Traps (NETs) Recently described; a less harmful version of the extracellular release of enzymes and other toxic

compounds. NETs consist of antimicrobial proteins decorating a mesh of chromatin, including histones. NETs form a physical barrier and scaffold to block microbial spread while minimizing damage to host

tissues. Neutrophils generally undergo apoptosis to release a NET using their nuclear chromatin. Eosinophils, in contrast, use the mitochondrial DNA and thus remain intact after releasing an EET.

7) Synthesis of Chemical Mediators of Inflammation

A chemical mediator is any messenger that acts on blood vessels, inflammatory cells or other cells to contribute to an inflammatory response.

General principles: o Originate from plasma or cells; when in plasma, are in an inactive state and must be activated

and when in cells, they are often within granules and need to be secreted or they are synthesized in response to a stimulus.

o Production of active mediators is triggered by microbial products or host proteins (eg cytokines). o Some have direct enzymatic activity; most require binding to specific receptors on target cells

for biologic activity.


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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.

Chemical Mediators Classified by Effect

Effect Mediator

Vasodilation Histamine Nitric Oxide Prostaglandins: PGI2, PGE2, PGD2

Increased Vascular Permeability

Histamine Complement: C3a & C5a (anaphylatoxins) Bradykinin Oxygen metabolites (ROS) Leukotrienes: LTC4, LTD4, LTE4 Platelet-activating factor (PAF)

Chemotaxis

Complement: C5a Leukotrienes: LTB4 & LTC4 Chemokines such as TNF, IL-1, IL-8 Bacterial products such as LPS

Fever IL-1, TNF, IL-6 Prostaglandins

Pain Bradykinin Substance P Prostaglandin (PGF2)

Tissue Damage Oxygen metabolites (ROS) 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.


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It is also important in allergic reactions as it promotes contraction of extravascular smooth muscles in the bronchi and stimulates stromal cells to synthesize and release eotaxins (chemotaxins for eosinophils).

The following agents can stimulate release of histamine from mast cells: o Ag (eg pollen) binding to IgE on mast cells o Anaphylotoxins (C3a and C5a) o Physical injury, mechanical trauma, heat, chemical agents o Snake venoms, toxins, bile salts, ATP o Histamine-releasing factors from neutrophils, monocytes, and platelets o Cytokines (IL-1, IL-8) o Neuropeptides, like substance P

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

Another important member of the vasoactive amines is substance P. Substance P is produced in some leukocytes and sensory nerve fibres (thus known as a neuropeptide); it has similar effects to those of bradykinin. Curiously, its release is also activated by capsaicin, which is present in chiles (hot peppers). Substance P release from nerve fibres and other cells is responsible for the burning sensation associated with eating or being in direct contact with chiles.


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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.

c) Arachidonic Acid Metabolites When cells are activated by diverse stimuli, their lipid membranes can be rapidly remodelled to

generate biologically active lipid mediators. These lipid mediators are like short-range hormones that are formed rapidly and exert their effects

locally and then are inactivated. Oxygenated arachidonic acid derivatives act in biologic & pathologic processes, one of which is

inflammation. Arachidonic acid is a 20-carbon polyunsaturated fatty acid that is derived directly from the diet or by

conversion from linoleic acid. Arachidonic acid is not free in the cell but esterified in membrane phospholipids; in order to be

released from phospholipids it must be activated by cellular phospholipases, particularly


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phospholipase A2 (via mechanical, chemical and physical stimuli or by other mediators). Following activation, biosynthesis of the metabolites of arachidonic acid occurs by one of two major

pathways: the cyclooxygenase pathway and the lipoxygenase pathway.

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

Drugs such as corticosteroids, aspirin and indomethacin have anti-inflammatory properties because they inhibit specific steps of arachidonic acid metabolism (see below).


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Leukotriene B4 1. Primary leukotriene synthesized by neutrophils, also made by macrophages 2. One of the most potent chemotactic agents for neutrophils and macrophages.

Leukotrienes C4, D4, E4

1. Cause intense vascular leakage from venules. 2. Mast cells, eosinophils and macrophages make C4.

o Lipoxins Secreted mainly by platelets Have both pro- and anti-inflammatory effects and can counteract leukotrienes Inhibit neutrophil chemotaxis and adhesion to endothelium but promotes macrophage

adhesion to endothelium. Lipoxins A4 causes vasodilation and counteracts leukotriene C4-induced

vasoconstriction

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.

e) Oxygen-Derived Free Radicals

The main oxygen free radicals are superoxide anion (•O2-) & hydroxyl radical (•OH) (see previous

notes on oxygen-dependent mechanisms of intracellular killing). When released into tissue (ie escape from inflammatory cells) they cause: o Endothelial cell damage with resultant increased vascular permeability. o Inactivation of antiproteases unopposed protease activity increased destruction of ECM o Injury to a variety of cell types (tumour cells, red cells, parenchymal cells).

f) Platelet Activating Factor (PAF)

PAF is of phospholipid origin, derived from the cell membranes of leukocytes, endothelial cells & platelets.

PAF has several inflammatory effects, including: o Platelet aggregation and release. o Bronchoconstriction and vasoconstriction (at high concentrations). o Vasodilation & increased vascular permeability (low concentrations); much more potent than

histamine. o Increased leukocyte adhesion to endothelium, chemotaxis, degranulation and oxidative burst.

g) Cytokines and Chemokines Cytokines are polypeptides produced by many cells (but principally activated macrophages and

lymphocytes) and they function to modulate the function of other cell types. Chemokines are cytokines that promotes leukocyte chemotaxis and migration across capillaries and postcapillary venules.

Cytokines are essential transmitters of cell-to-cell communication in many physiological and patho-physiological processes, but the main mediators of inflammation are listed below.

IL-1 and TNF-α

The “Master Cytokines”, produced by monocyte-macrophages. Biochemically and immunologically distinct proteins, but are similar in their biologic activities:

o On endothelial cells, they increase leukocyte adhesion (induction of surface antigens), stimulate the synthesis of PGI2 and PAF, and increase pro-coagulant activity (surface thrombogenicity).


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o They induce systemic acute phase responses, eg fever, neutrophilia, hemodynamic effects (shock).

o On fibroblasts, they induce proliferation, increased collagen formation, and increased collagenase & protease synthesis.

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).

h) Nitric Oxide (NO)

A tiny molecule produced mainly in endothelial cells, neurons and macrophages. NO relaxes smooth muscles in vessels (causes vasodilation). Cells need the enzyme nitric oxide synthase (NOS) and co-enzymes to form NO. There are isoforms of NOS which are either continuously produced (endothelial eNOS and neuronal

nNOS), or are cytokine-inducible (iNOS, esp in macrophages). When iNOS is up-regulated in macrophages (& others), as in sepsis, there is massive

vasodilation & shock. Superoxide anion can convert NO to its own free radical (peroxynitrite), which is bactericidal:

NO + •O2- → •ONOO (peroxynitrite)


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HOW DOES ACUTE INFLAMMATION END? 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.

*However, if the stimulus remains, chronic inflammation follows. Possible Outcomes of the Acute Inflammatory Response

a) Resolution (the ideal outcome) Destruction, dilution or inactivation of inciting stimulus. Mediators decay or are neutralized. Return of normal vascular permeability. Cessation of leukocyte infiltration and death (apoptosis) of neutrophils already present in tissue. Removal of excess fluid, leukocytes, foreign material, necrotic debris. Repair and resolution of inflammation. Return to normal structure and function.

* complete resolution is more likely if the area of inflammation, the amount of exudate and the numbers of inflammatory cells are small.

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.


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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). 1) Characteristics of Chronic Inflammation

Mononuclear inflammatory cells are the most numerous leukocytes. Tissue destruction is present; often prominent. Repair is underway through proliferation of fibroblasts and endothelial cells (angiogenesis, aka

neovascularization). 2) Causes of Chronic Inflammation

May progress from acute inflammation if acute process cannot be resolved because of persistence of the agent or interference with normal healing.

Some specific injurious agents which typically cause chronic inflammation, including: Certain viral infections (eg. caprine arthritis encephalitis virus, porcine circovirus type 2). Certain persistent microbial infections (eg. Mycobacterium spp. and fungi). Prolonged exposure to some toxic agents (eg. asbestos). Some autoimmune diseases (eg. lupus erythematosus).

3) Morphologic Appearance of Chronic Inflammation

a) Gross - Shrunken, firm to hard tissue, uneven surface, may be discolored.

b) Histology - Mononuclear infiltration (macrophages, lymphocytes, plasma cells) and tissue destruction. Attempts of healing via granulation tissue (fibrosis) & angiogenesis (neovascularisation).

4) Pathogenesis of Chronic Inflammation

Persistent release of chemical mediators induces: Abundant tissue destruction. Persistent increase in blood flow & increased vascular permeability. Recruitment of inflammatory cells, mostly macrophages, lymphocytes, plasma cells. Proliferation of:

parenchymal cells (epithelial). supportive cells (fibroblasts, capillary endothelial cells), ie granulation tissue.

*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.

CELLS INVOLVED IN CHRONIC INFLAMMATION 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


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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.

c) Multinucleated Giant Cells

Result from fusion of macrophages under the influence of IL-4 and IFN-γ. There are various types including:

o Langhans: nuclei located at periphery; found in most types of chronic inflammation. o Foreign body: nuclei scattered throughout the giant cell cytoplasm. o Touton: rosette of nuclei at the centre, can be in tumours of histiocytic (tissue

macrophage) origin or xanthomas (masses composed of lipids, foamy macrophages and giant cells; associated with defects in lipid or triglyceride metabolism).

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.

Actions of activated macrophages

Inflammation & Tissue Injury, due to the release of:

o reactive oxygen and nitrogen species o proteases o cytokines / chemokines o coagulation factors o arachidonic acid metabolites

Repair / Fibrosis, due to the production of:

o growth factors (PDGF, FGF, TGFß) o fibrogenic cytokines o angiogenesis factors o "remodelling" collagenases

Langhans Foreign body

Touton

Yale Rosen, MD “Atlas of Granulomatous Diseases”


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2) Lymphocytes and Plasma Cells

T and B lymphocytes migrate into inflammatory sites. T lymphocytes have a reciprocal relationship with macrophages which drives chronic inflammation.

o Macrophages present “processed” antigen fragments on their surface antigen-presenting macrophages interact with lymphocytes lymphocytes activation Lymphocyte-derived mediators are produced (eg. IFN-γ) activation of additional macrophages cycle of lymphocyte and macrophage stimulation persists until triggering antigen is removed or reaction otherwise modulated.

B lymphocytes are also stimulated by macrophages and T helper lymphocytes (CD4) to become plasma cells and produce antigen-specific antibodies.

GRANULOMATOUS INFLAMMATION Definition: A chronic inflammatory reaction which is histologically dominated by macrophages; typically epithelioid macrophages &/or multinucleated giant cells.

Granulomatous inflammation is a chronic process and is a distinctive pattern of chronic inflammation. It is a mechanism for dealing with indigestible substances and certain microorganisms that are difficult

to kill. The dominant cells are macrophages and lymphocytes. Cell-mediated hypersensitivity can accelerate development and intensity of 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).


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REPAIR AND FIBROSIS – HEALING

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.

Two tissue repair mechanisms:

Regeneration Replacement of cells by those of an identical type; it requires the tissue have the capacity for

parenchymal regeneration and the maintenance of the architectural framework (eg. renal tubular basement membranes).

Regeneration begins early in the inflammatory process; mediators often both pro-inflammatory & pro-repair.

Fibrosis Replacement by fibrous connective tissue when regeneration of the local tissue cannot be

accomplished. Results in an increase in connective tissue (fibroblasts and collagen) and new blood vessels. Granulation tissue

Exposed connective tissue that forms within a healing wound, often hemorrhagic. Microscopically, collagen and fibroblasts run parallel to wound surface in contrast to new blood vessels which perpendicular.

Excess granulation tissue = proud flesh; common in horses.

Return to normality at the end of a repair process depends on: The ability of the host to eliminate the inciting agent The extent of necrosis / tissue damage, particularly to the supporting connective tissue framework. How much of the exudate is removed / resolved. The ability of the injured tissue cells to regenerate (labile or stable vs permanent).

* 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. Tissue Proliferative Capability

Repair capability of tissues varies with the organ system; ie different cell types have varying regenerative ability:

1. Labile (continuously dividing) cells

Cells which continue to multiply throughout life to replenish cells lost due to normal turnover. Labile cells are continuously dividing (>1.5% of these cells are in mitoses) and dying; ie

regenerating from a population of adult stem cells. Tissues composed of labile cells regenerate rapidly after injury (if architecture not severely altered

& enough stem cells remain viable). Tissues made up of labile cells include: Epithelium of skin and mucous membranes (eg gut)

Lymphoid cells Haematopoietic cells

2. Stable (Quiescent) cells Cells in this category generally have long life spans and are capable of rapid division following

tissue damage. In these tissues, <1.5% of the cells are in mitosis in a normal adult.


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Tissues made up of stable cells include: Epithelial cells of the liver, kidney and lung Endocrine organs

Smooth muscle cells Fibroblasts Endothelial cells

3. Permanent (nondividing) cells Cells with very limited regenerative ability. None of these cells are in mitosis in a normal adult. Cells in this category may regenerate portions of the cell (eg axonal regeneration). Tissues made up of permanent cells include: Neurons

Cardiac muscle cells Lens epithelium

Fibrosis (Repair by Connective Tissue Proliferation) Repair begins within 24 hours of the occurrence of the injury/start of the inflammatory reaction. Sequential events include:

o Fibroblast migration to site of injury/inflammation. o Induction of fibroblast and endothelial cell proliferation. o Granulation tissue (fibroblasts + collagen & neovascularization) within 3-5 days. o Gradual increase in collagen & regression of vessels (scar) over weeks to months.

There are 4 components of repair by fibrosis:

1. Angiogenesis (neovascularization) - see diagram

Proteolytic degradation of parent vessel basement membrane & ECM (allows formation of capillary sprouts).

Migration of endothelial cells to angiogenic stimulus (via endothelial integrins binding to fibrin & fibronectin).

Proliferation of endothelial cells (esp. VEGF secreted by macrophages, platelets, endothelium & fibroblasts).

Maturation into capillary tubes with recruitment & proliferation of supporting pericytes & smooth muscles cells.

New vessels are leaky.

2. Migration and proliferation of fibroblasts Due to growth factors produced by activated endothelial cells and activated leukocytes

(esp macrophages). Main growth factors are: Platelet Derived Growth Factor (PDGF), Transforming Growth

Factor beta (TGF-β) & Fibroblast Growth Factor (FGF).


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3. Deposition of extracellular matrix (ECM) Fibroblasts synthesize ECM (especially collagen); starts from 3-5 days and continues

onward. Main growth factors involved are: TGF-β & PDGF

4. Maturation and reorganization of fibrous tissue

Appearance of granulation tissue and subsequent scar tissue formation. In well organized granulation tissue fibroblasts and collagen fibres run in bundles (black

arrows) perpendicular to long thin blood vessels (red arrows, neovascularisation). 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.


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Cutaneous Wound Healing

Healing by First Intention (primary union) o When there is close apposition in a wound (incision with scalpel blade) with full skin apposition. o A primary union where epithelial regeneration predominates over fibrosis.

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.

Day 5: - Neovascularization peaks as granulation tissue fills space. - Collagen fibrils become more abundant & begin to bridge the incision (horizontally oriented).

Week 2: - Collagen accumulation and maturation. - Diminished edema and leukocytes. - Blanching occurs as vascular channels regress.

Week 4: - Scar is composed of fibrous connective tissue with few inflammatory cells. - Tensile strength continues to increase with time.

Healing by Second Intention (secondary union) o When there is poor apposition of a cutaneous wound (ragged cuts of the skin). o A complex reparative process which usually develops in more extensive injuries.

More extensive inflammatory component (fibrin & leukocytes). More granulation tissue. Wound contraction occurs due to myofibroblasts. Usually results in an irregular scar (which generally contracts).


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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).

Granulation tissue is composed of four recognizable zones:

o Zone of necrotic debris and fibrin: superficial area of variable thickness.

o Zone of macrophages (clean-up) and in-growing capillaries (angiogenesis).

o Zone of proliferating capillaries and fibroblasts: budding young blood vessels grow from the more mature vessels in the deeper zone up into the wound. These vessels grow perpendicular to the surface of the defect.

o Zone of mature fibrous connective tissue: the oldest portion of the healing process (mature collagenous scar)

Exuberant Granulation Tissue (Proud Flesh) o In some cases (esp horses) there is production of exuberant (excessive) granulation tissue. o This is an abnormal way of repairing injury and can occur when there is:

Severe and prolonged tissue injury. Loss of tissue framework (basement membranes). Large amounts of exudates

Consequences of granulation / fibrosis

o Loss of functional parenchymal tissue. o Alteration of physical properties of tissue

Skin with a scar is more prone to tearing. Pulmonary fibrosis decreases compliance and thus increases the workload for

respiration.

Healing in Specific Tissues 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

Example of hepatic cirrhosis (see photo): After massive hepatic necrosis the remaining hepatocytes regenerate. When there is damage to the extracellular matrix framework, the results is formation of irregular hepatocyte nodules (nodular regeneration) separated by fibrous connective tissue (scars). This gives the liver a multinodular firm appearance, often referred to as cirrhosis.


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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.

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).

Example: (right) Cortical Tubules with tubulorrhexis (ie with destruction of basement membrane): as the basement membrane is disrupted, epithelial cells have no platform to proliferate on. Macrophages and fibroblasts infiltrate and proliferate. Fibrous connective tissue replaces the affected tubules and surrounds, sometimes suffocates, the surviving ones, which become atrophied. (left) Cortical Tubules without tubulorrhexis (ie without rupture of tubular basement membrane): the surviving tubular cells in the vicinity of the wound flatten (acquire a squamoid appearance) and migrate into the necrotic area along the basement membrane. Mitoses are frequent, and occasional clusters of epithelial cells project into the lumen. Within a week, the flattened cells are more cuboidal and differentiated cytoplasmic elements appear. Tubular morphology and function are normal by 3-4 weeks.


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6. Bone

Stages in fracture healing:

FIGURE 26-15 (Rubin) Healing of a fracture

A. Soon after a fracture is sustained (first 1 to 2 days), an extensive blood clot (hematoma) forms in the subperiosteal and soft tissue, as well as in the marrow cavity. The bone at the fracture site is jagged. B. The inflammatory phase (to end of week 1) of fracture healing is characterized by neovascularization and beginning organization of the blood clot. Because the osteocytes in the fracture site are dead, the lacunae are empty. The osteocytes of the cortex are necrotic well beyond the fracture site, owing to the traumatic interruption of the perforating arteries from the periosteum. C. The reparative phase (after the 1

st week) of

fracture healing is characterized by the formation of a callus of cartilage and woven bone near the fracture

site. The jagged edges of the original cortex have been remodeled and eroded by osteoclasts. The marrow space has been revascularized and contains reactive woven bone, as does the periosteal area. D. In the remodeling phase (after several weeks to months), during which the cortex is revitalized, the reactive bone may be lamellar or woven. The new bone is organized along stress lines and mechanical forces. Extensive osteoclastic and osteoblastic cellular activity is maintained. Eventually the woven bone is replaced by lamellar bone.

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.

inflammation and repair - university of prince...

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