Lecture 5 - Kapila - Infl Ii-mediators - 9 Sep 2006

Acute Inflammation-II Mediators of Inflammation Kusum Kapila September 2006

Acute inflammation

Time 4-6 hours to 3-5 days Vascular involvement Active hyperemia Edema, occ.fibrin thrombi

Neutrophils Cardinal signs of inflammation Lymphatics Role to remove exudate Can lead to inflammation.

Lymphangitis

The series of events in the process of inflammation in organs are: Vasodilation: leads to greater blood flow to the area of inflammation, resulting in redness and heat. Vascular permeability: endothelial cells become "leaky" from either direct endothelial cell injury or via chemical mediators.

Exudation: fluid, proteins, red blood cells, and white blood cells escape from the intravascular space as a result of increased osmotic pressure extravascularly and increased hydrostatic pressure intravascularly here PMN's that are marginated along the dilated venule wall (arrow) by process of diapedesis spill out into extravascular space.

Phases of Acute Inflammation 1- Initiation: 1- Stimulation (injury) with changes in microvasculature 2- Structural changes leading to fluid extra-vasation 3- Emigration of WBCs to the site of injury 2- Amplification: Both soluble mediators and cellular inflammatory systems are activated and amplified 3- Termination: Specific inhibition or dissipation of the mediators

Tissue injury Vasoactive mediators (eg. histamine)

Increased vascular permeability Edema

Production of  inflammatory  mediators

Chemotactic factors (eg. c5a)

Recruitment of inflammatory  cells Acute   inflammation PMNs

Chronic  inflammation Monos

CHEMICAL MEDIATORS OF INFLAMMATION

General Principles: Originate from plasma and cells. When in plasma are found in inactive state and,must be activated. When in cells are often within granules and are synthesized in response to a stimulus.

CHEMICAL MEDIATORS OF INFLAMMATION · Production of active mediators is triggered by microbial products or host proteins. · Most require binding to specific receptors on target cells for biologic activity. · Some have direct enzymatic activity, · One mediator can stimulate the release of other mediators by target cells- amplification · May have different affects on different cells. · Most are short lived. · Many have the potential to be harmful.

Inflammation Chemical Mediators of Inflammation • • • • • • • • • • •

Vasoactive amines Complement system Kinin system Coagulation pathway Fibrinolytic pathway Arachidonic acid metabolites Platelet activating factor Cytokines Nitric oxide Proteases – lysosomal constituents Oxygen-derived free radicals

Inflammation Sources of Inflammatory Mediators Inactive precursors in plasma, – –

Complement proteins (C3a, C5a) Coagulation proteins initiated by Hageman factor (FDPs)

Cell-derived Proteins sequestered in granules Membrane phospholipids (via arachidonic acid metabolism) Vasoactive amines (mast cells and platelets)

1 - Plasma Factors Four enzymatic cascade and interrelated systems, important in the inflammatory response are found within plasma. These systems include the complement, kinins coagulation system fibrinolytic system

Chemical Mediators of Inflammation 1.1 - Complement cascade system C3 is the critical control point, and interacts with both pathways-classic,alternate C3a and C5a are known as “anaphylatoxins”, and are capable of releasing histamine from mast cells, along with potent chemotactic abilities (C5a) Membrane attack complex (MAC) is the active agent of complement lysis and consists of C5,6,7,8,9.

Inflammation Chemical Mediators of Inflammation 1.2 - Kinin system Vasoactive peptides called kinins are generated by proteases called kallikrein Hageman factor is a potent activator of kallikrein Most important product is bradykinin Vascular dilation and increased permeability Release of histamine from mast cells Activate arachidonic acid cascade pain

Inflammation Chemical Mediators of Inflammation 1.3 - Coagulation pathway – fibrinogen to fibrin

Inflammation Chemical Mediators of Inflammation 1.4 - Fibrinolytic pathway – fibrin to FDP –

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Both systems are induced by activated factor XIIa (Hageman factor or tissue factor) The coagulation and fibrinolytic systems complement and counterbalance each other Most important molecules are fibrinogen, fibrin, thrombin, plasminogen, and plasmin

Hageman Factor - Dependent Pathways (Factor XII of intrinsic coagulation cascade) Activation by: -Contact with collagen -Activated platelets -Proteases from neutrophils Results in: -Blood clotting -Fibrinolytic system activated -Production of kinins -Activation of complement cascade -Feedback system

Inflammation 2 - Chemical Mediators released from Cells

Vasoactive amines 2.1- Histamine Found in mast cells, basophils and platelets Released in response to stimuli Promotes arteriolar dilation and venular endothelial contraction results in widening of interendothelial cell junctions with increased vascular permeability 2.2- Serotonin Vasoactive effects similar to histamine Found in platelets

Inflammation Chemical Mediators released from Cells The following agents can stimulate release of histamine from mast cells: * Physical injury, mechanical trauma, heat, chemical agents, Snake venoms, toxins, bile salts, ATP * Immune reactions involving binding of antibodies to mast cells * Fragments of complement called anaphylatoxins (C3a and C5a) * Histamine-releasing factors from neutrophils, monocytes, and platelets * Cytokines (interleukin-1, IL-8)

Inflammation Chemical Mediators released from Cells 2.3 - Arachidonic acid metabolites These lipid mediators are shortrange hormones -formed rapidly - exert their effects locally and are then inactivated. Synthesized from cell membrane phospholipids though the action of phospho-lipases (inhibited by corticosteroids) Form leukotrienes via 5lipoxygenase Form prostaglandins and thromboxane A2 via cyclooxygenase (COX-1 inhibited by aspirin and indomethacin )

Inflammation Chemical Mediators released from Cells 2.4 - Platelet activating factor -Bioactive phospholipids. -Produced by a variety of cells, including platelets, basophils, mast cells, neutrophils, monocytes,macrophages and endothelial cells * Platelet aggregation and release * Bronchoconstriction and vasoconstriction * Vasodilation and increased vascular permeability (100-10,000 X histamine) * Increased leukocyte adhesion to endothelium

Inflammation

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Cytokine Networks

local secretion of cytokines at the site of injury is the primary means of regulating the infl response The macrophage is the pivotal cell, secretes many different molecules, two most powerful being lipopolysaccharide (LPS – Gram negative bacteria) IFN-γ (viruses)

Inflammation Chemical Mediators of Inflammation 4 - Nitric oxide (NO) –

Formed by NO synthase (NOS) Constituitively expressed in endothelial and neuronal cells. Inducible NOS – induced in macrophages by TNF-α or IFN-γ

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Potent vasodilator Involved in the pathogenesis of septic shock

5 - OXYGEN-DERIVED FREE RADICALS -Hydrogen peroxide (H2O2) -Superoxide anion (O2-) -Hydroxyl radicals (OHC) Produce: * Endothelial cell damage with resultant increased vascular permeability. * Inactivation of antiproteases, thus leading to unopposed protease activity, with increased destruction of ECM. * Injury to a variety of cell types

Sequence of events 

Arteriolar dilatation histamine-from mast cells Nitrous oxide and prostaglandin PGD2, PGE2 increases amount of blood to tissue Opens previously closed capillaries Produces redness and heat Starts in arterioles continues through capillaries to post capillary venules.

Mechanisms of increased vascular permeability-1 Formation of endothelial gaps in venules – reversible, affects only venules C. Endothelial cell contraction - immediate transient response Histamine, Brady kinin, Leukotrienes Endothelial cell retraction - structural reorganisation of cytoskeleton - Takes time TNF, IL-1, IF-y

Mechanisms of increased vascular permeability-2

Direct injury to endothelial cell 

Immediate sustained response Arterioles, capillaries and venules Toxins/Burns/Chemicals

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Delayed prolonged response

Mechanisms of increased vascular permeability-3 Leukocyte Dependent Endothelial Injury Activated WBC’s aggregate and adhere to endothelium  Release substances which damage endothelium Toxic oxygen species + proteolytic enzyme Increased Transcytosis Vascular endothelial growth factor (VEGF) Leakage from new capillaries VEGF and histamine

Sequential involvement of adhesion molecules Rolling  Adhesion  Transmigration

Central Axial Stream

SELECTINS (E&P)

INTEGRINS & Ig-LIKE MOLECULES (ICAM, VCAM)

Qualitative and Quantitative Endothelial and PMN Changes

Adhesion Molecules involved in adhesion and transmigration 4 main groups Selectins E- selectin and P- selectin 2. Mucin – like glycoproteins like heparan sulfate 3. Integrin receptor family LFA – 1 and MAC - 1 4. Immunoglobulin superfamily ICAM-1, VCAM-1 1.

MAC-membrane attack complex:ICAM-intercellular adhesion molecule;VCAM-vascular cell adhesion molecule;LFA-lymphocyte function associated molecule

Selectins-Rolling P selectin -expressed on platelets and endothelium by redistribution to surface. -histamine, thrombin, PAF E selectin -expressed on surface endothelium of post capillary venules. -TNF, IL-1, cytokine

Rolling

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- Leucocytes and endothelium bind to one another -Not a strong bond. -Weak stimulus will break away.

Endothelial cells produces - PAF, IL-8 - Chemokines Produce stronger adhesion

Adhesion- Stronger binding 1.Integrins and immunoglobulin superfamily molecules -Leukocyte receptors β 2 integrins -MAC-1

-Endothelial receptors Inter Cellular Adhesion Molecule -1 Immunoglobulin superfamily

2.Chemical mediators which increase TNF IL-1 endotoxins

LEUKOCYTES - CHEMOATTRACTANTS Plasma-derived - C5a, C5a des-Arg - Fibrin degradation products Inflammatory Cell derived - LTB4, HETE’s - from arachidonic acid metabolism - PAF, cytokines – IL-8 Others - Bacterial - fMLP-like peptides, lipid products - Dead cells – necrotaxis Chemokines -Small proteins divided into families. - A form of cytokine.

Inflammation Inflammatory Cell Activation PMNs are activated by many substances: The Fc portion of IgM and IgG molecules C5a, C3b, and iC3b Leukotriene B4 Cytokines (TNF-α) Formylated chemotactic peptides derived from bacteria

Inflammation Activation of Inflammatory Cells Macrophages are activated by many substances: Lipopolysaccharide (LPS), found in Gram-negative bacteria Platelet activating factor (PAF) Cytokines produced by T-cells, particularly interferon gamma (IFN-γ) Fibronectin, a component of extracellular matrix

LYSOSOMAL CONSTITUENTS. 1.Neutrophils exhibit two major types of granules: -Specific - contain lactoferrin, lysozyme, alkaline phosphatase and collagenase -azurophilic - contain myeloperoxidase, cationic proteins, acid hydrolases, and neutral proteases -Cationic proteins increase vascular permeability and cause chemotaxis -Neutral proteases degrade ECM ■2.Monocyte granules contain acid hydrolases, elastase, collagenase and plasminogen activator

Morphology and function of inflammatory cells

Inflammation Systemic Manifestations Fever - clinical hallmark of inflammation - Endogenous pyrogens: IL-2 ,TNF-α Constitutional symptoms - malaise, anorexia,nausea Weight loss - due to negative nitrogen balance Hyperplasia of mononuclear phagocyte system

Inflammation Systemic Manifestations Leukocytosis - may be neutrophils, eosinophils, or lymphocytes Anemia –blood loss,chronic due to toxic depression of bone marrow Acute Phase Reactants - non-specific elevation of many serum proteins marked increase in ESR Amyloidosis – longstanding chronic infection

Systemic effects of Inflammation Acute phase reaction/response - IL-1 and TNF - Fever - Malaise - Anorexia Bone marrow - leukocytosis - IL-1 + TNF Lymphoid organs

Liver -IL-6, IL-1, TNF -Acute phase proteins C-reactive protein Lipopolysaccharide binding protein Serum amyloid A a-2 macroglobulin Haptoglobin Ceruloplasmin fibrinogen