INFLAMMATION

CONTENTS
INTRODUCTION HISTORICAL HIGHLIGHTS DEFINITION AND CAUSES ACUTE INFLAMMATION -VASCULAR CHANGES -LEUKOCYTE CELLULAR EVENTS CHEMICAL MEDIATORS CHRONIC INFLAMMATION SYSTEMIC EFFECTS OF INFLAMMATION

Introduction
War is the metaphor for inflammation. Both are necessary evils

The word inflammation is derived from the state of being inflammed. To inflamme means to set afire which conjurs up the color red , a sense of heat and often pain

The word is derived from the latin word enflammare

DEFINITION AND CAUSES

DEFINITION: Inflammation is defined as the local response of living mammalian tissues to injury due to any agent. It is body defense reaction in order to eliminate or limit the spread of injurious agent. Inflammation is defined as a complex reaction to injurious agents such as microbes and damaged, usually necrotic cells, that consists of vascular responses, migration and activation of leukocytes, and systemic reactions (Robins)

Purpose of inflammation
It is a body defense reaction to eliminate or limit the spread of an injurious agent as well as to remove the consequent necrosed cells and tissues. It is the channel which the immune system uses, both the innate and adaptive immune responses Innate immunity -inherent nonspecific antimicrobial response on exposure to a pathogen. Eg. Phagocytosis

Adaptive immunity- lymphocytes specifically recognize each pathogen and retains memory , and are able to combat it on reexposure to antigen.
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Historical Perspectives
The English word inflammation carries the same association with heat The earliest reference to inflammation in ancient medical literature is in the Smith Papyrus from around 1650 B.C. Egypt. The use of a symbol of a flame as the determinant shows that the ancient Egyptians associated inflammation with heat.

HISTORICAL PERSPECTIVES Ancient Greeks used the term flegmonh to mean inflammation, also indicating a hot thing (flegein to burn). The Greek term persists in our word phlegmon, used to describe certain internal inflammatory lesions
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FOUR CARDINAL SIGNS

Signs of inflammation were first described in the Egyptian papyrus in 3000BC

RUBOR(redness) TUMOR(swelling) CALOR(heat)

DOLOR(pain)

Fifth Cardinal Sign of Inflammation

Redness and Swelling with Heat and Pain ...and Loss of Function ...et Functio lsa was added by the
famous 19th century pathologist Rudolf Virchow
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The Cellular Theory of Inflammation

was advanced during the mid19th century through works of experimentalists Julius Cohnheim, who first observed vasomotor effects and leukocyte emigration in sites of inflammation and Elie

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 Contemporaneously, the work of Paul Ehrlich defining the role of complements and antibodies in host defenses gave rise to a. Humoral Theory of Inflammation Modern understanding of inflammation admits important roles for both cellular and soluble

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The components of acute and chronic inflammatory responses

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ACUTE INFLAMMATION
Acute inflammation is the immediate and early response to injury, Short duration, lasting from a few minutes up to a few days Characterized by fluid and plasma protein exudation and by a predominantly neutrophillic leukocyte accumulation. It has three major components a) Alteration in vascular caliber b)Structural changes in microvasculature c)Emigration of the leucocytes

These changes account for five classical signs of acute 14 inflammation.

Signs of inflammation

Redness (rubor) caused by hyperaemia Swelling (tumor) caused by fluid exudation Heat (calor) caused by hyperaemia Pain (dolor) resulting from release of bradykinin and PGE2 Loss of function- due to combined effects of the above
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STIMULI FOR ACUTE INFLAMMATION

Infections (bacterial, viral, parasitic) and microbial

toxins Trauma (blunt and penetrating) Physical and chemical agents (thermal injury, e.g., burns or frostbite; irradiation; some environmental chemicals) Tissue necrosis (from any cause) Foreign bodies (splinters, dirt, sutures) Immune reactions (also called hypersensitivity reactions)
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TERMINOLOGIES
The escape of fluid, proteins, and blood cells from the vascular system into the interstitial tissue or body cavities is known as exudation. An exudate is an inflammatory extravascular fluid that has a high protein concentration, cellular debris, and a specific gravity above 1.020. Transudate is a fluid with low protein content (most of which is albumin) and a specific gravity of less than 1.012.

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 Edema denotes an excess of fluid in the interstitial or serous cavities; it can be either an exudate or a transudate. Pus, a purulent exudate, is an inflammatory exudate rich in leukocytes (mostly neutrophils), the debris of dead cells and, in many cases, microbes.

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VASCULAR CHANGES
CHANGES IN VASCULAR FLOW AND CALIBER
Transient vasoconstriction(seconds) Vasodilatation involving arteriole first Local increase in blood flow

redness and warmth(erythema)

Increased permeability resulting in exudation of protein rich fluid into the extra vascular tissues
Red blood cells become concentrated increasing the viscosity and slowing the circulation(process called STASIS) Leucocytes (neutrophils) accumulate along the vascular endothelial surface(process 21 called MARGINATION)

 After adhering to endothelial cells the leucocytes squeeze between them and migrate through the vascular wall into interstitial tissue(process called emigration)

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INCREASED VASCULAR PERMEABILITY

In earliest phase, vasodilatation and increased blood flow increase intra vascular hydrostatic pressure

in filtration of fluid from the capillaries called Transudate.

flow of protein rich fluid and cells called Exudate into the interstitium ( reduces the intravascular osmotic pressure, while increasing the osmotic pressure of the interstitial fluid.)

Net result is out flow of water and ions into the extra vascular tissues ,accumulation is called Edema. How does the normally non penetratable endothelial layer become leaky during 24 acute inflammation ?

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FIVE MECHANISM ARE KNOWN

Endothelial contraction
- Lasts for (15-30 min)

cell

-Immediate transient response

-Capillaries and arterioles are unaffected

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Junctional retraction -reversible mechanism -occurs 4-6 hr after initial stimulus -persists for 24 hr or more -structural reorganization of cytoskeleton -induced by cytokine mediator(TNF&IL-1)

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Direct endothelial injury

-vascular leakage by causing endothelial cell necrosis and detachment - reaction is called immediate sustained response -direct injury may also induce a delayed prolonged leakage, begins after a delay of 2-12hr.lasts for several hours or even days -Attributed to apoptosis, and actions of cytokines.

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Leukocyte dependent endothelial injury

-due to leukocyte accumulation during inflammatory response -leukocyte may be activated, releasing toxic oxygen species and proteolytic enzymes which cause endothelial injury or detachment -injury restricted to sites where leukocytes can adhere to the endothelium(venules,and pulmonary capillaries)

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Increased transcytosis
-via an vesiculovacuolar pathway -augments venular permeability,f ollowing exposure to certain mediators (vascular endothelial growth factor VEGF)

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Leakage from new blood vessels

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 In acute inflammation, fluid loss from vessels with increased permeability occurs in distinct phases: (1) an immediate transient response lasting for 30 minutes or less, mediated mainly by the actions of histamine and leukotrienes on endothelium; (2) a delayed response starting at about 2 hours and lasting for about 8 hours, mediated by kinins, complement products, and other factors; and (3) a prolonged response that is most noticeable after direct endothelial injury,for example, after burns.
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LEUKOCYTE CELLULAR EVENTS

Extravasation of leukocytes from vascular lumen to extravascular space is divided into Margination and rolling Adhesion and transmigration between endothelial cells Miagration in interstial tissue toward a chemotactic stimulus

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MARGINATION AND ROLLING

In normal blood flow red and white blood cells travel along the central axis

As vascular permeability increases, fluid exits the vascular lumen and blood flow slows As a result leukocytes settle out of central column, marginating to vessel periphery

Subsequently they tumble on the endothelium, transiently sticking along the way, a process called rolling.
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The multistep process of leukocyte migration through blood vessels, for neutrophils

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Leukocyte

Adhesion

and

Transmigration

Regulated largely by the binding of complementary adhesion molecules on the leukocyte and endothelial surfaces, chemical mediators chemoattractants and certain cytokinesaffect these processes by modulating the surface expression or avidity of such adhesion molecules. The adhesion receptors involved belong to four molecular families the selectins, the immunoglobulin superfamily, the integrins, and mucin-like glycoproteins.
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Selectins

E-selectin (on endothelium) P-selectin (on endothelium & platelets; is preformed and stored in Weibel Palade bodies) L-selectin (leukocytes) Ligands for E-and P-Selectins are sialylated glycoproteins (e.g Sialylated Lewis X) Ligands for L-Selectin are Glycan-bearing molecules such as GlyCam-1, CD34, MadCam-1

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E-selectin(ELAM-1)-

Identified as cytokine-inducible endothelial glycoprotein that binds to both neutrophils and monocytes Not found in uninflammed endothelium Expressed within 30 mins of stimulation Peak concn. - 2 - 4 hrs Expressed for about 24 hrs. Induced by IL-1 and TNF secreted by macrophages
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P-SELECTIN Platelet activation dependent granule to external membrane protein (PADGEM) P-selectin contains the largest number of complement binding protein like sequences P-selectin is found in platelets upon activation with thrombin, histamine etc and in activated endothelium. Ligand lacto-n-fucopentaose III- sialyl LewisX Bind to neutrophils and monocytes Expressed within 10- 30 mins

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L-SELECTIN

Leukocytes use it to interact with carbohydrate molecules called vascular addressins (sialomucin CD34) on the luminal surface of the endothelial cells. SYNONYMS: Lectin adhesion molecule-1 and Leu8 and is believed to be intimately associated with lymphocyte binding to endothelium This molecule contains only 2 complement-binding extracellular domains and is found on lymphocytes, PMNs, and monocytes The expression of this molecule is associated with leukocyte cell activation and rolling.
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 Following adhesion to endothelial cells the expression of L-selectin is diminished and other leukocytic integrins that help in transendothelial migration are increased

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INTEGRINS

Family of transmembrane glycoprotein cell surface receptor adhesins with one alpha and one beta subunit Three domains- large extracellular domain 6 types of beta subunits and 11 different alpha subunits = 16 integrins Beta 2 subunit is most important in leukocyte adhesion Other functions include helping in diapedesis, leukocyte migration, T-cell macrophage interactions, clot formation, epithelial cell migration
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Integrins- and factors expression in adhesion

controlling

their

C5a induces increased surface expression of LFA-1, Mac-1 and P15095 (CD11/18) integrin complex on the surface of leukocytes. The ligand on endothelial cells for LFA and MAC-1 is ICAM-1 molecule on endothelium.

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 IL-1 stimulates leukocyte adhesion by inducing synthesis or increased surface expression of adhesive proteins on endothelial cells which can then bind to receptors on leukocytes- an endothelium dependent defect. TNF affects expression of both leukocyte and endothelial molecules.

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Integrins(a+bchain)

Heterodimeric molecules VLA-4 (b1 integrin) binds to VCAM-1 LFA1 and MAC1 (CD11/CD18) = b2 integrin bind to ICAM Expressed on leukocytes

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Endothelial adhesion molecule

Receptor on leukocyte

Function

ELAM-1

Sialyl Lewis X

PMN adhesion

ICAM-1

Integrins LFA-1 Mac -1 (CD11a,b/CD18)

Neutrophil, Lymphocyte adhesion

VCAM-1

Integrin VLA-4

Lymphocyte and monocyte adhesion

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Immunoglobulin family

Are expressed on activated endothelium Ligands are integrins on leukocytes Induction ICAM-1- 4-6hrs, max. 24hrs Leukocytes binds to ICAM- flatten over epithelium ICAM-1 (intercellular adhesion molecule 1) VCAM-1 (vascular adhesion molecule 1) 53 ICAM-2

ICAM-2 33kd, binds to leukocyte integrin a12 and a22 ICAM-1,2- localizes leukocytes to the site of injury ICAM-3 87 kd, 5 domains forms Head-tail arrangement For T cell activation VCAM-1 is induced on endothelial cells 54 by several inflammatory cytokines

PECAM-1 (CD31) cell surface glycoprotein of 130kd found on platelets, endothelial cells, monocytes and neutrophils and some Tcell subsets Localized at endothelial cell junctionsmay provide means by which endothelial cells may lose their adhesiveness and permit emigration Ligand is aVb3 integrin
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Mucin-like glycoproteins

Heparan sulfate (endothelium) Ligands for CD44 on leukocytes Bind chemokines

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 Rolling leukocytes become activated by chemokines such as monocyte chemoattractant protein 1 and RANTES, presented by the activated endothelial surface. The activated leukocytes can bind to other endothelial adhesion molecules, such as ICAM-2, and start transmigrating into the vascular intima. Prolonged endothelial stimulation 57 results in more of ICAM-1

Diapedesis
Neutrophils and monocytes create a passage between the endothelial cells. Diapedesis typically occurs in venules and is mediated by PECAM-1(CD31) A leukocyte leading edge process rich in F-actin, catenin and LFA-1 interacts with endothelial cell surface in creating this channel The endothelial zonula adherens junction undergoes rapid assembly and disassembly during the process
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Emigration
Adherent neutrophils throw out cytoplasmic pseudopods and cross the basement membrane by damaging it with secreted collagenases and escapes into the extravascular space. First 24 hours- PMNs escape 24-48 hours- monocytes and macrophages Neutrophils are shortlived. Monocyte migration is sustained Chemotactic factors for neutrophils and monocytes are activated at different phases of the response
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Interaction with cells of intercellular matrix

After crossing endothelium leukocytes have to interact with cells of intercellular matrix using VLA molecules of the 1 integrin group VLA-2,3 for collagen VLA-3,6 for laminin VLA-3,4,5 for fibronectin

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CHEMOTAXIS

AND

ACTIVATION

After extravasation leukocyte emigrate toward the site of injury along a chemical gradient in a process called chemotaxis Both endogenous and exogenous substances can act as chemotactic agent for leukocyte including: -soluble bacterial products, paricularly peptides with N-formyl methionine termini -components of complement system, particularly C5a -products of lipoxygenase pathway of arachidonic acid (AA)metabolism, leukotrieneB4 (LTB4) -cytokines

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Leukocyte activation

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Biochemical events in leukocyte activation

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PHAGOCYTOSIS
Process of engulfment of solid particulate material by the cells of the size visible to light microscope Cells performing phagocytosis are microphages (PMN) and macrophages Results in the eventual containment of the pathogen in a within a membrane- delimited structure -phagosome

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Timed sequential events in the formation of a phagosome

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STEPS

Recognition and (opsonisation) Engulfment stage Secretion (degranulation) Digestion (degradation)

attachment

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Recognition and attachment

Phagocytes are recognized and attracted to bacteria by chemokines released by bacteria and tissue proteins Opsonized by serum complement, immunoglobulin (C3b, Fc portion of IgG) Corresponding receptors on leukocytes (FcR, CR1, 2, 3) leads to binding Microorganisms get coated with opsonins from the serum to make a bond between bacteria and cell membrane of the phagocytes as phagocyte possesses receptor for opsonin

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Opsonization- coating the pathogen with a few recognizable ligands which enable the pathogen to bind to and ingest the pathogen

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OPSONINS

IgG-Fc fragments C3bgenerated by complement pathway Lectins-carbohydrate binding proteins in plasma binding to bacterial cell wall

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 The activation of the neutrophils makes them more efficient at phagocytosis and killing. Opsonisation of bacteria by complement and immunoglobulins renders them more readily phagocytosed. This entire process is orchestrated by a plethora of chemical mediators derived from injured tissues, bacteria, plasma proteins and leucocytes
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Engulfment stage

Opsonized particles bonded to the phagocyte is engulfed by formation of cytoplasmic pseudopods around the particle due to activation of actin filaments beneath the cell wall, enveloping it in a phagocytic vacuole.

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Degranulation stage
Preformed granule stored products of PMN azurophilic granules are fused to phagosomes phagocytosis triggers an oxidative burst engulfment and formation of vacuole which fuses with lysosomal granule membrane (phagolysosome) Granules discharge within phagolysosome and extracellularly (degranulation) Lysosomes discharged into intercellular space

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Killing/degradation
Killing by Hydrolytic enzymes

After the microorganisms are killed by antibacterial substances they are degraded by hydrolytic enzymes

OxygenOxygenDependent independent Bactericidal Bactericidal enzymes enzymes

Nitric oxide Mechanisms enzymes

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Oxygen-dependent

bactericidal

mechanisms

Require oxygen & redox potential of -160mv Within seconds of stimulation the neutrophils sharply increase their oxygen uptake (RESPIRATORY BURST) because of two biochemical events One electron reduction of molecular oxygen to superoxide radical oxidation of glucose via HMP shunt with resultant production of NADPH and reduced glutathionine (GSH) A respiratory burst oxidase is activated that catalyzes the transfer of electrons from NADPH to oxygen through an electron transport chain consting of a flavin and cytochrome 558 to create superoxide.

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 Extracellular superoxide is also converted to hydrogen peroxide using plasma protein ceruloplasmin H2O2 diffuses across cell membranes and is utilized by MPO enzyme as substrate with halide ions to form HOCl ions Microbicidal activity by forming toxic, reduced oxygen metabolites such as superoxide anion O2- & H2O2.is comparitively weaker than that of HOCl.

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Detoxification of hydrogen peroxide

Reduced glutathionine(GSH) is acted upon by enzyme glutathione peroxidase to detoxify H2O2.
It is also detoxified in the cytoplasm by catalase to water and oxygen.

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Oxygen-Independent Antimicrobial Activity

Bactericidal permeability increasing protein (BPI) causes phospholipase activation, phospholipid degradation and increased membrane permeability Lysozyme causes degradation of bacterial coat oligosaccharides Major basic protein (MBP) is cytotoxic component of eosinophil granules- parasites Defensins are pore-forming antibacterials. alpha defensins are small antimicrobial peptides( eg.HNP-1,2,3) ,rendering them susceptibile to cathepsins and ROS esp. for bacteria and fungi.

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 Inhibition of locomotion--- serum of certain patients, drugs- corticosteroids and phenylbutazone. Deficiencies in generation of chemotactic signals--complement system Disorders of phagocytosis- Opsonin deficiencies complement or IgG sickle cell disease failure of alternate pathway activation. Defects of engulfment- morphine analogues, diabetic ketoacidosis Disorders of lysosomal fusion steroids, colchicine, Chediak-Higashi syndrome

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Chemical Mediators
Mediators- in plasma or at local site of inflammation Induce effect by binding to specific receptors on target cells. Stimulate target cells secondary effector molecules amplify a particular response or have an opposing function Act on only one or very few targets, or widespread activity. Function is tightly regulated Quickly decay-----AA metabolites Eliminated -------antioxidants scavenge toxic O2 metabolites Inhibited------complement inhibitory proteins.
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Chemical mediators of inflammation

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Vasoactive amines Histamine- tissues- in mast cells adjacent to vessels

( basophils, platelets)

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 Neuropeptides small peptides, such as substance P and neurokinin A, Transit pain signals, regulate vessel tone and modulate vascular permeability Nerve fibres secrete neuropeptides Commonly seen in lung & GIT

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 Plasma proteases Three interrelated plasma derived factors- kinins, clotting system, complement activation of Hageman factor

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Increased vascular permeability and leukocyte emigration

leukocyte adhesion Increased permeabili ty, arteriolar dilation, bronchial smooth muscle

vascular permeability and chemotactic 88 for leukocytes