Lecture 7- Freedman- Inflammation

Acute Interstitial Pneumonia with *fibrin*

Compatible with Acute Respiratory Distress syndrome (ARDS)

Mechs of increased vascular permeability - #3

Delayed-prolonged leakage (*cytokines* play a role) • delay of 2-12 hours is common • lasts for hours or days • affects capillaries and venules • *example:* mild to moderate thermal injury, x-irradiation, UV exposure (sun burn), bacterial toxins

Chronic Inflammation

Prolonged response during which active inflammation, tissue destruction and repair happen at the same time often begins with low grade asymptomatic response Ex: chronic interstitial nephritis (cats), atherosclerosis (hypothyroid dogs). Rheumatoid arthritis, myco-bacterium, chronic lung & heart disease, etc

Overview/review of Acute inflammation

- Initiated by injury or agent, mast cells activated - Mediators affect vascular endothelial cells, increased vascular permeability, increased selectin expression on endothelium, arteriolar dilation, open capillary beds (histamine), blood flow which causes redness, warmth, swelling. - Plasma and leukocytes leave vasculature - Leukocytes are activated by locally produced mediators - Phagocytes are activated & try to engulf and kill inciting agent - Phagocytes release cytokines (e.g. IL-1, TNF) and lipid mediators - Leukocytes try to phagocytose agent, if successful, things return to normal. Main histologic and pathologic features of acute inflammation: - Neutrophils first, then macrophages - Plasma/fluid (exudate or transudate?)

Increased vascular permeability

Allowing protein-rich fluid (*exudate*) to escape. *Decreases* intravascular osmotic pressure. Together with increased blood flow/pressure this facilitates *fluid loss* from vasculature.

Acute Interstitial Pneumonia

Alveolar filled with inflammatory cells & proteinaceous fluid

Acute Inflammation

- rapid response to injurious agent that delivers mediators of host defense to the site of injury - initiated in the *microvasculature* - Initial steps are: 1. *alterations in capillary blood flow* (*histamine, NO*) 2. *changes in permeability* of microvasculature to *plasma proteins* and *leukocytes* [*starling's relationship*] 3. *stasis, transmigration of leukocytes* and *fluid leaks* from vasculature at *sites of injury* 4. *activation of leukocytes* by offending agent

Vasoactive amines- Serotonin

- similar action to Histamine - produced by platelets & enterochromaffin cells - *release from platelets after they aggregate* - following contact with collagen, thrombin, ADP, Ag-Ag complexes (*vascular injury*)

Responses to Leukocyte Activation

Initiation of effector functions to *rid host* of offending agent: - *arachidonic acid* production - *lysosomal enzyme* production (degradation enzymes like elastase & collagenase) but also antibacterial such as lysozyme and myeloperoxidase secretion - activation of *adhesion molecules* (integrins)

"Sub"acute inflammatory response

Many neutrophils, but also numerous macrophages in the lung. Evidence of Neo-vascularization (repair), no fibrosis

Arachidonic Acid con't

Metabolized into Inflammatory mediators by: - *cyclo-oxygenases* (COX1 &2) convert AA —> prostaglandins, thromboxane, and prostacyclines (PGE2, PGD2, PGF2a, PGI2, TXA2). These are the targets of NSAIDS, COX inhibitors, steroids. - *lipoxygenases* produce *leukotrienes* and *lipoxins* from AA

Chemotaxis

Migration of immune cells towards site of injury: - Chemo-attractants include *bacterial products, complement (C5a), lipoxygenase products (leukotriene B4), cytokines and chemokines*. - *pathogen-associated molecular patterns* (PAMPs), released by invading microorganisms, and *damage-associated molecular patterns* (DAMPs), derived from damaged and/or dead-cells, or in response to tissue and/or cellular stress. - Many chemotactic agents operate via *G-protein coupled receptors* (GPCR) to activate *phospholipase C* (PLC) and *PI3-kinase*. - *Ca2+ signals* and *activated GTPases* (e.g. rac/rho/cdc42) increase actin polymerization initiating *cell migration* via *filopodia extension.*

Avian aspergillosis

Multifocal granulomas not well organized (granulomatous) with Heteroptera, Epitheloid Macs, MN giant cells

caseous necrosis of the lung

No cellularity. Edema & inflammatory cell infiltrate in alveoli & surrounding the granuloma. Chronic inflammatory cells include macs, lymphocytes, and plasma cells. Fibrosis present in the wall of the granuloma

Acute necrosuppurative osteomyelitis (bone infection) - mouse

Normal tissue architecture disrupted by accumulation of necrotic debris & degenerate Neutrophils. Pink = *fibrin* accumulating

Endocarditis- Mitral valve

Note: organized thrombi

Oxygen-dependent mechs of killing & degradation microbes

O2 —[NADPH oxidate]—> O2 —[superoxide dismutase]—> H20 —[catalase]—> H2O + O2 Or H20 + 2Cl- —[myeloperoxidase]—> HOCl- [*hypochlorite...most efficient killing system in neutrophils*]

Cellular Reaction - Leukocyte extravasation & phagocytosis

Objective of inflammation = *deliver leukocytes* to the site of injury and to *activate them* 1. *Extravasation*= journey from vessel lumen - Normally RBC's form a *central column* and leukocytes are displaced *peripherally*. *Blood stasis and vessel dilation* during inflammation produces *greater leukocyte margination.* - Leukocytes in vessel *roll along endothelium* and *adhere to endothelium* (adjacent to sites of inflammation) which is activated by *cellular derived mediators.* - Following *adhesion*, pseudopods are inserted into the jxn. between endothelium. - Leukocytes *transmigrate* across vessel wall into interstitial spaces toward a chemotactic stimulus. So, *margination*, *rolling & adherence*, and finally *transmigration* Note: *TNF & IL-1* which are released by macrophage w/ microbes inside of it (infected Mac) which signals the leukocyte to come

SUMMARY

Overview, definition, causes [5 cardinal signs of inflammation: Rubor, Calor, Tumor, Loss of Function, Dalor] MECHS of inflammation: *Vascular changes*: blood flow, permeability, fluids ; *Cellular features*: cell types, extravasation, chemotaxis

Plasma Proteins - Clotting system

Goal= activate *thrombin* and formation of *fibrin* Intrinsic pathway: activated by *Hagemen factor, Factor XII*, synthesized by liver, circulates as inactive form, activated by collagen, BM or activated platelets (endothelial injury), *thrombin* (a protease) links Inflammation & clotting KNOW the image!!

Actinomycosis

Gram + bacteria. Causes "lumpy jaw" in soft tissue of mandible. The organism cannot be digested by macs and abscess & granuloma forms

Granulomatous Inflammation

subset of chronic inflammation and is characterized by the formation of granulomas. Granuloma= one or more isolated foci of granulomatous inflammation. Granuloma is originally defined as tiny, pale, granular foci in tissues resulting from hematogenous spread of the TB organism. A central histo feature of granulomatous inflammation = presence of epithelioid macrophages and multinucleated giant cells

acute vs chronic inflammation

*ACUTE* - rapid onset (seconds-mins) - short duration (mins-few days) - fluid & plasma proteins leak into tissues (*edema*) and (*leukocytes*) migrate into tissues. If unresolved, leads to *chronic inflammation* - NEUTROPHILS, EOSINOPHILS, ANTIBODIES *CHRONIC* - longer duration (days, weeks, months) - *lymphocytes and macrophages* present [mononuclear cells] - blood vessels proliferate - *fibrous tissue* deposition [*fibroblasts*] - tissue necrosis - lots of destruction and repair - scarring, fiber deposits, amyloidosis

Inflammatory exudate in heart

*Acute valvular endocarditis* - aortic valve showing *erythema* (rubor, calor, tumor?) and *fibrin* deposition Vs. *fibriotic CT* —> won't easily break off!!

Extravasation - con't (Leukocyte adhesion & transmigration)

*Adhesion* - mediated by molecules on leukocytes & endothelium: 1. *Selectins* (weak interaction, slow down causing *rolling*) - bind to glycoproteins on lymphocytes. —> *E-Selectins* confined to endothelium —> *P-Selectins* on endothelium and platelets —> *L-selectins* mostly on leukocytes 2. *Ig superfamily molecules* - 2 key *endothelial adhesion molecules* —> *ICAM-1* (intracellular vascular adhesion molecule) —> *VCAM-1* (vascular cellular adhesion molecule) 3. *Integrins* (strong interaction, *arrest*) - expressed by *leukocytes*, bind to ligands on other leukocytes, endothelium, and E.C. matrix. Integrate signals between cells or cells and ECM —> *b2 integrins* (LFA-1 and Mac-1) bind *ICAM-1* —> *b1 integrins* (VLA4) bind *VCAM-1* (1o endo. adhesion molecule) 4. *Mucin-like glycoproteins* - *heparan sulfates* are ligands for the leukocyte adhesion molecule *CD44*

Lipid-Derived Inflammatory Mediators

*Arachidonic Acid* - Lipid mediator of inflammation - Clinically/therapeutically important - aspirin and other NSAIDs control inflammatory responses by *inhibiting AA metabolism* - AA is a 20-carbon polyunsturated FA esterified in membrane FA's, primary dietary source is the phospholipid linoleic acid. - Generated by enzymatic cleavage from membrane phospholipids AA derived lipids are found in inflammatory exudates at sites of inflammation. - AA metabolites induce *vasodilation, vascular leakage, coaglulation, thrombosis, and pain.* - *phospholipase A2*- cleaves AA from *membrane precursors* of signaling intermediates & inflammatory mediators —> activated by mechanical, physical, and chemical stimuli, GPCR signaling induced by chemokines, cytokines, and other mediators - *PLA2* is activated by increased *Ca2+*, *kinases*

Important Aspects of AA metabolism

*COX-1* is constitutively expressed- AA production for daily needs *COX-2* is *induced* by factors like endotoxins, cytokines, and GF. Produces PG's involved in inflammatory reactions —> *target of Vioxx* COX-2 is constructively expressed in some tissues, like epididymis epithelium & defers duct. NSAID treatment might be correlated w/ temporary sterility

Lipoxygenase products - 5-lipoxygenase (5-LOX) in Neutrophils

*LTB4* and *5-HETE* (pro-inflammatory)— chemotactic molecules that also cause *leukocyte adhesion* to vascular endothelium. Generates oxygen free radicals. Induces release of lysosomal enzymes. *LTC4, LTD4, LTE4* - together, cause *intense vasoconstriction, bronchospasm, increases vascular permeability, leakage in venules* — more potent than histamine. Involved in *pathogenesis of Asthma* &is produced by neutrophils *Lipoxins, Resolvins, and Protectines* - help to *tamp down the inflammatory response*. Neutrophils produce lipoxin intermediates, which are converted to lipoxins by platelets. Facilitated by contact between Neutrophils and platelets. Lipoxins inhibit leukocyte recruitment & adhesion to endothelium

Mechs of increased vascular permeability - #4

*Leukocyte mediated* endothelial injury • leukocytes adhere early in inflammation • activation of leuks. causes release of toxic oxygen species (O2-) causing endothelial injury • acutely restricted to vascular sites of leukocyte adhesion.

Mechs of increased vascular permeability - #1

*Stimulus-induced formation of *endothelial gaps* in venules [caused by *histamine, bradykinin, leukotrienes, substance P*. - Duration is 15-20 mins - Small venules (<20-60mm), not capillaries or arteriole - many leukocyte events occur in venules [adhesion, emigration] - Gaps form between the vascular endothelium; contraction of cytoskeleton -*Cytokines* play a role... *IL-1, TNF, IFNg* - This effect is delayed/more prolonged (4-24hrs).

Products involved in inflammatory response (and coagulation)

*TXA2* - major product of *platelets*. Causes *platelet aggregation* and *vasoconstriction, coagulation,* is short lived. (KEY: aspirin and other NSAIDs lead to prolonged bleeding because they inhibit TXA2 production.) *PGI2* (prostacylin) - from vascular endothelium, potent vasodilator, inhibits coagulation, major product of endothelium.

Mechs of increased vascular permeability - #2

*direct endothelial injury*: - resulting in cell *necrosis or detachment* - direct endothelium damage - severe burns, lytic bacteria etc - OR substantial *leakage* over hours until thrombosis occurs - endothelial *cell detachment* allows platelet adhesion to the BM - capillaries, arteriole, and venules affected

Kinetics of Leukocyte Appearance at Inflammatory Sites

- *Neutrophils* - 6-24 hrs —> more abundant in blood —> respond more *rapidly* to chemokines —> attach more *firmly* to adhesion molecules in early inflammation (like P-selectin and E-selectin) —> after entering tissue, they are short-lived & do rapid apoptosis & disappear - *Monocytes* (macrophages) - 24hrs & beyond —> long-lived, slower response - *Lymphocytes* - >48 hours (*chronic inflammation*) New=Neutrophils Lymph=livin' the good life slowly

Histological hallmarks of fluid in skin

- *Rubor/Calor* due to *increased blood flow. *Hyperemia* is evident from dilated blood-filled vessels - *increased vascular permeability* to fluid & cells —> *Edema (tumor)* in dermis & epidermis. Epidermis is *thickened* & basal layers show *prominent space* between cells due to edema - *inflammatory cells* in the dermis. Inflammatory cells can also be seen marginating in the larger venule on the upper right dermis

Hyperemia- Increased vessel diameter & blood flow

- *vasodilation*: stimulus induced increase in blood flow —> *redness* (RUBOR) and *heat* (CALOR). Mediators of vasodilation = *histamine* and *NO* which relax smooth muscle - increase in vascular *permeability* —> extravasation of protein rich fluid (leads to EDEMA and swelling —> *TUMOR*) - fluid is lost from microvessels: concentrates blood, increases viscosity. Dilated BV are packed with RBC, which can lead to *stasis*. Arteriole pressure increases and mean capillary pressure increases b/c of *arteriole dilation*. Venous pressure increases but *osmotic pressure is reduced.* *protein leaks* across the venules - blood stasis —> *leukocyte* accumulation (initially neutrophils). Leukocytes bind to endothelium & *migrate out*

3. Progression to chronic inflammation

- Acute to chronic change when acute response is unresolved - Inciting cause is not cleared or the process evolves towards a chronic response with establishment of an autoimmune reaction (e.g. epitope spreading, molecular mimicry and exposure of antigens normally segregated from the immune system).

Inflammatory Mediators

- Produced by tissues and secreted into *plasma* (*complement, kinins*, etc. must be activated) or produced *locally* by cells (*histamine, lysosomal enzymes, etc*.) - Mediator production is triggered by *microbial* or *host products.* - Generally function by binding to a *specific host cell receptor* (some have inherent activity e.g. proteases, reactive oxygen species) - Mediators trigger production of other *pro and anti-inflammatory mediators.* - Each mediator typically has a *variety of targets* - Mediators are typically *short-lived*. They decay, are inactivated, are scavenged, or are inhibited rapidly. - Most mediators ALSO have the *potential to cause harm*

Type IV (delayed) hypersensitivity

- Type 4 (Cell-mediated) - Only reaction not mediated by antibody - Sensitized T-lymphocytes - Graph rejection, PPD reaction, poison ivy or oak - delayed, developing 24-48hours after exposure - classical examples = tuberculin reaction, and contact dermatitis - underlies granulomatous inflammation associated with TB, fungal, and protozoal infections - typically triggered by persistent antigens Characteristics: 1. DTH reactions are Ag specific & involve tissue infiltration of blood-derived monocytes & lymphomas. DTH does not depend on antibody 2. Can be transferred with cells, not serum 3. Dependent upon T-cells pic: events that give rise to granuloma formation & role of Th1 driven hypersensitivity (DTH) reaction

Vascular changes in acute inflammation

- blood flow *increases*, vessels *dilate* and become permeable to vascular elements, allowing them to enter/exit sites of injury - *increased vessel diameter/blood flow* - *increased vascular permeability*

2. Healing via connectve tissue replacement - fibrosis

- follows substantial tissue destruction e.g. liver chirrosis - occurs in tissues incapable of regeneration or when fibrinous exudate cannot be cleared, connective tissue lays down fibrous CT on scaffold of fibrinous exudate.

Vasoactive amines - Histamine

- produced by *mast cells* in CT next to vessels. Also made by *basophils* and *platelets* - release is triggered by *physical injury, trauma, hot, cold*, *immune rxns* (Ab binding; IgE), *anaphylotoxins* (C3a, C5a—> causes histomaine release & increased vascular permeability), *histamine-release proteins*, *neuropeptides* (like substance P), *cytokines* (IL-4, IL-8) - causes arteriolar *dilation*, increase in venule *permeability* - principle mediator of immediate *transient phase of increased vascular permeability* (endothelial H1 receptors— receptors for histamine to bind)

How do Mast Cells work?

- they express high affinity Fc receptors - preformed antibodies bind FcR - most type I hypersensitivity responses are mediated by IgE produced by B-cells - B-cells need T-helper cells (TH2) - bridging of IgE bound to mast cells triggers inflammatory mediator release Primary mediators: - biogenic amines (Histamine, serotonin, vasodilation, increase in vascular permeability, increase in SM contractions, increase in secretions) - enzymes (proteases, acid hydroplases can cause tissue damage, generate kinins and activate C) Secondary mediators: - lipids (AA products: leukotrienes [potent vasoactive agents], prostaglandins, PAF [platelet aggregation, histamine release, vascular permeability] - cytokines (TNF, IL-1, IL-4, etc: recruit & activate inflammatory cells like neutrophils & macs)

Plasma proteins - Kinin system

- vasoactive peptides, cleaved by kallikreins - kallikrein stimulates *bradykinin* production *Effects of Bradykinin*: 1. increase vascular permeability like histamine (tumor, calor) 2. dilation of blood vessels (rubor) 3. pain (dolor) 4. sm. muscle contraction 5. brady = slow to produce contraction of sm. muscule *Hagemann Factor* (Factor XII of clotting pathway) is activated to XIIa by collagen, basement membranes, negatively charged surfaces. *Factor XIIa* converts prekallikrein to kallikrein, which converts plasminogen to *plasmin*, which lyses fibrin and activates C3.

Types of Fluid found @ sites of inflammation

1. *Exudate* - fluid, proteins, and blood cells that escape into interstitial spaces or the body cavities. Specific gravity > 1.020. Stains *pink* b/c of protein. - due to *increase in vascular permeability* (CKD - chronic kidney disease) - purulent (pus) exudate is one *rich in leukocytes* (1o neutrophils) *types of exudates*: a. *Suppurative exudate* - composed of neutrophils and dead cells (pus). *Purulent* is a synonym of suppurative. An *abscess* is a localized form of suppurative inflammation b. *Fibrinous exudate* - increased vascular permeability during acute inflammation permits *leakage of plasma proteins* including *fibrinogen*, which is cleaved into *fibrin* and *polymerizes into clots*. This can occur in seconds. Can later be replaced by *fibrosis*, which is a *distinct process* involving deposition of *connective tissue by fibroblasts.* c. *Serous exudate* - lesions characterized by accumulation of fluid rich in *protein* on body surfaces —> e.g. *oozing of fluid* from burns. Blisters are an example of a serous exudate.

Types of Fluid found @ sites of inflammation con't

1. *Transudate* - fluid with *low protein content* (<1% albumen) with specific gravity < 1.012. - *Increased osmotic or hydrostatic imbalance* across vessel wall or a *decrease in colloid (oncotic) pressure* w/o an increase in permeability of vessel wall. - ultrafiltrate of blood plasma [holding protein back, but allowing fluid) *review* 2. *Edema* - general term for *increased fluid in the interstitium* with the only exception being the LUNG where excessive fluid in the alveolar lumen is also defined as edema. 4. *Effusion* - fluid in the *serous cavities.*

2 principle components of inflammation

1. *Vascular reaction* - involves *fluid* and *plasma proteins* (e.g. complement and fibrin), *blood vessels*, and *extracellular matrix elements* 2. *Cellular reaction* - *neutrophils, monocytes, eosinophils, lymphocytes, basophils, platelets, tissue mast cells, fibroblasts, and macrophages*. Important role for extracellular matrix such as fibrous proteins (*collagen and elastin*) *adhesive glycoproteins* (fibronectin, laminin) and *proteoglycans*

Functions of Complement

1. *cell lysis* by membrane attack complex (MAC) 2. Induction of microbe *phagocytosis* 3. Proteolytic fragments (C3a and C5a) activate *other cells* Fragment effects: A. *vascular* - C5a, C3a (analphylatoxins) — stimulate *histamine* release from *mast cells.* Increases *vascular permeability, vasodilation*, *C5a activated lipoxygenase* pathway of AA metabolism B. *leukocyte adhesion* - chemotaxis & activation (neutrophils, basophils, monocytes, and eosinophils) C. *Phagocytosis* - C3b and iC3b are *opsonins* on bacteria and increase phagocytosis by neutrophils & macrophages

Steps of Inflammatory Response

1. Damaged tissues release histamines, increasing blood flow to the area. 2. Histamines cause capillaries to leak, releasing phagocytes and clotting factors into the wound. 3. Phagocytes engulf bacteria, dead cells, and cellular debris. 4. Platelets move out of the capillary to seal the wounded area.

Systemic Effects due to inflammation

1. Fever: response to bacterial pyrogens, increased prostaglandins synthesis in hypothalamus. Leukocytes (macs) release IL-1 and TNF which are endogenous pyrogens. Increase COX activity and PG synthesis from AA. 2. Acute phase proteins produced: plasma protein synthesized in liver (increase 100 fold by inflammatory stimuli including IL-1, IL-6, TNF). C-reactive protein, fibrinogen, serum amyloid A protein (SAA). Prolonged increase in these factors can cause pathology (for ex, increase in SAA —> amyloidosis in kidney, brain, other organs) 3. Leukocytosis: accelerated release from bone marrow. IL-1 and TNF —> increase in # of immature cells (LEFT shift). Eventually, bone marrow output increases. Bacteria —> neutrophilia; viruses —> lymphocytosis. Bronchial asthma, hayfever, parasitic infections —> eosinophilia 4. Disseminated intravascular coagulation: LPS —> activated macrophages —> IL-1 and TNF production —> DIC & septic shock. (Hypoglycemia, cardiovascular failure). Can also lead to Neutrophil-mediated endothelial damage, causing respiratory, kidney and bowel failure

Characteristics of granulomatous inflammation

1. Inflammatory response characterized by predominance of epithelioid macrophages & variable # of lymphocytes or other leukocytes surrounding an indigestible organism, particle, or central necrotic area 2. Macs usually cluster in a characteristic semi-spherical formation around the causative agent. Epithelioid macs are larger pale cells w/ a single nucleus. Multinucleated giant cells may also be seen. These cells arise from chronically stimulated macs @ site of inflammation 3. Fibroplasia (fibrosis): often seen around/within granulomas 4. Delayed Type Hypersensitivity (DTH) reactions are the driving cause of these lesions (this is a form of immunopathology) Pyogranulomatous inflammation: epitheloid macrophages forming granulomas with neutrophils in center of granuloma & pus

Cells found in chronic inflammatory lesions

1. Lymphocytes- Ab & cell-mediated immune reactions 2. Macrophages & lymphocytes interact in a bidirectional way. These reactions play an important role in chronic inflammation. 3. Plasma cells- develop from activated B-lymphocytes. Produce Ab's against persistent antigen in the inflammatory site, or against altered tissue components 4. Eosinophils- activated by IgE in parasitic infections. These eosinophil granules contain major basic protein, which is toxic to parasites & mammalian epithelial cells. 5. Mast cells- widely distributed in CT. Fc receptors bind the Fc portion of IgE Ab. When this is bound & a specific antigen is recognized, the mast cells release mediators like histamine and products of AA oxidation

Morphological Hallmarks of Chronic Inflammation

1. Mononuclear cells infiltrate — (Macs & Lymphocytes). *Macs*= dominant player in chronic inflammation!! Macs are derived from blood monocytes, activated by cytokines, bacterial products, and/or necrotic cells. Monocytes 1/2 life= 1 day. Macrophage 1/2 life = months/years. 2. Tissue destruction- induced by a persistent offending agent 3. Attempts at healing- connective tissue/angiogenesis/fibrosis (scar formation)

Causes of Chronic Inflammation:

1. Persistent infections by certain microbes...like mycobacterium, viruses, fungi, and parasites. These are low toxicity, but evoke a DTH response, sometimes takes a pattern of granulomatous reactions 2. Prolonged exposure to toxic agents: like non-degradable inanimate material, atherosclerosis, chronic inflammation of the arterial wall 3. Autoimmunity - self-perpetuating immune reactions against self-tissues, chronic tissue damage, and inflammation (lupus, rheumatoid arthritis)

Forms of Acute Inflammation

1. Serous effusion - outpouring of thin fluid. - skin blister, plasma of mesothelial secretion in peritoneum, pleura 2. Fibrinous inflammation (NOT fibrous) - more severe, greater vascular permeability - fibrinogen passes vascular barrier - large leak, procoagulant stimulus - typical of body lining/cavity inflammation - fibrin is eosinophilic thread like material 3. Catarrhal inflammation - typical of mucosal surfaces, exudate resulting from overproduction of mucus mixed with neutrophils and other leukocytes. - Resolution: fibrinolysis, macrophage replacement with fibrous connective tissue (organization). In pericardium, pleura see thickening, opaque fibrous thickenings or tags.

The 4 mechs of increased vascular permeability

1. Stimulus induced formation of endothelial gaps in venules 2. Direct *endothelial injury * 3. Delayed-*prolonged leakages* (cytokines play a role) 4. *Leukocyte-mediated* endothelial injury *more than one mech typically operates during injury*

Outcomes of acute inflammation

1. complete resolution 2. healing by connective tissue 3. chronic, persistent inflammation that does not recede

5 cardinal signs of inflammation

1. rubor (redness) 2. tumor (swelling) 3. color (heat) 4. dolor (pain) 5. functio laesa (loss of function)

Inflammatory mediators, con't

10. oxygen free radicals: produced by NADPH oxidase system. products include superoxide (O2-), hydrogen peroxide (H2O2) and hypochlorite (HOCl-). - Effects: increase chemokine, cytokine and leukocyte adhesion. Amplifies inflammatory response Goal: destroy phagocytosed microbes (hypochlorite) Pathology: 1. endothelial damage 2. inactivate anti-proteases 3. injury to other cell types Endogenous anti-oxidants: 1. ceruloplasm - Cu2+ containing protein 2. transferrin 3. superoxide dismutase 4. catalase 5. glutathione peroxidase 11 . Neutrophil Extracellular Traps - innate cellular mechanism for defense against microorganisms (bacteria in particular).

Plasma Proteins - Complement system

2 actions: 1. *directly lyse cells* and 2. *activate other immune cells* - causes *increased vascular permeability, chemotaxis, and opsonizes pathogens* - inactive in plasma, when activated, C proteins become *proteolytic enzymes* that degrade other complement proteins - complement activation —> C3 cleavage - C functions are mediated by C3 products, other complement proteins, and the *membrane attack complex* (MAC) *Classical pathway* —> Ab on microbe; C binds to Ab opsonized molecule *Alternate pathway* —> C3 binds direct to microbe surface molecules including LPS & polysaccharides *Lectin pathway* —> mannose-binding lectins (Do we need to know more detail than this?)

Consequences of Neutrophil & Macrophage activation...*Phagocytosis*

3 steps of Phagocytosis: [NOTE: during phagocytosis, granule contents may be released into tissues & cause additional tissue damage & inflammation!!] 1. *Recognition & attachment*: - *mannose* (microbial cell wall) and *scavenger receptors*—> recognize elements of gram + and gram - bacteria - opsonized particles have *increased rate* of phagocytosis—> Ig-antibody, C3, plasma lectins opsonize 2. *engulfment* - cytoplasm "flows" around particle - fusion of lysosomes with phago. vacuoles 3. *killing & degradation* - *oxygen DEPENDENT* - Increased O2 consumption, glycogenolysis, and glucose oxidation is required for production of *ROIs* including superoxide (O2-), hydrogen peroxide (H2O2) & hypochlorite (HOCl-) - *Myeloperoxidase* makes *hypochlorite* (bleach) from hydrogen peroxide. Most efficient killing system in neutrophils. - microbial reactive oxygen intermediates are produced within phagocytic vesicles 3b. *killing & degradation* - *oxygen INDEPENDENT* leukocyte granules also contain: a. *lysozyme* - hydrolyzes N-acetyl glucosamine, bact. wall b. *lactoferrin* - Fe binding protein c. *defensins* - cationic arginine rich peptides toxic to bacteria d. *major basic protein* - parasites e. enzymes - e.g. elastase f. *bacterial permeability protein* (PL degradation) After killing, acid hydrolases degrade microbes within *phagolysosomes*

Forms of acute inflammation, con't

4. Suppurative/purulent Inflammation - large amounts of pus with neutrophils, necrotic cells, edema. - pyogenic bacteria are generally the cause. - abscess - collection of purulent inflammatory material deep in organ or contained space. - central mass of pus/necrotic tissue surrounded by neutrophils. - vascular dilation, parenchymal and fibroblastic proliferation - can be walled off and replaced with connective tissue

Inflammatory mediators, con't

5. Platelet activating factor (PAF) - phospholipid with glycerol backbone. elaborated by platelets, basophils, mast cells, neutrophils, mac's, endothelium. signals via GPCR. Induces platelets aggregation, vasoconstriction, bronchospasm, but also increased vascular permeability with 100-10,000x more potenty than histamine. PAF signaling system can trigger inflammatory and thrombotic cascades. increases leukocyte adhesion to endothelium, chemotaxis, degranulation and oxidative burst activity. 6. Cytokines- IL-1 and TNF - major pro-inflammatory cytokines with systemic effects. Produced by activated macrophages. Production is induced by endotoxin (LPS), microbial products binding to TLR, immune complexes, physical injury, other inflammatory stimuli. Activates endothelium, leukocytes, fibroblasts. Induces Acute Phase Reactions

Inflammatory mediators, con't

7. Chemokines: bind to seven transmembrane domain GPCR's - a chemokines C-X-C - b chemokines C-C - g chemokines C - chemo-attractants, stimulate leukocyte recruitment during inflammatory response, but also normal migration of cells through tissues. 8. Nitric Oxide: causes vasodilation, sm. muscle relaxation; released from vascular endothelium. decreases platelet aggregation/activation and leukocyte adhesion. NO is microbiocidal, reactive nitrogen kills bugs. NO production increases during host response. synthesized from L-arginine by NOS (nitric oxide synthase) - eNOS (endothelial) - nNOS (neuronal) - iNOS (inducible) - induced in macrophages and other cell by cytokines (TNF and IFNg) Functions of NO: 1. reduces platelet adhesion 2. relaxes vascular SM 3. decreases leukocyte adhesion 4. toxic to bugs

Inflammatory mediators, con't

9. lysosomal components: neutrophils + macs - small (specific) secondary granules - secreted (see image of MMP9 in neutrophils). MMP9 is a class of zinc-metalloproteinases...involved in the degradation of the ECM. - large (azurophilic) primary granules - 1o into phagosome lysosomal enzymes can exacerbate tissue damage - acid proteases - bacteria and debris - neutral proteases - extracellular components (collagen, BM, fibrin, elastin, cartilage). - also cleaves C3 and C5 releasing anaphylatoxins. - activity controlled by protease inhibitors in serum and tissue

Overview of Inflammatory Mediators

A. *Vasoactive amines* - increase vascular permeability. *Histamine, Serotonin* B. *Plasma Proteins* - *complement proteins*, *clotting system* (thrombin, fibrinogen, factor XIIa, fibrin), *kinin system* (*bradykinin*) C. *Plasma membrane/lipid derived mediators*- Arachidonic acid derived mediators, Prostaglandins, Thromboxane, Prostacyclines, Leukotrienes, Platelet activating factor D. *Immune Cell Factors* - Cytokines (IL-1, IL-6), Chemokines, Nitric oxide & oxygen-free radicals, Lysosomal products

Fibrinous pericarditis

A. Deposits of fibrin on the pericardium B. A pink meshwork of fibrin exudate (F) overlies the pericardial surface (P)

activated macrophages

A. TISSUE INJURY: - toxic oxygen metabolites - proteases - neutrophil chemotactic factors - coagulation factors - AA metabolites - Nitric oxide B. FIBROSIS: - growth factors (PDGF, FGF, TGF-beta) - fibrogenic cytokines - angiogenesis factors (FGF) - "remodeling" collagenesis

KNOW the key stuff here

Activation by *factor XIIa*

Anti-inflammatory drugs target Arachidonic acid metabolsim

COX Inhibitors - aspirin, NSAIDS. inhibit prostaglandin synthesis, aspiring acetylates COX-1 and COX-2. COX-2 inhibitors target inflammation specifically. also, prolong bleeding time by inhibiting TXA2 formation LOX inhibitors - used in asthma in cats & recurrent airway obstruction in horses. May be useful in treatment of asthma where leukotrienes play a role in pathogenesis Glucocorticoids - decrease expression of genes encoding COX-2, PLA2 directly, IL-1, TNF, and iNOS. Increase expression of anti-inflammatory genes, inhibit AA release from membranes

Horse skin

Eosinophilia Granuloma in horse skin due to invading parasite

Local & systemic effects of IL-1 and TNF

Produced by macrophages... has systemic effects. Pro-inflammation

Issue with immune response?

Substances that kill pathogens also lead to tissue *injury* *lysosomal enzymes* secreted into EC milieu - regurgitation during feeding - indigestible materials deposited on immovable surfaces (e.g. glomerular basement membrane). *reactive oxygen species* (ROS), and products of *arachidonic acid metabolism* can also induce collateral damage

T-cells exiting high endothelial venules

T-cells (red) exiting HEVs (green) in a lymph node via lucent areas which appear to be *gaps* in vessel

NOT to be confused with...fibrous adhesions on a canine spleen

The end result of healing. Note: grey tags, highly adherent, tough CT.

Macrophages

The products of activated mac's eliminate injurious agents, and initiate repair. Macs are responsible for much of tissue injury in chronic inflammation (ROI's, NO, extracellular matrix proteases) Also, induce fibroblast activation and proliferation (IL-1 and TNF), collagen deposition and angiogenesis tissue destruction is a hallmark of chronic inflammation Other substances that can promote chronic inflammation: 1. Necrotic tissue: perpetuates the inflammatory cascade 2. Activated kinin, coagulation, complement, and fibrinolytic systems 3. Ongoing tissue destruction can activate the inflammation cascade 4. Acute and chronic inflammation often CO-EXIST

Leukocyte Activation

Triggered by *microbes, necrotic cell components, Ag-Ab complexes, cytokines, chemokines* 1. *Toll like receptors* (TLR - homologues of drosophila Toll) recognize *pathogen derived molecules* (LPS:endotoxin), LTA, dsRNA, CpG DNA, proteoglycans, viral RNA (pathogen associated molecular patterns (PAMPS), and DAMPs. 2. *GPCR* bind *microbial products and cellular mediators* produced in response to tissue injury. These are: fMLP, chemokines, complement C5a, lipid mediators (PAF), arachidonic acid, prostaglandin E, leukotriene B4, etc. • Modulate *adhesion and leukocyte migration* from the blood • increase *respiratory burst* activity • activate G-proteins which increase Ca2+, activate PKC, stimulate cytoskeleton, cause degranulation, cytokines 3. *Cytokines*- receptors on phagocytes bind these (like *IFNg which is secreted by lymphocytes & activates macrophages). Also, *interleukins*. This is a "complex symphony" not well understood. 4. *Opsonin* receptors (target opsonization). *Opsonin* = anything that binds & coats a pathogen making it more recognizable to the immune system. Ex: - *antibodies* - bind Fcg receptors (FcgR) on phagocytes cells - *complement* - C3 binds to C-receptors via the classic mech (antibody dependent). Many bacteria activate C (independent of antibodies) - *lectins* - plasma proteins including mannose-binding lectin, fibronectin, fibrinogen, C-reactive protein coat microbes & are recognized by *phagocytic receptors*

1. Complete Resolution

mediators decay quickly, tissue regeneration, vascular permeability returns to normal. decrease in leukocyte infiltration, neutrophils die, edema and proteins removed 1. return of normal vascular permeability 2. edema & proteins drain --> lymphatics or by pinocytosis into macrophages 3. phagocytosis of apoptotic neutrophils & of necrotic debris 4. disposal of macrophages (macs also produce factors that initiate repair. Note central role of macs in resolution!!) WHY does this happen? - mediators have short 1/2 life - production & release of TGF-beta from macrophages - production & release of IL-10 - production of anti-inflammatory lipoxins - downreg of pro-inflammatory molecules (like leukotrienes) - upreg of anti-inflammatory molecules like IL-1 - apoptosis of pro-inflammatory cells - cleavage of chemokines by matrix MMPs might lead to production of anti-inflammatory factors KEY: inflammatory response must be actively terminated