Chemical mediators of inflammation

Prof. Chandu de SilvaDepartment of Pathology

Faculty of Medicine, Colombo

Chemical mediators1. Chemical mediators may be circulating in plasma or

may be produced locally at the site of inflammation by cells.

2. Most mediators induce their effects by binding to specific receptors on target cells.

3. Mediators may stimulate target cells to release secondary effector molecules.

4. Mediators may act only on one or a few targets or may have widespread activity.

5. Mediator function is tightly regulated. Once activated and released from the cell, most mediators quickly decay, are inactivated by enzymes, eliminated or are inhibited.

6. A major reason for the checks and balances is that most mediators have the potential to cause harmful effects.

Chemical mediators

CHEMICAL MEDIATORS OF INFLAMMATION

Originating from cells Present in Plasma

Performed

1) Amines-Histamine

Serotonin

2) Lysosomal enzymes

Newly Synthesized

1) Arachidonic acid

metabolites

2) Cytokines

3) Platelet activating factor

4) Nitric oxide

1) Complement system

2) Kinin system

3) Clotting / Fibrinolytic systems

Complement system

• Has 20 component proteins, together with their cleavage products.

• Mediate biologic reactions, all of which serve in the defense against microbial agents.

CLASSICAL PATHWAY ALTERNATE PATHWAY

Ag-Ab complexes bind to C1

Activated C1

C4+C2

C3 Convertase

(C4b2a)

• Increase Vascular

permeability

• Vasodilation

Bacterial endotoxins, Aggregated globulins

C3 C3b

C3

direct cleavage by plasmin, bacterial proteases C3bBb (C3

convertase)

C3a C3b

C5

C5 convertase classical Pathway (C4b2a3b)

C3bBb3b (aternate path C5 convertase)

C5a C5b C5b67 Chemotaxis

C5b-9 (Membrane attack complex)

Membrane lysis of cells

Productionof oxygen metabolites

• Chemotaxis

• Adhesions of leukocytes to endothelium

• Leukocyte activation

• Activate Arachidonic Acid pathway

• C3b acts as an opsonin.

• C3 and C5 can be activated by several proteolytic enzymes present in the inflammatory infiltrate (plasmin, lysoxmal enzymes).

Complement system

CLOTTING – FIBRINOLYTIC SYSTEM

Hageman factor (Factor XII)

Negatively changed surfaces (collagen, basement membrane)

Activated Hageman Factor (Factor XIIA)

Cofactor HMWK (high molecular weight kininogen)

KININ SYSTEM CLOTTING SYSTEM

FIBRINOLYTIC SYSTEM

KININ SYSTEM

Prekalikrenin Kalikrein Chemotaxis

Converts C5toC5a

Highmolecular Weight kininogen (HMWK)

Bradykinin

Increase vascular permeability Pain Dilatation of blood vessels Contraction of smooth muscle

FIBRINOLYTIC SYSTEM

Plasminogen Plasmin Lyse fibrin clots

Activates factor XII Cleaves C3 to produce C3 fragments Degrades fibrin to fibrin split products

Increase vascular permeability

CLOTTING SYSTEM

XI XIA

X XA

Prothrombin Thrombin

• leukocyte adhesion

• fibroblast proliferation

Fibrinogen Fibrin

Fibrinopeptides

• Increase vascular permeability

• chemotaxis

ARACHIDONIC ACID METABOLISM • Arachidonic acid metabolites are also called eicosanoids.

Cell membrane phospholipids

Phospholipase

Arachidonic Acid

Cyclooxygenase pathway

Lipoxygenase Pathway

Chemical, physical & other mediators like C5a

Blocked by corticosteroids

Cyclooxygenase pathway

O2 Blocked by aspirin/ Indomethacin, COX-1 & COX-2 inhibitors PGG2

PGH2+(OX) – Free radical of oxygen

Prostacyclin PGI2

Inhibitor of platelet aggregation Vasodilatation Potentiate oedema

ThromboxaneTXA2

Vasoconstriction Platelet aggregation

PGE2

PGF2

PGD2

pain + fever vasodilatation

Potentiate oedema

Major metabolite in mast cells, causes vasodilatation

Arachidonic Acid

Lipoxygenase Pathway O2 Lipoxin A4 & B4

Vasodilatation Inhibit chemotaxis

5HPETE HETE

LTA4

Leukotriene

LTB4 Chemotaxis

LTC4 LTD4 LTE4

Vasoconstriction Bronchoconstriction Vascular Permeability

• HPETE = Hydroperoxy eicosatetraenoic acid

• HETE = Hydroxy eicosaaatetraenoic acid

• SRS – A is a mixture of LTC4 & LTD4

Prostaglandins • Are best regarded as autocoids or local short range

hormones, which are formed rapidly, exert their effects locally and decay spontaneously or are destroyed enzymatically.

• Prostaglandins also cause pain & fever.

• Arachidonic acid metabolites can pass from one cell to another

• Different cells cooperate with one another to produce different eicosanoids

• There are two forms of cyclooxygenases called COX-I and COX-2.

• COX-1 is found in the gastric mucosa and mucosal prostaglandins produced by COX-1 are protective against acid induced damage.

• Thus inhibition of cyclooxygenase by aspirin and NSAIDs predisposes to gastric ulceration.

• To prevent this highly selective COX-2 inhibitors are now available

Cyclooxygenase pathway

Histamin and serotonin Histamin is present in basophils, platelets and mast cells.

It is released in response to

a variety of stimuli such as

1. Leukocyte derived histamine release factors

2. Physical agents

3. Immunologic reactions

4. C3a, C5a

5. Cytokines

• Histamine causes vasodilatation and is the principal mediator of immediate phase reaction.

• It is inactivated by histaminase.

• Serotonin is found within platelets.

• It is released during platelet degranulation and has actions similar to histamine.

Histamin and serotonin

Lysosomal constituents

1.Cationic proteins Increase vascular permeability

Chemotaxis of monocytes

Inhibition of movement of neutrophils and eosinophils

2.Acid proteases Degrade bacteria and debris within phagolysosomes

3.Neutral proteases Degrade extracellular components

(eg; elastase, collagenases, Cleave C3 and C5 directly

cathepsin)

• These harmful proteases are held in check by a system of antiproteases in serum and tissue fluids

eg; alpha-1 antitrypsin inhibits neutrophil elastase.

• Deficiency of alpha-1 antitrypsin lead to sustained activity of leukocyte proteases.

Neutral proteases

Platelet activating factor [PAF]

1.Sources

Mast cells/Basophils

Neutrophils

Monocytes/Macrophages

Endometrium, Platelets

Functions

Platelet activation,

Release of histamine from platelets

Increase vascular permeability

Vasoconstriction, bronchoconstriction

Increased leukocyte adhesion to endothelium

Leukocyte degranulation

Stimulates synthesis of other mediators like eicosanoids

Chemotaxis

Platelet activating factor [PAF]

Cytokines • Cytokines are polypeptide products of activated

lymphocytes (lymphokines) and monocytes (monokines).

• Many non lymphoid cells too produce these.

• Their secretion is transient and tightly regulated

• Their effects tend to be pleiotrophic (different cells are affected differently by the same cytokine).

• They can act on the same cell that produces them (autocrine effect), on other cells in the immediate vicinity (paracrine effect) or systemically (endocrine effect).

Types of cytokines1. Those regulating lymphocyte function such as

activation, growth and differentiation.

e.g.- IL-2-stimulates lymphocyte growth and transforming growth factor β inhibit lymphocyte growth

2. Cytokines involved in innate immunity- TNF and IL-1

3. Cytokines that activate inflammatory cellsduring cell mediated immune responses, such as interferon ɣ (IFN- ɣ) and IL-12

4. Chemokines that have chemotactic activity for various leukocytes.

5. Cytokines that stimulate haematopoiesis-

Granulocyte-monocyte colony stimulating factor (GM-CSF) and IL-3

Types of cytokines

Endotoxin, Bacterial products, immune complexes, toxins, physical injury, other cytokines

Macrophage (and other cell) activation Autocrine, paracrine

& endocrine effects IL-I/TNF (Interleukion 1, & Tissue Necrosis Factor act synergistically)

Acute phase reactions

Endothelial effects

Fibroblast effects

Leukocyte effects

Acute phase reactions 1. Fever2. Increased sleep

(slow wave sleep)3. Decreased appetite4. Increased synthesis of acute phase proteins5. Haemodynamic effects

decrease vascular resistance, hypotension, shock, increase heart rate

3. Neutrophilia 4. Release of ACTH & Corticosteroids

Endothelial effects

1. Leukocyte adhesion

2. Stimulation of PAF

3. Increase procoagulant activity decrease antico-agulant activity of endothelium making it potentially thrombogenic.

Leukocyte effects Increase cytokine secretion

(IL-1,IL-6)

Fibroblast effects Collagen synthesis

Proliferation of fibroblasts, release of proteolytic enzymes by mesenchymal cells

Nitric oxide (NO) NO is a short lived, soluble free radical gas produced

by endothelial cells, macrophages and specific

neurones in the brain

• Causes vascular smooth muscle relaxation.

• Antagonism of platelet aggregation and adhesion.

• Reduction of leukocyte recruitment at inflammatory sites.

• Cytotoxic to certain microbes and tumour cells.

• Uncontrolled NO production by activated macrophages in septic shock can lead to massive peripheral vasodilatation and shock.

Oxygen derived free radicles

These include superoxide , OH, H2O2 . Their production is

dependent on the activation of the NADPH oxidative

system

They are released by neutrophils and macrophages

They cause,

1. Endothelial cell damage

2. Increase vascular permeability

3. Inactivation of antiproteases, and activation of proteases

4. Injury to other cell types – tumour cells, red cells, parenchymal cells

Serum, tissue fluids and target cells possess

anti oxidant protective mechanisms

Eg.ceruloplasmintransferinsuperoxide dismutasecatalaseglutathione peroxidas

Summary of chemical mediators of inflammation 1. Vascular permeability Histamine

C3a, C5aBradykininLeukotrienes-

LTD4,LTC4,LTE4Platelet activating factor

2. Chemotaxis C5aLTB4Bacterial products,

chemokines (IL-8) 3. Acute phase reactions IL-1,TNF

4. Tissue destruction Lysosomal products,O2 derived free radicles,

NO

5. Vasodilatation Prostaglandins, NO

6. Fever IL-1, IL-6,TNF,prostaglandins

7. Pain Prostaglandin, Bradykinin

• The different mediator systems although discussed separately are intimately intertwined and act together

• With all the mediators, there seems to be an intelligent system of checks and balances

Systemic effects of inflammation 1) Fever

Infections, toxins Immune complexes, neoplasia,

IL-1/TNF IL-6

Vascular receptors in the thermoregulatory center of the hypothalamus

Prostoglandins (E)

Vasomotor center

Sympathetic nerve stimulation

Vasoconstriction of skin vessels

Reduced heat dissipation

fever

2. Leukocytosis• usually in infflammation - 15,000 to 20,000

cells/ml• in leukemoid reactions 40,000 to 100,000

cells/ml• IL-T/TNF accelerate release of cells from

bone marrow. This leads to a rise in number of more immature neutrophils in the blood (“ shift to the left”).

Systemic effects of inflammation

CSF (colony stimulating factor) causes

proliferation of precursors in bone marrow

• Most bacterial infections neutrophilia.

• Infectious mononucleosis and mumps lymphocytosis.

• Parasitic infestations eosinophelia

• Typhoid and viral infections leucopenia

3. Acute phase reactions• Increase in slow- wave sleep

• Decreased appetite

• Hypotension

• Increased degradation of proteins

• Synthesis of acute – phase proteins by the liver

C reactive protein

Serum amyloid A

Complement

Coagulation proteins

• Most of these are mediated by IL-1, IL-6 and TNF

Inflammatory reaction

Cardinal features of inflammation

Tests for specific aetiological factors

Culture, gram stained smears

Serum antibody levels, complement levels

Inflammatory mediators

Fever

Biopsy Examination of inflammatory exudate

Increase in total number of neutrophils in peripheral blood

Presence of toxic granules

Presence of immature band forms (shift to the left)

Increase of certain plasma proteins (C-reactive protein, fibrinogen, haptoglobulin)

ESR Measurement of C-reactive protein

Diagnosis of acute inflammation