Biology of Disease CH0576 The Inflammatory Response II Dr Stephen Todryk

Endothelial cells

Oedema

Bradykinin Plasminogen Thrombin C3 of complement Plasma derived mediators Hageman Factor (factor XII)

Histamine Leukotriene B4 Nitric Oxide Prostaglandin IL-8 TNF  Cell derived mediators

Situations / Diseases involving inflammation

Wounds, burns, infections

Situations / Diseases involving inflammation Wounds, burns, infections Arthritis, Inflammatory Bowel Disease Allergy / hypersensitivity, dermatitis Atherosclerosis

Inflammatory Response Body's rapid reaction to trauma - Increase of blood flow to the affected area (vessel dilation and increased permeability) - Release of chemical mediators to attract white blood cells (neutrophils) - Increase flow of plasma - Recruitment of monocytes to remove damaged tissue / clean up the debris / microbes.

This macrophage is ingesting chains of coccoid bacteria. Dr Gordon Beakes © University of Newcastle upon Tyne

Inflammatory Response Acute / Chronic Signs : swelling, redness, heat, pain, loss of function Causes Functions / Results (beneficial and harmful) containment Components / steps Mediators e.g. Oedema – swelling due to fluid

Chemical mediators Physical effects Cellular effectors

CHEMICAL MEDIATORS OF INFLAMMATION Definition: Any messenger molecule that acts on blood vessels, inflammatory cells, or other cells to contribute to an inflammatory response Exogenous - Endotoxins e.g. LPS from gram -ve trigger complement activation and other inflammatory response mediators Endogenous - Plasma Leukocytes Endothelial cells Fibroblasts

Acute Inflammation - Chemical Mediators The spread of an acute inflammatory response following injury to a small area of tissue suggests chemical substances are released from injured tissues and disseminate towards uninjured areas These chemicals are : ENDOGENOUS CHEMICAL MEDIATORS Chemical mediators can be split into two groups CELLULAR OR PLASMA DERIVED

Acute Inflammation – Chemical Mediators Endogenous mediators can be derived from molecules normally present in plasma in inactive form OR Are released at injury site by various cell types that may store preformed mediator molecules in granules e.g. histamine or can rapidly switch on mediator synthesis machinery to produce e.g. metabolites of arachidonic acid Their activities result in vasodilation, increased vascular permeability, emigration of neutrophils and chemotaxis

Acute Inflammation - Chemical Mediators

Acute Inflammation - Cellular Mediators HISTAMINE Histamine is the major pre-formed chemical mediator in acute inflammation. It is stored in and released from MAST CELLS, BASOPHILS, EOSINOPHILS and PLATLETS. The pharmacologic effects of histamine are a) Transient dilation of arterioles. b) Increased vascular permeability in venules. c) Primary cause of early increase in vascular permeability in the first hour after injury.

HISTAMINE Mast cells and Basophils can be made to degranulate their contents by a range of different mechanisms including: a) Physical agents such as cold or trauma to the tissue b) Exposure to cationic proteins derived from platelets and lysosomal granules c) Immune mechanisms mediated by IgE d) Complement components C3a and C5a.

Histamine Mast Cell degranulation of histamine and other pre- formed mediators (heparin and tryptase) - triggered most often by cross-linking of IgE receptors on cell surface (e.g. pollens; foods) - fusion of the granule membranes with the cell membrane - granules are released and are rapidly solubilised in the surrounding aqueous environment

Acute Inflammation - Cellular Mediators Arachidonic Acid Derivatives PROSTAGLANDINS (PG) THROMBOXANES (Tx) and LEUKOTRIENES are all metabolites of the long chain unsaturated fatty acid ARACHIDONIC ACID, a molecule stored in membranes of many cell types Release of Arachidonic acid can be initiated by a variety of stimuli including histamine and interaction of Neutrophils and monocytes with damaged tissues or cells. Arachidonic acid is metabolised via two pathways:

Arachidonic Acid Derivatives 1.CYCLO-OXYGENASE PATHWAY This pathway of arachidonic acid metabolism produces the PROSTAGLANDINS and THROMBOXANES There are two cyclo-oxygenase enzymes: COX-1 COX-2 that generate three important chemical mediators

Arachidonic Acid metabolites via COX Pathway Prostacyclin (PGI 2 ) inhibits platelet aggregation and causes vasodilation Stable prostaglandins (PGE 2, PGF 2 and PGD 2 ) all act on vascular tone and increase permeability; PGE 2 also causes pain Thromboxane A 2 aggregates platelets and causes vasoconstriction

Arachidonic Acid Derivatives 2.LIPOXYGENASE PATHWAY This pathway produces all the LEUKOTRIENES (LT) Occurs mainly in neutrophils LTC 4, LTD 4 and LTE 4 are potent vasoconstrictors, cause smooth muscle contraction and an increase in venule specific vascular permeability and are 1000x more potent than histamine at ↑ permeability LTB 4 stimulates the adhesion of neutrophils to endothelial cells and is a potent chemotactic agent

Arachidonic Acid Derivatives The important role that the various metabolites of Arachidonic acid play in inflammation is well illustrated by the fact that many of non-steroidal anti-inflammatory drugs act by inhibiting various points in the two pathways discussed E.g. aspirin, ibuprofen, indomethacin Aspirin exerts it powerful anti-inflammatory effect by inhibiting the first step in prostoglandin synthesis Pharmaceutical companies are very keen to have successful COX-2 inhibitors in the marketplace

To summarise: Some anti-inflammatory properties interfere with arachidonic acid metabolism –Corticosteroids interfere with phospholipase –Aspirin interferes with cyclooxygenase Arachidonic Acid Derivatives NSAIDS; Ibuprofen

Acute Inflammation - Cellular Mediators Platelet Activating Factor (PAF) PAF is a phospholipid synthesised by mast cells and basophils and also by platelets, neutrophils, monocytes and endothelial cells. PAF is a mixture of molecular species. PAF dilates blood vessels, increases platelet aggregation, induces phagocytosis, triggers arachidonic acid metabolism and promotes leukocyte chemotaxis.

Acute Inflammation - Cellular Mediators Cytokine Network Cytokines have multiple physiological roles and act to mediate communication between organs including those that are generated during inflammation Cytokines are small protein products of activated leukocytes, particularly lymphocytes and monocytes and interact with specific receptors on target cells Cytokines may form a concentration gradient and are therefore thought to be important controllers of directed cell migration (CHEMOTAXIS ; chemokines)

Acute Inflammation - Cellular Mediators Cytokines with a role in inflammatory response include : IL-1 , IL-8, TNF , IL-6

Acute Inflammation - Cellular Mediators Nitric Oxide (NO) NITRIC OXIDE - a small molecule locally synthesised by the endothelium and macrophages from L-Arginine by the action of the enzyme NITRIC OXIDE SYNTHASE (NOS) Nitric oxide diffuses into smooth muscle and is a potent vasodilator and increases vascular permeability Nitric oxide has a very short half life (3-4 seconds) NO, the nitric oxide radical is the active compound produced from several vasodilator drugs employed commercially.

Nitric Oxide Nitric oxide can be highly toxic and used by the body to kill invading cells (bacteria) or used by the body as an intracellular signal. Thus it may have both a protective and damaging effect. Nitric oxide is sometimes referred to ENDOTHELIUM DERIVED RELAXING FACTOR (EDRF). Endothelial cells also produce PGI 2 and ENDOTHELIN. Endothelin is a low MW peptide that induces prolonged constriction of vascular smooth muscle.

Nitric Oxide

Acute Inflammation Plasma Derived Mediators Plasma contains four enzymatic cascade systems which produce various inflammatory mediators Each cascade occurs as a series of inactive precursor molecules which, when triggered, react as a series of sequential proteases The four cascade systems are : 1. The complement system. 2. Coagulation factors. 3. The kinin system. 4. The fibrinolytic system.

Plasma Derived Mediators The Alternative Pathway of Complement Activation The complement system is a cascade of enzymatic proteins. It can be activated during acute inflammation in a number of ways: a) Tissue necrosis b) During infection - A variety of viral, bacterial, fungal and parasite material can activate complement e.g. Yeast cell walls (Zymosan), endotoxins from gram -ve bacteria and polysaccharides c) Products of the kinin, coagulation and fibronolytic systems.

Alternative Pathway of Complement Activation Complement can be activated via 3 pathways- Classical, lectin and Alternative pathway Antibodies do not have role in the Alternative pathway The first three components of the classical pathway are bypassed Six proteins perform the functions of initiation, recognition and amplification of the pathway These are : C3, B, D, H, I and P This results in the formation of the activator bound C3/C5 convertase

Alternative Pathway of Complement Activation 1. C3 undergoes spontaneous cleavage to C3a and C3b in plasma 2. Unless it binds to a cell surface, C3b is rapidly inactivated 3. C3b forms a complex with Bb and factor P. This is a C3 convertase which deposits many more molecules of C3b on the pathogen. 4. Some molecules of C3b bind to the existing C3b, Bb complex to form C3b 2,Bb which is a C5 convertase 5. C5 is split into C5a and C5b Proteins: C3, B, D, H, I and P

Alternative Pathway of Complement Activation C3a and C5a are ANAPHYLATOXINS - cause smooth muscle contraction and increased vascular permeablity. This effect is amplified by the release of further histamine from mast cells and basophils. C3a and C5a are also powerful chemotactic agents.

Acute Inflammation - Plasma Derived Mediators COAGULATION FACTORS THE HAGEMAN FACTOR (coagulation factor XII) Factor XII is activated by: 1. contact with a variety of negatively charged surfaces e.g. basal lamina, enzymes of bacterial origin 2. Platelets 3. Proteases from inflammatory cells

Plasma Derived Mediators COAGULATION FACTORS Activated Factor XII can activate a number of systems – it converts PLASMINOGEN to PLASMIN, PREKALLIKREIN to KALLIKREIN and activates the KININ system Products from this sequence can themselves activate Factor XII by a feedback mechanism resulting in amplification

Plasma Derived Mediators

Acute Inflammation - Plasma Derived Mediators THE KININ SYSTEM Kinins are small peptides, 9-11 amino acids long, including BRADYKININ and LYSYL BRADYKININ. Both are powerful vasodilators, increase vascular permeability and mediate pain THE FIBRINOLYTIC SYSTEM Plasmin is responsible for the lysis of fibrin into fibrin degradation products which have local effects on vascular permeability.

Acute Inflammation Cellular/Plasma Derived Mediators These complex interactions bring about increased vascular permeability at the LOCAL site of damage. After this the next phase of the acute inflammatory response occurs. This is cellular recruitment into the inflammatory site. The cells which are recruited at the site vary with time. This has led to the classification of the inflammatory cells into acute and chronic categories.

Acute Inflammation - Cellular Recruitment The main cellular events in acute inflammation, all brought about by chemical mediators are :- 1.Normally inactive endothelial cells are activated to allow the adhesion of neutrophils. 2.Normally inactive circulating neutrophils are activated to enhance their capacity for phagocytosis and killing. 3.Neutrophils develop the ability to move actively in a directional fashion from the vessels towards the site of tissue damage.

Acute Inflammation - Cellular Recruitment During the first 24 hours following injury the main cells to accumulate are neutrophils due to the production of high concentrations of chemotactic factors at the site of injury The main cell to mediate the effects of acute inflammation is the NEUTROPHIL If tissue damage is slight there will be adequate neutrophils in the peripheral circulation

Acute Inflammation - Cellular Recruitment If tissue damage is extensive stores of neutrophils,including some immature forms, are released from the bone marrow to increase number of neutrophils in blood To maintain a supply of short lived neutrophils, growth factors derived from the inflammatory process, stimulate the division of myeloid precursors in the bone marrow.