Inflammation

and Repair - 2

Dr.CSBR.Prasad, M.D.

V2-May,2011

Revision of lecture-I

The nomenclature used to describe

inflammation in different tissues employs

the tissue name and the suffix “-itis”

e.g

Pancreatitis

Meningitis

Pericarditis

Arthritis

…itis

• Pneumonitis

• Nephritis

• Cystitis

• Balanitis

• Posthitis

• Panniculitis

• Proctitis

• Dactylitis

• Typhlitis

• Pleurisy

• Paronychia

• Bacterial vaginosis

• Cellulitis

What are the cardinal signs of

inflammation?

• Rubor = redness

• Tumor = swelling

• Calor = heat

• Dolor = pain • (described by Celsus 1st. Century AD)

• Functio laesa = loss of function – (added by R. Virchow)

Which sign was introduced by

Virchow?

• Functio laesa = loss of function

Which is the fifth sign of

inflammation?

• Functio laesa = loss of function

Factors that control the fluid

balance between the intravascular

and extravascular compartments

• OP and HP

What happens to total white count

in acute inflammation?

• Usually elevated

Name THREE elements which

make the inflammation possible?

1. Vessels

2. Plasma components

a) Immunoglobulins

b) Complement components

3. Leucocytes

Name THREE differences between

Necrosis and Apoptosis?

In Apoptosis:

• Structures are membrane bound

• Changes occur before death [Until the last moment the cell is alive]

• Absence of inflammation

• ATP dependent process

What makes inflammation a

reality?

Blood vessels

Why we need inflammation?

• To remove infectious / injurious agents

• To remove dead tissue form the body

• It’s protective.

Name some instances where

inflammation is damaging?

• Laryngitis

• Inflammation of the 4th ventricular area

• Encephalitis / Meningitis

Name the acute inflammatory

cells?

• Pyogenic infections – Neutrophil

• Allergic inflammation – Eosinophil

• Viral infections – Lymphocyte

Name the chronic inflammatory

cells?

• Lymphocytes

• Macrophages

• Plasma cells

How do you differentiate acute from

chronic inflammation?

• Onset / duration

• Type of inflammatory cells

FIGURE 2-1 The major

local manifestations of

acute inflammation,

compared to normal.

(1)Vascular dilation and

increased blood flow

(causing erythema and

warmth)

(2)Extravasation and

extravascular deposition

of plasma fluid and

proteins (edema);

(3)Leukocyte emigration

and accumulation in the

site of injury.

Exudation

The escape of fluid, proteins, and blood cells

from the vascular system into the

interstitial tissue or body cavities is known

as exudation

Terms

• Exudate

• Transudate

• Edema

• Pus

Differences between

Transudate and Exudate

Transudate • Usually seen in

congestive states

• Increased HP

• Sp. Gr: <1.020

• Proteins: <2 gm/dl

• Few leucocytes

Exudate • Seen in inflammatory

states

• Increased vascular

permeability

• Sp. Gr: >1.020

• Proteins: >2 gm/dl

• Many leucocytes

Vascular Events

Vascular events

1. Changes in Vascular Flow and Caliber

2. Increased Vascular Permeability

(Vascular Leakage)

3. Responses of Lymphatic Vessels

Vascular events

1. Changes in Vascular Flow and Caliber

Begins early in inflammation

Consists of the following:

Vasoconstriction (transient – a few seconds)

Vasodilation (first – arterioles, then vascular bed)

Histamine, NO

Vasodilation with increased vascular permeability

Vascular events

1. Changes in Vascular Flow and Caliber

2. Increased Vascular Permeability o Contraction of endothelial cells resulting in increased

interendothelial spaces o [histamine, bradykinin, leukotrienes, substance P]

o Immediate transient response occurs rapidly / short-lived (15–30 minutes)

o Delayed prolonged leakage [begins after a delay of 2 to 12 hours] may be caused by contraction of endothelial cells or mild endothelial damage. Eg: sunburn

o Endothelial injury, resulting in endothelial cell necrosis and detachment

o Direct damage to the endothelium - in burns, or by the actions of microbes, Neutrophils that adhere to the endothelium

o Transcytosis, VEGF

o Newly formed capillaries are leaky

Vascular events

Normal

tight

Gap

junction

Contraction

Causing

vascular

leakage

Vascular events

Vascular events

Vascular events

Vascular events

Vascular events

Although various mechanisms can cause

vascular leak, combination of mechanisms

discussed earlier may play a role in a

given setting

Vascular events

1. Changes in Vascular Flow and Caliber

2. Increased Vascular Permeability

3. Responses of Lymphatic Vessels

• Normally interstitial fluid is drained by lymphatics

• Lymphatics proliferate to handle the increased load

• May carry injurious substances away from the site

• In this process they may get inflammed - Lymphangiitis

Cellular Events

REACTIONS OF LEUKOCYTES IN

INFLAMMATION

• A critical function of inflammation is to deliver leukocytes to the site of injury and to activate the leukocytes to eliminate the offending agents.

• Most important leukocytes in inflammatory reactions are the ones capable of phagocytosis, namely neutrophils and macrophages.

• These leukocytes ingest and kill bacteria and other microbes, and eliminate necrotic tissue and foreign substances.

• Leukocytes also produce growth factors that aid in repair.

• Collateral damage: When strongly activated, leucocytes may induce tissue damage

The processes involving leukocytes

in inflammation consist of:

Recruitment

Recognition

Removal

The inflammatory response consists of

TWO main components:

1. Vascular reaction and

2. Cellular reaction

A critical function of the vascular

inflammatory response is to

deliver leukocytes to the site of

injury in order to clear injurious

agents

Recruitment of Leukocytes to Sites

of Infection and Injury

Extravasation of leukocytes is a coordinated

event of:

• Margination

• Rolling

• Adhesion to endothelium

• Diapedesis (Transmigration)

• Chemotaxis

In order for leukocytes to leave the

vessel lumen, endothelial cells need to

be activated and upregulate adhesion

molecules that can interact with

complementary adhesion molecules on

leukocytes

Up-regulation of adhesion molecules on

endothelial cells is induced by an array

of inflammatory mediators such as

TNF, IL-1, histamine and others

produced by tissue resident

inflammatory cells

Four families of adhesion molecules are involved in leukocyte migration

Selectins

E-selectin (on endothelium)

P-selectin (on endothelium &

platelets; is preformed and stored in

Weible 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 Immunoglobulin family

ICAM-1 (intercellular

adhesion molecule 1)

VCAM-1 (vascular adhesion

molecule 1)

Are expressed on

activated endothelium

Ligands are integrins on

leukocytes

Integrins (a + b chain)

Heterodimeric molecules

VLA-4 (b1 integrin) binds to

VCAM-1

LFA1 and MAC1

(CD11/CD18) = b2 integrin

bind to ICAM

Expressed on leukocytes

Mucin-like glycoproteins

Heparan sulfate (endothelium)

Ligands for CD44 on

leukocytes

Bind chemokines

Recruitment of Leukocytes to Sites

of Infection and Injury

Steps in extravasation:

• Margination

Redistribution of leucocytes from the central

column to the periphery – more closer to

endothelium

Movement of neutrophils from the center o the blood vessel to their

perimeter is called as margination.

Margination

Rolling

The two pools

of circulation

in peripheral

blood

Adhesion - Pavementing

Flattening and adhesion of PMNs to

endothelium is called as ADHESION -

Pavementing which precedes

transmigration across endothelium into

extravascular space

Chemotaxis

• Once outside the blood vessel, a

neutrophil is guided towards an infection

by various diffusing chemotactic factors.

Examples include the chemokines and

the complement peptide C5a, which is

released when the complement system is

activated either via specific immunity or

innate immunity.

This is a diagram showing the effect of chemokine concentration

gradient on chemotaxis direction. The attracted cell moves through

the gradient toward the higher concentration of chemokine.

Details of Leukocyte Chemotaxis:

Neutrophil adheres via integrin binding

Integrin binds to FN in ECM

Actually recognizes RGD sequence (arg-gly-asp) in FN (not shown in figure)

RGD is also found in other ECM proteins

Integrin is recycled

Adhesion molecules • They are complimentary molecules present on

leucocytes and endothelium

• Adhesion molecule

• Ligand (binding site)

• Helps in attachment of leucocytes to endothelium

• Their expression is enhanced by cytokines secreted by the altered cells

• IL-1

• TNF

• Chemokines

Adhesion molecules

Weak Adhesion: Selectins - The initial rolling

interactions

Types: L, E and P

Firm Adhesion: Integrins – permanent margination

of leucocytes. Present on leucocytes

Lignads for integrins: ICAM, VCAM

Endothelial - Leukocyte Adhesion Molecules

Complimetary molecules

Endothelial Leukocyte Major Role

P-selectin Sialyl-Lewis X–modified

proteins

Rolling (neutrophils, monocytes,

T-lymphocytes)

E-selectin Sialyl-Lewis X–modified

proteins

Rolling and adhesion (neutrophils,

monocytes, T lymphocytes)

GlyCam-1, CD34 L-selectin Rolling (neutrophils, monocytes)

ICAM-1 CD11/CD18 (β2) integrins

(LFA-1, Mac-1)

Adhesion, arrest, transmigration

(neutrophils, monocytes,

lymphocytes)

VCAM-1 VLA-4 (β1) integrin Adhesion (eosinophils, monocytes,

lymphocytes)

Endothelial - Leukocyte Adhesion Molecules

Relocation / Synthesis

Leukocyte Migration through

Endothelium

Diapedesis:

• Commonly occurs in post capillary venules

• Facilitated by PECAM-1 / CD31

Containment to the site of injury

• CD44

• PECAM-1

Importance of studying leucocyte

adhesion molecules

Genetic deficiencies occur involving these

molecules:

• Leucocyte adhesion deficiency type-1 • Deficient synthesis of LFA-1 and Mac-1 integrins

• Leucocyte adhesion deficiency type-2 • Absence of ligand for E- and P-selectins

Features: Recurrent bacterial infections as a

consequence of impaired leukocyte adhesion

and defective inflammation

Integrins & Disease

• Humans: Leukocyte adhesion deficiency

• Genetic defect

• Can't make Beta2 Subunit

• WBCs can't stick to endothelium; an

essential initial step in fighting infections

and inflammation

• Leads to persistent bacterial infections

This 10-month-old patient with severe leukocyte adhesion

deficiency type I (LAD I) developed a cervical adenitis

caused by Klebsiella pneumoniae. Following incision and

drainage, wound healing took 4 months.

Labial ulceration from

which Escherichia coli was

cultured in an 8-month-old

girl with leukocyte

adhesion deficiency type 1

(LAD I). Note the thin

bluish scar at the superior

aspect of the labia from an

earlier cellulitis.

This 3-year-old girl had

leukocyte adhesion

deficiency type I (LAD I)

with complete absence of

CD18 expression. Note the

typical gingivostomatitis,

which was culture-negative

for any pathogen.

Omphalitis

E N D

Inflammation

Recruitment of Leukocytes to Sites

of Infection and Injury

The journey of leukocytes from the vessel

lumen to the interstitial tissue, called

EXTRAVASATION

Inflammatory Paracrines

• What causes the characteristic sequence of events in acute inflammation? Various cells at the site of tissue damage or of a specific immune response release regulatory molecules that act locally as paracrines.

• Macrophages and lymphocytes are important sources of inflammatory paracrines. As we have discussed, macrophages release IL-1 and TNF-alpha, which have powerful, widespread effects.

• Also important are mast cells, which are found throughout the body, especially under epithelia. Mast cells are filled with large vesicles containing histamine and other inflammatory paracrines (They also release PG D2, several LTs and TNF-alpha, described below). Factors associated with tissue damage can trigger the exocytosis. But sometimes it is a specific immune response that triggers the release of the inflammatory paracrines.

• Also, various arachidonic acid derivatives are important. Both prostaglandins (notably PG D2) and leukotrienes (LT) can be important, depending on the tissue. Note the effectiveness of aspirin and various NSAIDs in quieting inflammation.

• Complement peptides, C3a and C5a

• Various other molecules including nitric oxide, certain platelet products, kinins, and certain other substances we will not discuss (serotonin, etc)

What types of molecules trigger

inflammation?

• Inflammatory paracrines

Name some cells that release the

paracrine molecules.

• Mast cells

• Macrophages

• Almost every cell can release AA

derivatives

What is complement C5a?

When the complement system is activated,

a small peptide C5a is cleaved from the

protein C5. C5a readily diffuses, causing

chemotaxis and inflammation in general

What change caused by inflammatory

paracrines results in edema in the affected

area?

Increased capillary permeability

Suppose tissue damage triggers the release of a

prostaglandin that causes inflammation in the

area. What specific molecule does the tissue

damage activate that starts the synthesis of the

prostaglandin?

Phospholipase A2

Other enzymes, including COX, then convert

the arachidonic acid to the prostaglandin

The Role of Chemokines in Homing to Inflammation

Tissue Distribution of Chemokines

Introduction to

Chemokine

Families and the

Cells They Affect

Ligands

• In biochemistry and pharmacology, a ligand (Latin ligare = to bind) is a substance that is able to bind to and form a complex with a biomolecule to serve a biological purpose.

• In a narrower sense, it is a signal triggering molecule, binding to a site on a target protein.

• The binding occurs by intermolecular forces, such as ionic bonds, hydrogen bonds and Van der Waals forces. The docking (association) is usually reversible (dissociation). Actual irreversible covalent binding between a ligand and its target molecule is rare in biological systems. In contrast to the meaning in metalorganic and inorganic chemistry, it is irrelevant whether the ligand actually binds at a metal site, as it is the case in hemoglobin.

• Ligand binding to a receptor alters the chemical conformation, that is the three dimensional shape of the receptor protein. The conformational state of a receptor protein determines the functional state of a receptor. Ligands include substrates, inhibitors, activators, and neurotransmitters. The tendency or strength of binding is called affinity.

• Radioligands are radioisotope labeled compounds and used in vivo as tracers in PET studies and for in vitro .