Chapter 3 - Inflammation and Repair Flashcards

Question

Leukocyte migration through endothelium

Answer 1

**The next step in the process of leukocyte recruitment is migration of the leukocytes through endothelium, called *transmigration*, or *diapedesis*.** This occurs mainly in postcapillary venules. PECAM-1 (platelet endothelial cell adhesion molecule) (a.k.a. CD31) is expressed in intercellular junctions of endothelial cells, and mediates leukocyte migration through endothelium. Leukocytes then pierce basement membrane by secreting collagenases, and enter extravascular space.

Answer 2

**After exiting the circulation, leukocytes move in the tissues toward the site of injury by chemotaxis**. Exogenous chemoattractants: bacterial products (peptides with N-formylmethionine terminal aa) Endogenous chemoattractants: Cytokines (IL-8), components of complement systme (C5a), arachadonic acid metabolites (leukotriene B4). All of these bind to specific 7-transmembrane G protein-coupled receptors on leukocytes --\> activate messengers that increase cytosolic calcium and activate small guanosine triphosphates (Rac/Rho/cdc42 family) --\> polymerization of actin at leading edge of cell and localization of myosin filaments at the back --\> leukocyte moves by extending filopodia **The nature of the leukocyte infiltrate varies with the age of the inflammatory response and the type of stimulus.** Usually in acute inflammation, neutrophils predominate the first 6-24 hours, replaced by monocytes in 24-48 hours. Exceptions: - Pseudomonas infection continuously recruits neutrophils for days - Viral infections may have lymphocytes arrive first - Some hypersensitivity reactions are dominated by lymphs, macs, plasma cells - Allergic reactions often are mostlyeosinophils

Answer 3

Nature of leukocyte infiltrates in inflammatory reactions. The photomicrographs show an inflammatory reaction in the myocardium after ischemic necrosis (infarction). A, Early (neutrophilic) infiltrates and congested blood vessels. B, Later (mononuclear) cellular infiltrates. C, The approximate kinetics of edema and cellular infiltration. For simplicity, edema is shown as an acute transient response, although secondary waves of delayed edema and neutrophil infiltration can also occur.

Answer 4

**Recognition of microbes or dead cells induces several responses in leukocytes that are collectively called *leukocyte activation.*** Activation results in increased cytosolic [Ca2+], enzyme activation (protein kinase C, phospholipase A2)

Answer 5

Leukocyte activation. Different classes of cell surface receptors of leukocytes recognize different stimuli. The receptors initiate responses that mediate the functions of the leukocytes. Only some receptors are depicted (see text for details). LPS first binds to a circulating LPS-binding protein (not shown). IFN-γ, Interferon-γ; LPS, lipopolysaccharide.

Answer 6

1. Recognition and attachment of the particle to be ingested by the leukocyte 2. Engulfment, with subsequent formation of a phagocytic vacuole 3. Killing or degradation of the ingested molecule

Answer 7

Phagocytosis and intracellular destruction of microbes. Phagocytosis of a particle (e.g., a bacterium) involves binding to receptors on the leukocyte membrane, engulfment, and fusion of the phagocytic vacuoles with lysosomes. This is followed by destruction of ingested particles within the phagolysosomes by lysosomal enzymes and by reactive oxygen and nitrogen species. The microbicidal products generated from superoxide () are hypochlorite (HOCl−) and hydroxyl radical (−OH), and from nitric oxide (NO) it is peroxynitrite (OONO−). During phagocytosis, granule contents may be released into extracellular tissues (not shown). MPO, Myeloperoxidase; iNOS, inducible NO synthase; ROS, reactive oxygen species.

Answer 8

**Mannose receptors, scavenger receptors, and receptors for various opsonins bind and ingest microbes.** The macrophage *mannose receptor* is a lectin that binds terminal mannose and fucose residues on glycoproteins/glycolipids found in microbial cell walls (mammalian cells contain terminal sialic acid or N-acetylgalactosamine). *Scavenger receptors* bind and endocytose oxidized or acetylated LDL particles that can't interact with conventional LDL receptors. Macrophage scavenger receptors also bind microbes. Phagocytosis efficiency is greatly enhanced when microbes are opsonized with complement (C3b) or antibodies (IgG), for which phagocytes express high-affinity receptors

Answer 9

After a particle is bound to phagocyte receptors, cytoplasmic pseudopods flow around it and the membrane pinches off to form a vesicle (phagosome) that encloses the particle. The phagosome then fuses with a lysosomal granule, which discharges contents into the new phagolysosome.

Answer 10

**Killing of microbes is accomplished by reactive oxygen species and reactive nitrogen species (mainly derived from NO), and these as well as lysosomal enzymes destroy phagocytosed debris.** 1. **Reactive oxygen species (ROS)** - Produced by rapid assembly and activation of NADPH oxidase (a.k.a. phagocyte oxidase), which oxidizes NDADPH, reducing oxygen to superoxide (O2\*). In neutrophils, this accompanies phagocytosis and is called *respiratory burst*. In response to activating stimuli, plasma membrane and cytosolic components translocate to phagosomal membrane and assemble into NADPH oxidase, which produces ROS *within the lysosome, and phagolysosome*. - O2\* is then converted to H2O2 by spontaneous dismutation. MPO in neutrophilic granules uses Cl- to convert H2O2 to hypochlorite (OCl₂^-- hypochlorite, bleach) which destroys microbes by halogenation - H2O2 also is converted to hydroxyl radical (-OH) - MPO deficiency leads to only minimal increase in susceptibility to infection - emphasizes redundancy of microbicidal mechanisms - These ROS are implicated in tissue damage with inflammation - Antioxidants (superoxide dismutase, catalase, glutathione peroxidase, ceruloplasmin, iron-free transferrin) are in serum, tissue fluids, and host cells 2. **Nitric oxide** - NO is a soluble gas produced from arginine with nitric oxide synthase (NOS) - endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutively expressed at low levels to maintain vascular tone and as a neurotransmitter, respectively - inducible NOS (iNOS) is involved in microbicidal killing and is induced when macrophages and neutrophils are activated by cytokines (e.g. IFN-y) or microbial products - NO reacts with O2\* to generate highly reactive free radical peroxynitrite (ONOO-) - nitrogen-derived free radicals, like ROS, attack and damage lipids, proteins, nucleic acids of microbes 3. **Lysosomal enzymes and other lysosomal proteins** - Neutrophils and monocytes contain lysosomal granules that contribute to microbial killing and tissue damage - Neutrophils have smaller *specific* (secondary) granules with lysozyme, collagenase, gelatinase, lactoferrin, plasminogen activator, histaminase, alkaline phosphatase) - They also have larger *azurophil* (primary) granules with MPO, bactericidal factors (lysozyme, defensins), acid hydrolases, a variety of neutral proteases (elastase, cathepsin G, proteinase 3) + Acid proteases - degrade bacteria and debris within phagolysosomes, which are acidified by membrane H+ pumps + Neutral proteases can degrade various extracellular components (collagen, BM, fibrin, elastin, cartilage) resulting in tissue damage + Neutrophil elastase can degrade virulence factors of bacteria - Both types of granules can fuse with phagosomes or release contents into extracellular space - Excessive tissue damage from proteases are controlled by *antiproteases* (e.g. a1-antitrypsin is major inhibitor of neutrophil elastase, a2-Macroglobulin) - Defensins: cationic arginine-rich granules toxic to microbes - Cathelicidins: antimicrobial proteins in neutrophils - Lysozymes: hydrolyzes muramic acid-N-acetylglucosamine bond in bacteria coat - Lactoferrin: iron-binding protein in specific granules - Major basic protein: cationic protein of eosinophils, cytotoxic to many parasites

Answer 11

**NETs are extracellular fibrillar networks that provide a high concentration of antimicrobial substances at sites of infection and prevent the spread of the microbes by trapping them in the fibrils.** They are produced by neutrophils in response to infectious pathogens and inflammatory mediators. They consist of a meshwork of nuclear chromatin that binds granule proteins. This leads to death of the neutrophils involved NETs have been seen in blood during sepsis - formation thought to be dependent on platelet activation

Answer 12

* *Leukocytes are important causes of injury to normal cells and tissues under several circumstances:** (1) Part of normal defense reaction against infection, when adjacent tissue suffers collateral damage. In infections that are tough to clear (tuberculosis, certain viral diseases) the host response can be more damaging than the infection (2) When inflammatory response is inappropriately directed at host tissue (i.e. autoimmune diseases) (3) When the host reacts excessively against usually harmless environmental substances (i.e. allergic disease) In all these situations, **mechanisms by which leukocytes damage normal tissues are the same as mechanisms involved in microbicide** (i.e. ROS, NO, lysosomal enzymes)

Answer 13

1. Produce cytokines that can amplify or limit inflammatory reactions, growth factors that stimulate endothelial and fibroblast proliferation, and synthesis of collagen and enzymes for tissue repair (mainly macrophages) 2. T lymphocytes contribute to acute inflammation (Th17 cells produce IL-17, which amplifies neutrophil response)

Answer 14

Inflammation declines after offending agents are removed because mediators are produced in rapid bursts and have short half-lives and neutrophils die quickly in tissue by apoptosis As inflammation develops, stop signals are built in that actively terminate the reaction (e.g. switch from proinflammatory leukotrienes to antiinflammatory lipoxins, liberation of antiinflammatory cytokines like TGF-b, IL-10

Answer 15

**Mediators of inflammation are substances that initiate and regulate inflammatory reactions.** **1. Most important mediators of acute inflammation are vasoactive amines, lipid products (prostaglandins, leukotrienes), cytokines (including chemokines), and products of complement activation.** **2. Mediators are either secreted by cells or generated from plasma proteins**. Cell-derived mediators are either stored in granules and can be rapidly exocytosed (e.g. histamine in mast cells) or are synthesized de novo (e.g. prostaglandins, leukotrienes, cytokines) in response to a stimulus. **Sentinels that detect invaders and damage in tissues (macrophages, dendritic cells, mast cells) are the major producers of acute inflammation mediators.** Platelets, neutrophils, epithelial and endothelial cells also elaborate some. *Plasma-derived mediators* (e.g. complement proteins) are produced mostly in the liver and circulate as inactive precursors. **3. Active mediators are produced only in response to various stimuli**. Stimuli include microbial products, stuff from necrotic cells **4. Most of the mediators are short-lived.** They decay quickly or are inactivated by enzymes, or are scavenged/inhibited. **5. One mediator can stimulate the release of other mediators**.

Answer 16

**The two major vasoactive amines are histamine and serotonin.** These are stored as pre-formed molecules in cells and are among the first mediators released in inflammation. Mast cells in perivascular connective tissue are the richest source of histamine; it is also found in basophils and platelets. Mast cells degranulate, releasing histamine due to (1) physical injury, (2) binding of antibodies to mast cells - allergic reactions, (3) products of complement called *anaphylatoxins* (C3a, C5a) **Histamine causes dilation of arterioles and increases permeability of venules.** It is the principal mediator of the immediate transient phase of increased vascular permeability, producing interendothelial gaps in venules. Serotonin is a preformed vasoactive mediator in platelets and certain neuroendocrine cells (e.g. GI tract). It is a neurotransmitter in GI tract and a vasoconstrictor.

Answer 17

**The lipid mediators *prostaglandins* and *leukotrienes* are produced from arachidonic acid (AA) present in membrane phospholipids, and stimulate vascular and cellular reactions in acute inflammation.** With mechanical, chemical, physical, or other (e.g. C5a) stimuli, phospholipases (e.g. phospholipase A2) are activated by increased cytoplasmic [Ca2+], and various kinase activation AA-derived mediators (a.k.a. eicosanoids) are synthesized in 2 pathways: (1) cyclooxygenases (COX): generate prostaglandins (2) lipoxygenases (LOX): Produce leukotrienes and lipoxins

Answer 18

**Prostaglandins (PGs) are produced by mast cells, macrophages, endothelial cells, and many other cell types, and are involved in the vascular and systemic reactions of inflammation.** Generated by: (1) COX-1: constitutively expressed in most tissues (e.g. fluid and electrolyte balance in kidneys, cytoprotection in GI tract), also produced in response to inflammation (2) COX-2: Induced by inflammatory stimuli to create pro-inflammatory prostaglandins, but otherwise is low or absent in normal tissue Most important prostaglandins for inflammation: **PGE₂, PGD₂, PGF_2a, PGI₂ (prostacyclin), and TxA₂ (thromboxane A2)** Some are tissue specific: - TxA2 is produced in platelet with thromboxane synthase: vasoconstriction, platelet aggregation - PGI2 is produced in vascular endothelium with prostacyclin synthase: vasodilation, inhibitor of platelet aggregation, promotes vascular permeability - PGD2, and PGE2 (more widely distributed) are produced in mast cells: vasodilation, increased permeability of postcapillary venules --\> edema - PGF2a stimulates contraction of uterine and bronchial smooth muscle - PGD2 is a chemoattractant for neutrophils PGs also involved in pain and fever pathogeneses

Answer 19

**Leukotrienes are produced by leukocytes and mast cells by the action of lipoxygenase and are involved in vascular and smooth muscle reactions and leukocyte recruitment.** 5-LOX predominant one in neutrophils: converts AA to 5-hydroxyeicosatetraenoic acid) 5-HPETE, which is chemotactic for neutrophils and is the precursor of the leukotrienes LTB4 is a potent chemotactic agent and activator of neutrophils LTC4, LTD4, LTE4 cause intense vasoconstriction, bronchospasm, increased venule permeability

Answer 20

**Lipoxins are also generated from AA by the lipoxygenase pathway, but unlike PGs and LTs, the lipoxins suppress inflammation by inhibiting the recruitment of leukocytes.** Require two cell types for production: leukocytes (mostly neuts) produce intermediates in lipoxin synthesis, that are then converted to lipoxins by platelets interacting with the leukocytes

Answer 21

Production of arachidonic acid metabolites and their roles in inflammation. Note the enzymatic activities whose inhibition through pharmacologic intervention blocks major pathways (denoted with a red X). COX-1, COX-2, Cyclooxygenase 1 and 2; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid.

Answer 22

**1. Cyclooxygenase inhibitors** (e.g. NSAIDs): inhibit COX-1 and COX-2, inhibiting PG synthesis. Selective COX-2 inhibitors can be 200-300 more potent in COX-2 inhibition than COX-1. **2. Lipoxygenase inhibitors:** useful to treat asthma **3. Corticosteroids:** reduce transcription of genes for COX-2, phospholipase A2, proinflammatory cytokines (e.g. IL-1, TNF), and iNOS **4. Leukotriene receptor antagonists** - useful in treating asthma

Answer 23

**Cytokines are proteins produced by many cell types (principally activated lymphocytes, macrophages, and dendritic cells, but also epithelial, endothelial, connective tissue cells) that mediate and regulate immune and inflammatory reactions.**

Answer 24

**TNF and IL-1 serve critical roles in leukocyte recruitment by promoting adhesion of leukocytes to endothelium and their migration through vessels.** Produced mainly by activated macrophages and dendritic cells - TNF also produced by T lymphs and mast cells - IL-1 is produced by some epithelial cells Secretion stimulated by microbial products, immune complexes, foreign bodies, physical injury, and a variety of other inflammatory stimuli, TNF and IL-1 generation induced by signaling through TLRs (generation of active IL-1 requires inflammasome)

Answer 25

**1. Endothelial activation**: increased expression of adhesion molecules, mostly E- and P-selectins and integrin ligands, increased production of cytokines chemokines, growth factors, eicosanoids, increased procoagulant activity **2. Activation of leukocytes and other cells**: TNF augments neutrophil responses to other stimuli and stimulates microbicidal activity of macrophages, in part by inducing NO production. IL-1 activates fibroblasts to synthesize collagen and stimulates synovial and other mesenchymal proliferation. IL-1 also stimulates Th17 responses **3. Systemic acute-phase response**: They induce APR, including *fever*. They're also implicated in sepsis from disseminated bacterial infection. Sustained TNF production --\> cachexia due to increased lipid/protein mobilization

Answer 26

Major roles of cytokines in acute inflammation. PDGF, Platelet-derived growth factor; PGE, prostaglandin E; PGI, prostaglandin I.

Answer 27

**Chemokines are a family of small (8-10 kD) proteins that act primarily as chemoattractants for specific types of leukocytes.** Classified according to arrangement of cysteine (C) residues: (1) C-X-C group (e.g. IL-8): In response to microbial products, IL-1, TNF, these are secreted by activated macs and endothelial cells, causing activation and chemotaxis for neutrophils (2) C-C group (e.g. MCP-1, eotaxin, MIP-1a, RANTES): Chemoattractant, works for monocytes, eosinophils, basophils, lymphocytes (3) C group (lymphotactin): specific for lymphocytes (4) CX₃C group (fractalkine): 2 forms - an endothelial form promoting adhesion of monocytes and T cells, and soluble form that is chemoattractant for same cells Bind to seven-transmembrane G protein-coupled receptors with overlapping ligand specificities Two main functions of chemokines: * *1. In acute inflammation:** *Inflammatory chemokines* induced by microbes stimulate leukocyte attachment to endothelium and their migration to site of damage * *2. Maintenance of tissue architecture:** *homeostatic chemokines* are constitutively produced, organizing various cell types into different anatomic regions (e.g. T and B zones of spleen, LNs)

Answer 28

IL-6 (made by macs and others) is involved in local and systemic acute inflammation IL-17 promotes neutrophil recruitment Type I interferons inhibit viral replication, and contribute to systemic manifestations of inflammation

Answer 29

**The complement system is a collection of soluble proteins and membrane receptors that function mainly in host defense against microbes and in pathologic inflammatory reactions.** Consists of \> 20 protiens (e.g. C1 - C9), and functions in innate and adaptive immunity against microbes. Inactive complement proteins circulate, and are activated in an enzymatic cascade. Cleavage products of complement proteins are elaborated that increase vascular permeability, chemotaxis, and opsonization * *The critical step in complement activation is proteolysis of C3. This happens one of three ways:** 1. Classical pathway - triggered by C1 fixation to IgM or IgG combined with antigen 2. Alternative pathway - triggered by microbial surface molecules (e.g. LPS), complex polysaccharides, cobra venom, others 3. Lectin pathway - plasma mannose-binding lectin binds carbs on microbes to activate C1 **All three of these lead to formation of C3 convertase, which splits C3 into C3a and C3b.** C3a is released, C3b covalently binds to cell/molecule to be activated. This results in C5 convertase which cleaves C5 into C5a and C5b; C5b stays attached and binds C6-C9 to form membrane attack complex (MAC, multiple C9s)

Answer 30

1. **Inflammation**: C3a, C5a, and less C4a (a.k.a. anaphylatoxins) stimulate histamine release from mast cells, increasing vascular permeability and causing vasodilation. C5a also chemotactic for neuts, monos, eos, basos and it activates LOX pathway in neuts and monos --\> release of more mediators **2. Opsonization and phagocytosis**: C3b inactive C3b fix to microbe cell wall, promoting their phagocytosis by neuts and macs **3. Cell lysis:** Deposition of MAC on cells makes them permeable to water and results in death, also works on bacteria with thin walls (e.g. Neisseria)

Answer 31

**Activation of complement is tightly controlled by cell-associated and circulating regulatory proteins.** **- C1 inhibitor (C1 INH)** blocks C1 activation; inherited deficiency is cause of *hereditary angioedema* **- Decay accelerating factor (DAF) and CD59:** 2 proteins linked to GPI anchor. DAF inhibits C3 convertase formation. CD59 inhibits MAC formation

Answer 32

The activation and functions of the complement system. Activation of complement by different pathways leads to cleavage of C3. The functions of the complement system are mediated by breakdown products of C3 and other complement proteins, and by the membrane attack complex (MAC).

Answer 33

1. Platelet-Activating Factor (PAF): phospholipid-derived mediator that causes platelet aggregation, and has inflammatory effects (vasodilation at low concentration, vasoconstriction and bronchoconstriction at higher concentrations). Produced by platelets, basos, mast cells, neuts, macs, endotehlial cells 2. Products of coagulation: Coagulation and inflammation are linked processes. Protease-activated receptors (PARs) are activated by thrombin and expressed on platelets and leukocytes. 3. Kinins (e.g. HMW bradykinin): Vasoactive peptides derived from kininogens by the action of kallikrein enzymes. **Bradykinin increases vascular permeability and causes contraction of smooth muscle, vasodilation, and pain.** 4. Neuropeptides (e.g. substance P, neurokinin A): secreted by sensory nerves and leukocytes, may help initiate and regulate inflammation. Substance P helps transmit pain, regulate blood pressure, stimulate hormone secretion, and increase vascular permeability.

Answer 34

**The morphologic hallmarks of acute inflammatory reactions are dilation of small blood vessels and accumulation of leukocytes and fluid in the extravascular tissue.**

Answer 35

**Serous inflammation is marked by the exudation of cell-poor fluid** into spaces created by cell injury or into body cavities lined by mesothelium (i.e. effusions). Typically the fluid does not have organisms and only has a few leukocytes.

Answer 36

**A fibrinous exudate develops when vascular leaks are large or there is a local procoagulant stimulus** (e.g. cancer cells). Large molecules (i.e. fibrin) leak out and are deposited extracellularly. Conversion of fibrinous exudate to scar tissue is called organization

Answer 37

**Purulent inflammation is characterized by the production of pus, an exudate consisting of neutrophils, the liquefied debris of necrotic cells, and edema fluid.** Most frequent cause of pus is bacterial infection ## Footnote **Abscesses are localized collections of purulent tissue.**

Answer 38

**An ulcer is a local defect, or excavation, of the surface of an organ or tissue that is produced by the sloughing (shedding) of inflamed necrotic tissue.**

Answer 39

**All acute inflammatory reactions typically have one of three outcomes:** **1. Complete resolution**: This is the usual outcome with a limited, short-lived injury that caused little tissue destruction in a regenerative spot. **2. Healing by connective tissue replacement (scarring, or fibrosis).** This occurs after substantial tissue destruction, when tissues incapable of regeneration are involved, or when there is abundant fibrin exudation into cavities that can't be cleared. Connective tissue grows into the area, converting it to a mass of fibrous tissue (i.e. organization) **3. Progression to chronic inflammation**: Occurs when the acute response cannot be resolved either due to persistence of the agent or interference with normal healing processes.

Answer 40

Outcomes of acute inflammation: resolution, healing by fibrosis, or chronic inflammation. The components of the various reactions and their functional outcomes are listed.

Answer 41

**Chronic inflammation is a response of prolonged duration (weeks or months) in which inflammation, tissue injury, and attempts at repair coexist in varying combinations.** This may follow acute inflammation, or begin insidiously as a low-grade, smoldering response without an acute phase.

Answer 42

**1. Persistent infections**: Some organisms are tough to eradicate (e.g. mycobacteria, viruses, fungi, parasites), and often evoke a delayed-type hypersensitivity response. **2. Hypersensitivity diseases**: Excessive and inappropriate activation of the immune system (e.g. autoimmune diseases, allergic diseases **3. Prolonged exposure to potentially toxic agents, either exogenous or endogenous**: (e.g. exogenous: silica, endogenous: endogenous cholesterol and other lipids in atherosclerosis)

Answer 43

**1. Infiltration with mononuclear cells (e.g. macs, lymphs, plasma cells)** **2. Tissue destruction** **3. Attempts at healing by connective tissue (i.e. fibrosis, angiogenesis)**

Answer 44

A, Chronic inflammation in the lung, showing all three characteristic histologic features: (1) collection of chronic inflammatory cells (\*), (2) destruction of parenchyma (normal alveoli are replaced by spaces lined by cuboidal epithelium, arrowheads), and (3) replacement by connective tissue (fibrosis, arrows). B, In contrast, in acute inflammation of the lung (acute bronchopneumonia), neutrophils fill the alveolar spaces and blood vessels are congested.

Answer 45

**The dominant cells in most chronic inflammatory reactions are macrophages, which contribute to the reaction by secreting cytokines and growth factors that act on various cells, by destroying foreign invaders and tissues, and by activating other cells, notably T lymphocytes.** **Macrophages are tissue cells derived from hematopoietic stem cells in bone marrow and from progenitors in the embryonic yolk sac and fetal liver during early development.** Half-life of monocytes in blood is about 1 day, life span of tissue macrophages is months to years * *There are two major pathways of macrophage activation:** * *1. Classical macrophage activation:** - induced by microbial products (e.g. LPS), and IFN-y which engage TLRs - M1 (classically activated) macs produce NO and ROS and upregulate lysosomal enzymes to enhance microbicidal ability and secrete cytokines for inflammation * *2. Alternative macrophage activation:** - induced by cytokines (e.g. IL-13, IL-4 from T lymphs) - main function of M2 macs is tissue repair (secrete GFs for angiogenesis, fibroplasia, collagen synthesis) * *The products of activated macrophages eliminate injurious agents and initiate tissue repair, but are also responsible for much of the tissue injury in chronic inflammation due to these functions:** (1) Macs ingest and eliminate microbes and dead tissue (2) Macs initiate tissue repair and are involved in scar formation and fibrosis (3) Macs secrete inflammatory mediators (e.g. TNF, IL-1, chemokines) and eicosanoids (4) Macs display antigens to T lymphs and respond to signals from T lymphs, in a feedbackloop necessary for defense

Answer 46

Maturation of mononuclear phagocytes. A, In the steady state, some tissue macrophages, including microglia and alveolar macrophages, may be derived from embryonic precursors and populate the tissues. The development of macrophages from hematopoietic precursors and monocytes may be more prominent when tissue macrophages need to be increased or replenished, as after injury and during inflammation. B, The morphology of a monocyte and activated macrophage.

Answer 47

Classical and alternative macrophage activation. Different stimuli activate monocytes/macrophages to develop into functionally distinct populations. Classically activated macrophages are induced by microbial products and cytokines, particularly IFN-γ. They phagocytose and destroy microbes and dead tissues and can potentiate inflammatory reactions. Alternatively activated macrophages are induced by other cytokines and are important in tissue repair and the resolution of inflammation.

Answer 48

**Microbes and other environmental antigens activate T and B lymphocytes, which amplify and propagate chronic inflammation.** Major function of these lymphocytes is adaptive immunity, but they are often present in chronic inflammation, and when activated, cause persistent and severe inflammation. * *By virtue of their ability to secrete cytokines, CD4+ T lymphocytes promote inflammation and influence the nature of the inflammatory reaction.** Three subsets of these: (1) Th1 cells produce IFN-y, activating macs classically (2) Th2 cells secrete IL-4, IL-5, IL-13, recruiting eos and alternatively activating macs (3) Th17 cells secrete IL-17, which are important for neutrophil recruitment **Activated B lymphocytes and antibody-producing plasma cells are often present at sites of chronic inflammation.**

Answer 49

**1. Eosinophils**: abundant in immune reactions mediated by IgE and in parasitic infections.They have major basic protein in their granules, which is toxic to parasites and can lyse mammalian epithelial cells (contribute to tissue damage in allergies) **2. Mast cells**: widely distributed in CT, participate in acute and chronic inflammation **3. Neutrophils:** characteristic cells of acute inflammation, but many forms of chronic inflammation may have many neutrophils (e.g. persistent microbes, osteomyelitis). Sometimes called *acute on chronic*

Answer 50

Macrophage-lymphocyte interactions in chronic inflammation. Activated T cells produce cytokines that recruit macrophages (TNF, IL-17, chemokines) and others that activate macrophages (IFN-γ). Activated macrophages in turn stimulate T cells by presenting antigens and via cytokines such as IL-12.

Answer 51

**Granulomatous inflammation is a form of chronic inflammation characterized by collections of activated macrophages, often with T lymphocytes, and sometimes associated with central necrosis.** Two types of granulomas: 1. **Foreign body granulomas:** incited by inert foreign bodies in absence of T cell-mediated responses. Epithelioid and giant macrophages are apposed to the surface of the foreign material **2. Immune granulomas**: caused by a variety of agents capable of inducing a **persistent T cell-mediated response.** Macs activate T cells to produce cytokines (e.g. IL-2), which activates other T cells, and IFN-y, which activates macrophages. **Tuberculosis is the prototype of a granulomatous disease caused by infection and should always be excluded as the cause when granulomas are identified.**

Answer 52

**Inflammation, even if it is localized, is associated with cytokine-induced systemic reactions that are collectively called the acute-phase response.** **The cytokines TNF, IL-1, IL-6 are important mediators of the APR; other cytokines, notably type I interferons, also contribute to the reaction.** APR consists of several clinical and pathologic changes: * *1. Fever**: Substances that induce fever are called pyrogens. Bacterial products (e.g. LPS) stimulate leukocytes to release **IL-1, TNF** (endogenous pyrogens) that increase conversion of AA to prostaglandins; in the hypothalamus, these PGs (mostly PGE2) reset the body temperature set point at a higher level. * *2. Acute-phase proteins** (e.g. CRP, fibrinogen, SAA): plasma proteins, mostly synthesized in the liver, whose concentrations increase several hundred-fold as part of the response to inflammatory stimuli. Their synthesis is stimulated by cytokines (IL-1 or TNF for SAA, IL-6 for CRP and fibrinogen). These have beneficial effects during inflammation, but too much in chronic inflammation causes secondary amyloidosis (especially SAA). * *3. Leukocytosis**: common feature of inflammation, can reach up to 100,000 / uL (i.e. leukemoid response). **4. Others:** Increased pulse and blood pressure, decreased sweating, chills, rigors, anorexia, malaise

Answer 53

**Repair, sometimes called healing, refers to the restoration of tissue architecture and function after an injury.** * *Repair of damaged tissues occurs by two types of reactions: 1. Regeneration:** Proliferation of residual cells and maturation of tissue stem cells. Only certain tissues are capable of this (epithelia of skin and intestines, liver), and the framework needs to remain intact. * *2. Connective tissue deposition (scar formation):** In tissues that cannot regenerate, or if damage is severe enough, fibrous tissue is laid down, which provides structural stability enough that the tissue can usually function. *Fibrosis:* extensive deposition of collagen as a consequence of chronic inflammation.

Answer 54

Mechanisms of tissue repair: regeneration and scar formation. Following mild injury, which damages the epithelium but not the underlying tissue, resolution occurs by regeneration, but after more severe injury with damage to the connective tissue, repair is by scar formation.

Answer 55

**1. proliferation** **2. growth factors** **3. extracellular matrix**

Answer 56

**1. Labile (continuously dividing) tissues (e.g. hematopoietic cells, surface epithelia (e.g. skin, oral, vagina, cervix, exocrine ducts, GI), transitional epithelium):** These are continuously being lost and replaced by maturation from tissue stem cells and proliferation of mature cells. These tissues can readily regenerate after injury as long as the pool of stem cells is preserved. **2. Stable tissues (e.g. parenchyma of most solid tissues (liver, kidney, pancreas), endothelial cells, fibroblasts, smooth muscle cells):** These cells are quiescent (in the G0 stage of cell cycle), and have minimal proliferative activity in the normal state. They are capable of dividing in response to injury or loss of tissue mass. Limited regenerative capacity after injury (exception = liver) **3. Permanent tissues (e.g. neurons, cardiac muscle cells)**: Cells are terminally differentiated and nonproliferative postnatally. Injury to the brain or heart is irreversible and results in a scar.

Answer 57

1. Macrophages - main source; epithelial and stromal cells - some 2. (1)Several GFs bind to ECM proteins and are displayed at high conc. (2) Cells use integrins to bind ECM proteins, transduces signals to drive cell proliferation

Answer 58

That's all

Answer 59

1. In labile tissues, regeneration is rapid if BM is intact. 2. Hematopoietic cell proliferation secondary to reduced numbers of blood cells is driven by colony-stimulating-factors (CSFs) 3. Tissue regeneration can occur in stable organs, but is slow (exception = liver) 4. Restoration of normal tissue structure can only occur if the residual is structurally intact (e.g. surgical reserction); if tissue is damaged by infection or inflammation, regeneration is incomplete and accompanied by scarring.

Answer 60

**The human liver has a remarkable capacity to regenerate, as demonstrated by its growth after partial hepatectomy.** **Regeneration of the liver occurs by two major mechanisms:** * *1. Proliferation of remaining hepatocytes:** - In humans, resection of up to 90% of the liver can be corrected by proliferation of hepatocytes - **hepatocyte proliferation in teh regenerating liver is triggered by cytokines and GFs in three stages:** (1) *priming* phase: IL-6 and other cytokines produced by Kupffer cells and act on hepatocytes to ready them to receive and respond to GFs (2) *growth factor* phase: HGF, TGF-a, others, act on primed hepatocytes to stimulate cell metabolism and entry into the cell cycle. This wave of hepatocyte proliferation is followed by nonparenchymal cell proliferation (e.g. Kupffer, endothelial, stellate cells) (3) *termination* phase: hepatocytes return to quiescence, likely involving TGF-b * *2. Liver regeneration from progenitor cells**: - When hepatocyte proliferation is impaired (e.g. chronic liver injury or inflammation), progenitor cells contribute to repopulation (in rats these are called *oval cells*). - Some of these reside in specialized niches called *canals of Herring*, where bile canaliculi connect with larger bile ducts

Answer 61

Liver regeneration by proliferation of hepatocytes. Following partial hepatectomy, the liver regenerates by proliferation of surviving cells. The process occurs in stages, including priming, followed by growth factor-induced proliferation. The main signals involved in these steps are shown. Once the mass of the liver is restored, the proliferation is terminated (not shown).

Answer 62

1. If the injury is severe or chronic, resulting in damage to parenchyma and CT framework 2. If nondividing cells are injured Scarring patches the tissue, where regeneration restores it. The term scar is most often used in connection to skin wound healing, but may also describe the replacement of any parenchymal cells in any tissue by collagen (e.g. heart after myocardial infarction)

Answer 63

**1. Angiogenesis**: formation of new blood vessels, which supply nutrients and O2 for repair process. New vessels are leaky because of VEGF, which causes part of the edema that persists in wound healing after inflammatory response has resolved **2. Formation of granulation tissue:** Fibroblast migration and loose CT deposition, together with the vessels and interspersed leukocytes form granulation tissue. Granulation tissue progressively invades injury site **3. Remodeling of connective tissue**: Maturation and reorganization of the connective tissue forms the stable scar.

Answer 64

Steps in repair by scar formation. Injury to a tissue, such as muscle (which has limited regenerative capacity), first induces inflammation, which clears dead cells and microbes, if any. This is followed by the formation of vascularized granulation tissue and then the deposition of extracellular matrix to form the scar.

Answer 65

**Macrophages play a central role in repair by clearing offending agents and dead tissue, providing growth factors for the proliferation of various cells, and secreting cytokines that stimulate fibroblast proliferation and connective tissue synthesis and deposition.** Repair begins within 24 hours of injury with emigration of fibroblasts, and fibroblast and endothelial proliferation. By 3-5 days, granulation tissue is apparent.

Answer 66

**Angiogenesis: process of new blood vessel development from existing vessels.** Steps of angiogenesis 1. Vasodilation in response to NO and increased permeability induced by VEGF 2. Separation of pericytes from abluminal surface and breakdown of BM to allow formation of a vessel sprout 3. Migration of endothelial cells toward site of injury 4. Endothelial cell proliferation just behind the 'tip' of the migrating cells 5. Remodeling into capillary tubes 6. Recruitment of periendothelial cells (pericytes for small capillaries, smooth muscle cells for larger vessels) 7. Suppression of endothelial proliferation and migration and deposition of BM Angiogenesis is regulated by: 1. **Growth factors** - VEGF (mainly *VEGF-A*) --\> stimulate migration, proliferation of endothelial cells, increased vascular permeability, vasodilation via NO production, lumen formation - FGFs (mainly *FGF-2*) --\> stimulate endothelial proliferation, promotes mac and fibroblast migration to injury site, stimulates epithelial migration to cover skin wounds - Angiopoietins 1 and 2 (*Ang 1 , Ang 2*) --\> GFs that play a rule in angiogenesis and structural maturation of vessels. *Ang1* interacts with Tie2, a tyrosine kinase receptor on endothelial cells for stabilization 2. **Notch signaling**: - regulates sprouting, branching of new vessels with VEGF 3. **ECM proteins**: - Participate in vessel sprouting through interactions with endothelial integrins - Provide scaffold for vessel growth 4. **Enzymes in ECM (e.g. MMPs):** - Degrade ECM to permit remodeling and vascular tube extension

Answer 67

Angiogenesis. In tissue repair, angiogenesis occurs mainly by sprouting of new vessels. The steps in the process, and the major signals involved, are illustrated. The newly formed vessel joins up with other vessels (not shown) to form the new vascular bed.

Answer 68

**The laying down of connective tissue occurs in two steps: (1) migration and proliferation of fibroblasts into the site of injury, and (2) deposition of ECM proteins produced by these cells.** These proceses are orchestrated by PDGF, FGF-2, TGF-b, which come from inflammatory cells (mainly M2 macs) at the site of injury and in granulation tissue. * **Transforming growth factor-b (TGF-b)* is the most important cytokine for synthesis and deposition of connective tissue proteins.** - produced by most cells in granulation tissue (mainly M2 macs) - TGF-b levels are regulated by activation of latent TGF-b, rate of active TGF-b secretion, and integrins in ECM that affect TGF-b activity - TGF-b stimulated fibroblast migration and proliferation, increases synthesis of collagen and fibronectin, and decreases ECM degradation due to MMP inhibition - TGF-b involved in scar formation, and also in fibrosis in lung, kidney, liver following chronic inflammation - TGF-b is anti-inflammatory, inhibiting lymphocyte proliferation

Answer 69

A, Granulation tissue showing numerous blood vessels, edema, and a loose extracellular matrix containing occasional inflammatory cells. Collagen is stained blue by the trichrome stain; minimal mature collagen can be seen at this point. B, Trichrome stain of mature scar, showing dense collagen, with only scattered vascular channels.

Answer 70

**The outcome of the repair process is influenced by a balance between synthesis and degradation of ECM proteins.** After its deposition, CT in the scar is modified and remodeled. Degradation of collagen and other ECM components is accomplished mostly by matrix metalloproteinases. - Interstitial MMPs (-1, -2, -3) cleave fibrillar collagen, gelatinases (MMP-2, -9) degrade amorphous collagen and fibronectin, and stromelysins (-3, -10, -11) degrade a variety of ECM constituents. MMPs are produced by fibroblasts, macs, neuts, synoviocytes, some epithelial cells. Tissue inhibitors of metalloproteinases (TIMPs) are produced by mesenchymal cells to shut down MMPs after ECM is remodeled. ADAM (a disintegrin and metalloproteinase) enzymes are anchored to plasma membrane and cleave/release extracellular domains of cytokines and GFs (e.g. TNF, TGF-b, EGFs) Neutrophil elastase, cathepsin G, plasmin, and other serine proteases can also degrade ECM.

Answer 71

**1. Infection**: clinically one of the most important causes of delays in healing. It prolongs inflammation and increases local tissue injury. **2. Diabetes**: metabolic disease that compromises tissue repair for many reasons. **3. Nutritional status:** Profound effects (e.g. protein deficiency, Vit. C deficiency inhibit collagen synthesis) **4. Glucocorticoids:** Anti-inflammatory effects, inhibiting TGF-b, diminishing fibrosis. Can be used strategically (e.g. corneal infections) to decrease inflammation **5. Mechanical factors:** Increased pressure or torsion **6. Poor perfusion**: due to arteriosclerosis and diabetes or obstructed venous drainage (e.g. varicose veins) **7. Foreign bodies**: Steel, glass, bone **8. Type and extent of tissue injury**: Nondividing cells cannot have complete restoration. Extensive injury, even in dividing cells, likely leads to loss of function **9. Location of the injury:** Inflammation in tissue spaces (e.g. pleural, peritoneal, synovial) develops extensive exudates. Subsequent repair may occur by *resolution*, digestion of exudate by leukocytes and resorption.

Answer 72

**When the injury involves only the epithelial layer, the principal mechanism of repair is epithelial regeneration** Three processes: inflammation, cell proliferation, and maturation of the scar. Steps in first intention healing of a surgical incision: 1. Blood clot formation with RBCs, fibrin, fibronectin, complement proteins, which stops bleeding and is a scaffold for cells which come due to GFs, cytokines, chemokines. VEGF release increases permeability and edema 2. Within 24 hours, neutrophils are present, which release proteolytic enzymes to clear debris. Basal cells at cut edge increase mitoses. In 24-48 hours, epithelial cells from both ends are migrating and proliferating along dermis, depositing BM until they eet. 3. By day 3, neutrophils replaced by macs, granulation tissue invades incisions pace. Epithelial cell proliferation continues 4. By day 5, neovascularization peaks and granulation tissue fills the space. Leaky vasculature causes edema. Fibroblasts come in due to TNF, chemokines, PDGF, TGF-b, FGF and proliferate, producing ECM components. Surface epithelium recovers thickness and starts keratinizing. 5. Second week - more collagen accumulation and fibroblast proliferation; diminished leukocytes, edema, vascularity. Blanching begins - collagen deposition 6. After first month, scar is a cellular connective tissue devoid of leukocytes, and covered by normal epidermis.

Answer 73

**When cell or tissue loss is more extensive, such as in large wounds, abscesses, ulceration, and ischemic necrosis (infarction) in parenchymal organs, the repair process involves a combination of regeneration and scarring.** Inflammatory reaction is more intense, abundant granulation tissue develops, large scar formation with wound contraction by myofibroblasts Differences from first intention: 1. Fibrin clot is larger --\> more exudate and necrotic debris, more inflammation 2. Much more granulation tissue formed 3. Provisional matrix with fibrin, fibronectin, type II collagen is replaced in 2 weeks by matrix of type I collagen. 4. WOund contraction occurs by myofibroblasts

Answer 74

Steps in wound healing by first intention (left) and second intention (right). In the latter, note the large amount of granulation tissue and wound contraction.

Answer 75

Healing of skin ulcers. A. Pressure ulcer of the skin, commonly found in diabetic patients. The histologic slides show a skin ulcer with a large gap between the edges of the lesion B. a thin layer of epidermal re-epitehlialization and extensive granulation tissue formation in the dermis C. Continuing re-epithelialization of the epidermis and wound contraction

Answer 76

Carefully sutured wounds have about 70% of the strength of normal skin. When sutures are removed 1 week after, wound strength os about 10% of normal, but increases rapidly over 4 weeks. Tensile strength recovery results from excess collagen synthesis over degradation for the first 2 weeks, and modifications of collagen fibers later on. Ultimately, w ound strength reaches about 70-80% of normal by months and that is it.

Answer 77

Fibrosis - excessive deposition of collagen and other ECM components in a tissue (same as scar) Basic mechanisms are the same as in the skin during tissue repair (very dependent on TGF-b) Fibrotic disorders (e.g. liver cirrhosis, systemic sclerosis, idiopathic pulmonary fibrosis, end-stage kidney disease) cause tremendous functional impairment

Answer 78

Mechanisms of fibrosis. Persistent tissue injury leads to chronic inflammation and loss of tissue architecture. Cytokines produced by macrophages and other leukocytes stimulate the migration and proliferation of fibroblasts and myofibroblasts and the deposition of collagen and other extracellular matrix proteins. The net result is replacement of normal tissue by fibrosis.

Answer 79

* *1. Inadequate formation of granulation tissue or formationo of a scar can lead to two types of complications: (1) wound dehiscence:**rupture of a wound; frequently increased abdominal pressure after abdominal surgery * *(2) ulceration:** Can happen because of inadequate vascularization during healing **2. Excessive formation of the components of the repair process can give rise to hypertrophic scars and keloids.** Hypertrophic scar - raised scar. Keloid - so much collagen that it goes beyond boundaries of original wound and does not regress. **3. Exuberant granulation**: Granulation tissue which protrudes above level of surrounding skin, blocking re-epithelialization. Desmoid - low grade neoplasia that arises from exuberant fibroblast proliferation from scar site 4. Contraction exaggeration --\> deformities of wound and surrounding tissues

Answer 80

Keloid. A, Excess collagen deposition in the skin forming a raised scar known as keloid. B, Note the thick connective tissue deposition in the dermis.