Veterinary PathophysiologyStudent’s Lectures, 5th Semester

Department of Internal Medicine,Faculty of Veterinary Science

Szent István University

Pathophysiology of defence mechanisms 4.Inflammation

DEFENCE MECHANISMS DEVELOPED DURING THE PHYLOGENESIS (Handouts p 28)

HISTORICAL BACKGROUND

Aulus Cornelius Celsus (1.század): "De Re Medicina": rubor (redness), dolor (pain), calor (heat), tumor (swelling)

Galenus (AD 130-200), Virchow (1821-1902): functio laesa (loss of function)

Hunter, Cohenheim: importance of vascular factors

Mechnicov: phagocytosis

Dale, Laidlow, Lewis: histamine - notion of "mediators"

Schwartzmann: endotoxin caused inflammation

Arthus: Immune Complex Mediated Hypersensitivity (Type III.)

Menkin: discovered ancestors of the "peptid mediators" Menkin`s "exudine"=bradykinin

Ratnoff: significance of Hageman (XII.)-factorDe Duve: lysosomes

Ex Libris of The International Club of Inflammation

Calor Rubor Tumor Dolor Functio

laesa

Three major parts of the inflammatory response:

1.Hemodynamic changes2.Permeability changes3.Cells involved

Hemodynamic changes

1.Transient vasoconstriction: lasts for a few seconds - neurogenic mechanism

2.Vasodilation: begins in a few minutes and lasts hours or longer, release of histamine form mast cells, generation of nitric oxide (NO) and prostaglandins (PGD2, PGE2) – causing acute local active hyperemia

3.Increased volume of blood flow in the area (a result of vasodilation)

4.Increased vascular permeability (histamine, leukotrienes C4, D4, E4,

bradykinin, and platelet activating factor (PAF) cause contraction of endothelial cells resulting gaps in the small vessels. Complement factors (C5a and C3a) indirectly participate by inducing release of histamine by mast cells.

5.Rate/speed of flow decreases (caused by vasodilation in the area)

6.Margination of leukocytes along vessel walls: a decreased rate of flow results in decreased shear force on white blood cells (WBC) as they contact the vessel wall.

7.Exudation: WBCs migrate out into the tissue as does the serum.

Permeability changes

Vascular leakage after local injury can occur: 1. directly; as an effect of any kind of structural injury to the

microvasculature affecting all types of vessels. 2. indirectly; as an effect of biochemical susbtances that appear in and

around the site and affect primarily the venules.

Permeability can be: 1. passive process - passage of fluid between the endothelial cells; 2. active process - endothelial cells create endocytotic vesicles, and

discharge the contents on the other side by active transport within the cell istelf.

Development of edema can occur by two major mechanisms: 1. purely hydrostatic; 2. permeability effected by biochemical mediators.

Permeability changes Exudation

Permeability effected by biochemical mediators:Biochemical mediators are stored and released from cells (ie. histamine and serotonin from mast cells, basophil granulocytes and platelets), or can be synthesized rapidly.

Plasma kinins (bradykinin - activated or cleaved enzymatically by plasma proteinases including Hageman factor /XIIa/)

Complement factors (C5a, C3a - induce mediator release from leukocytes

Leukotrienes (LTC4, LTD4, LTE4, LTB4 - produced through the arachidonic acid cascade)

Prostaglandins (cause vasodilatation and vascular leakage - PGE2, PGI2, vascular leakage - thromboxane A2 )

Cytokines (IL1, TNFα - are synthesized in leukocytes) – activate tissue factor

Platelet activating factor (PAF - affects both endothelium and leukocytes)

The following mediators cause contraction of endothelial cells resulting in gaps in the small vessels:

(a) histamine (from mast cells), leukotrienes C4, D4, E4, and bradykinin.(b) platelet activating factor (PAF)(c) serotonin (in rodent mast cells)(e) C5a, C3a indirectly participate by inducing release of histamine by mast cells

They exert their effects by direct effect on microvasculature: histamin, LTC4, LTD4, LTE4by use of the leukocytes as an intermediary: cytokines.

Cells involved

neutrophil, eosinophil and basophil granulocytes, mast cells, monocytes and macrophages, lymphocytes and plasma cells.

The production of myeloid cells, such as neutrophils, eosinophils, basophils, mononcytes are regulated primarily by IL-3, granulocyte and monocyte colony stimulating factor (GM-CSF), and G-CSF, M-CSF.

Neutrophil granulocytes:Azurophil granules – ie. myeloperoxidase, elastase, cathepsin, proteinase, collagenaseSpecific secondary granules – ie. lysozyme, lactoferrin, aminopeptidase, matrix metalloproteinases Tertiary granules - acid phosphatase, arlylsulfatase, gelatinase“Secretory vesicle” - adhesion molecule, alkaline phosphatase, urokinase plasminogen activator

Eosinophil granulocytes:Collagenases (MMP-1, -8)ElastaseCationic proteins -major basic proteinEosinophil cationic proteinHistaminaseArylsulfatase BPhospholipase C Eosinophil derived neurotoxinHageman FactorKininaseLysosomal enzymesOxygen productsPeroxidase (myeloperoxidase)Lysophspholipase Plasminogen activator, plasminogenProstaglandins (PGE1, PGE2)Zinc

Monocytes and macrophages

Functions:

Antimicrobial function

Clean up debris

Recruit other leukocytes to tissue

Acute phase response

Influence of the activities of other cells

Regulation of the immune response

Secretory products:Cytokines,chemokines, interferons, growth factors, free radicals, lipid mediators of

inflammation, complement components, lymphocyte mediating factors, mononuclear

cell factor, angiogenic factor, fibroblast mediating factors, fibronectin, chemotaxins,

procoagulant and fibrinolytic factors, lysosomal enzymes, binding proteins (ie.

transferrin, ferritin, transcobalamin II.)

Mast cells and basophil granulocytesMast cells are the major sources of histamine, reactions to parasites.

Lymphocytes and plasma cellsAntibody constitutes one of the major opsonins, sources of the potent lymphokines, IFN-γ, TNF-α, and IL-2

PlateletsADP, ATP, serotonin, PF4, PAF, fibronectin, fibrinogen, factor VIII-related antigen, factor V, epinephrine, histamine, Ca2+, complement-cleaving protease (resulting in C5a), TXA2, platelet derived growth factor, fibroblast growth factor (FGF), transforming growth factor (TGF), cathepsin, arylsulfatase, β-glucuronidase, phagocytic and bactericidal properties

Endothelial cells and fibroblastsLeukocytes must first adhere to endothels and then migrate through the microvasculatureFibroblasts are important cells by virtue of the fact that they can synthesize an array of cytokines

1. Margination

Resident tissue macrophages are stimulated to synthesize and secrete chemoattractants and activators of cells, such as cytokines and chemokines, attraction of leukocytes to the tissue, it is attributable to adhesion molecules on the surface of leukocytes and endothelial cells.

Cellular events(1) margination, (2) pavementing, (3) emigration, (4) chemotaxis, (5) phagocytosis, (6) synthesis of mediators

2. Pavementing

Adhesion molecules are stored in granules and move to the surface when the cells are stimulated. During inflammatory process biochemical mediators are diffusing throughout the tissue in that area. β2-integrins are always present on the surface of the leukocytes, but are quickly upregulated in adehesiveness when leukocytes are stimulated. These integrins involve CD11/CD18. Leukocyte-endothelial cell bonding is strong and the leukocytes can be firmly arrested at the site. Disease: Bovine leukocyte adhesion deficiency (BLAD) - calves from 1 to 4 months old. Calves had severe ulcers on oral mucous membranes, gingivitis, severe periodontitis, chronic pneumonia, and stunted growth associated with severe neutrophilia.

3. Emigration

Emigration refers the process by which leukocytes escape from their location in the blood to reach perivascular tissues

4. Chemotaxis

This phenomenon is a directional migration in response to a gradient of chemoattractants

CHEMOTACTICAGENTS:

endogenous

from plasma: complement-system,fibrinogen

from cells: leucotriens (LT),interleukins (IL-1, IL-8),platelet activating factor (PAF)

exogenous(ie.: bacteria, viruses, parasites)

Chemotaxins

Endogenous:Plasma-derived chemotaxins

C5a, C5a-des Arg, fibrinopeptides (fibrin degradation products)

Inflammatory cell-derived chemotaxinsarachidonic acid derivatives: LTB4, HETEs, platelet-activating factor (PAF), chemokines

Exogenous: Other factors-derived chemotaxins(1) Bacterial chemotaxins, including N-Formyl-Met-Leu-phe- (FMLP-) like

peptides, (2) Dead cells themselves (necrotaxins). (3) Superoxide anion radical (O2.-) (4) Endotoxin (lipopolysacharides)

Mechanism of chemotaxisMechanism of chemotaxis

1.1. Binding of chemoattractant to the cell surface. Binding of chemoattractant to the cell surface. 2.2. Release of calcium from intracellular stores and membrane Release of calcium from intracellular stores and membrane

translocation of calcium. This leads to increase in cytosolic Catranslocation of calcium. This leads to increase in cytosolic Ca2+2+. It is . It is necessary for triggering both degranulation and changes in membrane necessary for triggering both degranulation and changes in membrane polarity. It also influences the submembranous actin-myosin network. polarity. It also influences the submembranous actin-myosin network.

3.3. Leukocytes crawl. Proper locomotion requires reversible adhesiveness Leukocytes crawl. Proper locomotion requires reversible adhesiveness to a surface. to a surface.

4.4. The cells undergo characteristic morphological changes that begin with The cells undergo characteristic morphological changes that begin with increased surface membrane ruffling within 30 seconds of stimulation. increased surface membrane ruffling within 30 seconds of stimulation.

5.5. Transient dose-dependent leukocyte aggregation may occur within the Transient dose-dependent leukocyte aggregation may occur within the first few minutes of stimulation. first few minutes of stimulation.

6.6. Responding leukocytes assume a characteristic polarized orientation Responding leukocytes assume a characteristic polarized orientation during locomotion with broad-spreading lamellipodia at the leading during locomotion with broad-spreading lamellipodia at the leading edge.edge.

7.7. In a gradient of chemoattractant, populations of cells migrate toward the In a gradient of chemoattractant, populations of cells migrate toward the source of the chemotactic factor. source of the chemotactic factor.

8.8. Leukocytes easily become deformed as they migrate through narrow Leukocytes easily become deformed as they migrate through narrow places. places.

Effects: • white blood cell migration increases (causing temporary neutropenia!)• cell adhesion increases• lysosomal enzyme liberation increases• oxygen consumption, oxigen radical formation increase

Inhibitors: neutrophil immobilizing factor (NIF),leukocyta immobilizing factor (LIF),lysosomal proteases (elastases, proteases) etc.

5. Phagocytosis

CHEMOTACTICCHEMOTACTIC neutrophils` neutrophils` thanthan by by AGENTSAGENTS monocytes` migration increases monocytes` migration increases

connection develops between the phagocyte-receptor and the connection develops between the phagocyte-receptor and the opsonised pathogenopsonised pathogen

phagocytosis (phagosoma develops)phagocytosis (phagosoma develops)

digestion (phagolysosoma develops)digestion (phagolysosoma develops)

OO22-independent killing-independent killing OO22-dependent killing-dependent killing via lysosomal enzymes via lysosomal enzymes ooxidative burstxidative burstie. ie. Cathepsin G Cathepsin G via reactive metabolitesvia reactive metabolites

(O(O22• • HH22OO22, OH, OH••))

halids: (-OCl, -OBr, -OI)halids: (-OCl, -OBr, -OI)Decreased by:Decreased by: increased adreno-cortical function (Cushing`s increased adreno-cortical function (Cushing`s syndrome, syndrome, glucocorticoidglucocorticoid therapy) therapy)

OOxidative burstxidative burst

6. Synthesis of mediators

Biochemical mediator might be any messenger that acts on blood vessels, inflammatory cells, or other cells to contribute to an inflammatory response.

Chemical mediators of acute inflammation

I. Compounds of the immediate (early) phase of exsudation: (0 - some hours, mainly plasma is exudated)

1. Histamine2. Serotonin

II. Compounds of the delayed (late) phase of exsudation:a) Hageman-factor dependent pathways Coagulation factors Fibrinolytic factors Kinins Complement systemb) Lipid mediators

Histamine

(from mast cells and

basophil granulocytes)

Affects: pericyte H2 receptors: relaxation endothel H1 receptors: contraction

Consequences: - vasodilation and capillary permeability increases - pain develops - immediate (anaphylactic, Type 1)

hypersensitivity reaction

Inhibitors: antihistamines, adrenalin (epinephrine), PGE2

Vasoactive effect of histamine

Pericyte

Pericyte – EM picture

2. Serotonin /5-HT/ (from thrombocytes)

Effects: Vasoconstriction of smooth muscles - permeability increases

bronchial muscles contraction

importance in development of asthma bronchiale in humans intestinal peristalsis increases diarrhoea

As a monoamine neurotransmitter, involves many sensory and higher centers in the brain including memory, appetite, sleep, and learning; known to many as the "feel good" hormone, serotonin, along with endorphins, GABA, and dopamine

Inhibitors: LSD, tissue (not MAO) enzymes.These mediators provoke also emigration and activation of vasoactive

peptides, responsible for the delayed phase of exudation

Histamine Serotonin(5-hydroxytryptamine, 5-HT)

Size 111 Da 176.2 Da

Formation preformed mediator, formed by the decarboxylation of the amino acid L-histidine

preformed mediator, formed from dietary tryptophan, oxidation and decarboxylation

Location in granules of mast cells, basophil neutrophils, and in some species, the platelets

in certain cells (ie. neural cells) of gastrointestinal tract, brain, lung, platelets, in rodents: mast cells and basophils

Inactivation by histaminase or oxidative deamination. by monoamine oxidases

Release in response to immunological processes (like antigenic chalange of IgE antibody sensitized cells, exposure to complement components C3a and C5a), physical injury, mechanical trauma, irradiation, heat, chemical agents, like toxins, snake venoms, melittin from bee venom, trypsin, bile salts, ATP

a) form platelets occurs during the platelet-release reaction triggered by stimuli such as thrombin, trypsin, collagen, antigen-antibody complexes, globulin-coated surfaces, snake venoms, epinephrine, PAF, and ADP; b) from neurons occurs due to various stimuli

Function in the acute, immediate, active phase of increased vascular permeability; produces increased vascular permeability and has contracting effects on vascular and bronchial smooth muscles; dilates arterioles; vascular permeability is increased by the formation of gaps between the margins of adjacent endothelial cells of venules by contarcting them; it stimulates stromal cells to synthesize and release eotaxins, which are chemikones with potency for chemotaxis of eosinophils

a) has effects on smooth muscles, blood vessels, and nerves in different regions of the body and can mediate the sensation of pain; b) as a monoamine neurotransmitter, involves many sensory and higher centers in brain ie.memory, appetite, sleep, and learning; known as the "feel good" hormone, serotonin, along with endorphins, GABA, and dopamine, form the biological process known as the reward cascade

II. Compounds of the delayed exudative phase

Mainly cells are exuded on the effect of chemotaxis

Hageman-factor-dependent pathways

(plasma contact activating systems)Activation of factor XII caused by contact with collagen, activated platelets, or various negatively charged surfaces (including glass) results in

(1) blood clotting, (2) fibrinolytic system, (3) generation of kinins, (4) activation of the complement cascade

Effects:

further increase of vasodilation and permeability, chemotaxis, pain

(similar effects to histamine)Inhibitors: "protease inhibitors": - 2 macroglobulin

- antitripsin, antithrombin III., - kininases (mainly from endothelial cells, especially in the lungs)

Hageman (XII.) factor responsible for coagulation and activates kallikrein and plasmin

Kallikreinogen (prekallikrein) kallikrein (enzyme),

activates kininogens (HMWK) to kinins (globulins), ie. bradykinin

Rosenthal factor (thrombocytic antecedent XI.) and factor XII. plasmin activator – triggers fibrinolysis

Complement activation by factor XII.

Collagen activates the Hageman factor dependent pathwayCytokines (IL1, TNFα) activate Tissue factor

Blood clotting

Fibrinolytic system

Kinins

Protoype: Bradykinin(MW = 1059.6 D) is a nonapeptide

Act on peripheral vessels to cause either vasodilation or vasoconstriction - depending on local conditions, and they cause bronchoconstriction.

Increase vascular permeability by the same mechanism as histamine.

Along with the prostaglandins, major mediator of the pain of acute inflammation by virtue of its effects on afferent nerve fibers.

The term “brady-” indicates that it is slow to produce a full contraction of smooth muscle in comparison to histamine.

25 plasma proteins found in the globulin-fraction (mainly enzymes)

Essential humoral (circulating) inflammatory substances.

This enzyme system is derived from the liver, macrophages and intestinal mucosa.Inter-related with the kinin and coagulation systems.

The complement proteins circulate as precursor soluble proteins (zymogens) that are hypoactive. The activated complement components are enzymes, and they interact sequentially in an ordered way in the classical pathway of antibody-directed cell lysis.

A variety of enzymes from other sources such as plasmin, kallikrein, and the lysosomal enzymes of leukocytes, can activate some of complement components.

Complement system

They increase local symptoms (release of histamine, chemotaxis) and decrease generalised symptoms

The primary targets of most complement-mediated reactions are the biological membranes, and the ultimate result is perforation of the membranes with holes, thus leading to cytolysis.

Activation of the system: cascade-like

• "Classical" way of activation: immune mediated

• "Alternative" way of activation: not immune mediated (exogenous agents i.e. bacterial lipopolysacharide or polisacharide molecules)

The by products of the cascade, such as C5a and C3a, can cause activation of cellular functions resulting in enhanced vascular permeability, smooth muscle contraction, chemotactic attraction of leukocytes, release of mediators from mast cells, immune adherence, degranulation and release of lysosomal enzymes form neutrophils, and enhanced phagocytosis.

Activation the complement system

Complement protein

binding to WBC, mastocytes receptors

activation - membrane-lysis

liberation of other mediators

inflammatory reaction increases

proteolysis active peptid fragments (enzymes)

C , C are the most important

opsonisation (IgM, IgG)

removal of macromolecules, immune complexes

C 1-9 3a 5a

The interrelations between immune reactions and enzyme cascade systems

PAF

coaulation

system

Immune complex

complement system

damage to vascular

endothelium

activated Hageman

factor XII

pre-kallikrein

activators

fibrinolytic

system

C2b

kinin

kinin

system

PAF, platelet-activating factor.

From: Robinson & Huxtable: Clinicopathologic Principles for Veterinary Medicine

Lipid mediatorsLipid mediators

phospholipases (A2 ) Inhibitors: glycocorticoids

membrane phospholipids

arachidonic acid, C20:4 (eicosa-tetraeonic acid)

Lipoxygenase pathway Cyclo-oxygenase pathway

non - corticoid type anti- inflammatory agents (ibuprophen, indo- methacine, salicylates)

epoxide formation endoperoxid formation

peroxide-derivates leucotriens, LT (from WBC-s)

prostaglandins, PG (from prostate, kidney, blood vessels) Thromboxane, TX (from platelets) prostacyclines (from blood vessels)

Eicosanoids

Secreted and Cytosilic PLA-s

COX1 is responsible for regulating blood flow by inducing moderate, physiological variations in vasodilatation, for normal stimulation of cells of the gastric mucosa to produce mucus, and other.COX1 needs to be available and functional for everyday activities, that is, “housekeeping” activities.

COX2 is transcriptionally regulated ie. by nuclear factor kappaB (NFкB), and becomes present in “unusual” circumstances, as in inflammation.The COX2 allows generation of more of prostaglandins and thromboxane than what would normally be produced at a particular tissue, and this contributes to the inflammatory process.

Effects of lipid mediators:

vasodilation Attention:increased vascular permeability antiinflammatorychemotactic effects on neutrophils (LTB4) PG-s also exist!

pain (PGE2, PGI2),

smooth muscle contraction (LTC4, LTD4, LTE4)

- histamine-.like effects

Prostanoids are associated with the pain during inflammation. They can cause vasodilatation, can assist with other mediators in producing increased vascular permeability and edema.

A little amount of prostaglandins causes contraction of smooth muscle in animals.

Potential therapeutic uses include prevention of conception, termination of pregnancy, prevention or alleviation of gastric ulcers, control of inflammation and of blood pressure, and relief of asthma and nasal congestion.

Prostanoids

Synthesis of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which is the precursor of potent leukotrienes.

Leukotriene B4 (LTB4) is a potent inflammatory mediator and induces leukocyte (especially) neutrophil chemotaxis, aggregation, increased adhesiveness, lysosomal enzyme release, and superoxide anion generation.

Leukotrienes can produce vasoconstriction. They are more potent than histamine in producing increased vascular permeability and attraction of leukocytes.

They are important regulators in many diseases involving inflammatory or immediate hypersensitivity reactions, such as asthma. Moreover, they are 100-1000 times more potent than histamine or prostaglandins as constrictors of the bronchial airway musculature.

Leukotrienes

Role of Omega-3 and Omega-6 Fatty Acid Metabolism

Some foods contain arachidonic acid per se, and many foods (sunflower oil, corn oil) contain common fatty acids such as linoleic (namely, -6 fatty acid), which will yield arachidonic acid upon metabolism, and it can be incorporated into cell membrane phospholipids for later use in the arachidonic acid cascade.

In contrast, other oils (hemp oil, flaxseed oil and green leaves) contain α-linoleic acid (namely, ώ-3 fatty acid), and this fatty acid is metabolized to eicosapentaenoic acid (EPA, that is found in marine fish oil) and further metabolized to docosahexaenoic acid (DHA). When they persistent in sufficient amounts in the diet, they will become incorporated into cell membrane phospholipids, displacing some of the arachidonic acid.

When arachidonic acid cascade is activated EPA and DHA will compete with arachidonic acid for entry into the cascade, that is, compete for binding sites on the COX and 5-LOX enzymes. Then less arachidonic acid is metabolized and less proinflammatory prostaglandins and leukotrienes are generated.

SYSTEMIC REACTIONS OF ACUTE INFLAMMATION

"ACUTE PHASE RESPONSE"

A harmonized defence mechanism of the whole organism in order to localise inflammation and to repair the original healthy state.

Monocytes mainly interleukin-1 and 6 (citokines)

- acute phase proteins- fever- neutrophilia- catabolic procedures in muscles- activation of lymphocytes (T and B)- stress mechanism develops

ACUTE PHASE PROTEINS (APPs)

There are positive (+) and negative (-) APPs.- fibrinogen increases coagulation- C-reactive protein (CRP) opsonization- serum amyloid-A protein (SAA) increases amyloid production- haptoglobin captures Hb- fibronectin opsonization- ceruloplasmin: (ferroxidase)- transferrin Fe+++ transport- lactoferrin (from neutrophils) Fe+++ transport

ACUTE PHASE PROTEINS IN PLASMA (Eckersall, 1993)

Acute Phase

Fibrinogen

C3 (complement)

C-reactive protein

Ceruloplasmin

Protein SAA

Haptoglobulin

-antitrypsin*

-acid glycoprotein*

Re1ativeIncrease in Electrophoretic

Inflammation Migration50%

50%

1000X

50%

1000X

2-3X

2-3X

2-3X

FunctionCoagulation; forms fibrin polymers

Fulcrum for C cascade; leads to cell or bacterial lysis(membrane phase) and liberation of chemotactic frag-ments (Quid phase)

Initiates C-dependent opsonization (facilitates phago-cyosis)

Copper transport

? (forms amyloid fibrils)

Hemoglobin transport

Inhibition of proteases in serum

Blocks binding of microorganisms to cells

*Well-characterized only for man.

Protein

1

1

2

2

2

1

1

DEVELOPMENT OF CHRONIC INFLAMMATION

Cause:

- prolonged or repeated action of mild irritant (ie. silica)

- secondary immune mechanism (autoimmune) develops

- deficient acute phase response

Characteristics: continous cell migration(mainly mononuclear cells),fibroblast appearance collagen

production increasesrecapillarisation

regeneration reparation