INFLAMMATION ( Pathology )

                                                INFLAMMATION

Introduction

¨ A complex reaction in vascularized CT provoked by pathological stimuli. It is the reaction of blood vessels which leads accumulation of fluid and leukocytes in the extra vascular tissue.

¨ Inflammation serves to destroy, dilute or wall off the injurious agent and sets in motion a series of events that heals and reconstitutes the damaged tissue.

¨ Repairing begins early in the inflammation and completes after the injurious agent is neutralized. During repair the injured tissue is replaced by regenerative native parenchyma cells, by filling with fibroblasts (scar tissue) or both.

¨ Inflammation is a protective response with ultimate goal is to rid of injurious agent and its consequence (necrotic cells and debris) but it may have a potential harmful effects e.g. anaphylaxes reaction, deformation resulting from scars.

Acute inflammation
Is the immediate and early response to the injurious agent and the major role is played by vascular phenomena.

There are 3 major components:

a) Vascular caliber changes that leads increased blood flow at the zone of inflammation.

b) Structural changes in the microvasculature permitting plasma proteins and leukocytes to leave the circulation

c) Emigration of leukocytes from the microcirculation and their accumulation in the site of injury.


Exudates are a fluid rich in proteins and blood cells from the circulation to the interistium and body cavity. It contains high protein concentration, cellular debris, WBC and specific gravity of more than 1020. When the exudates contains large number of neutrophils and parenchyma cell debris we call it purulent exudates or simply pus.
Transudate is a fluid of low protein content (mostly albumin) and a specific gravity of less than 1012. it is due to hydrostatic imbalance across the vascular wall but the permeability is conserved.


I.Vascular change (caliber and flow)

a) After vasoconstriction lasting few seconds, vasodilatation occurs. The dilation involves first the arterioles and then in opening of new capillary bed in the area with a resulting increase in blood flow and hence heat and redness.

b) The increase in permeability of the microvasculature accompanied by outpouring of protein rich fluid in the interstitium results in increase in the blood viscosity in the small vessels and the consequent stasis.

c) The stasis amplifies the physiologic peripheral lining of the leukocytes until the whole wall of the vessel is covered with leukocytes phenomenon called pavementing.



II.Increased vascular permeability or vascular leakage.

This leads the escape of exudates in to the interstitial space which is the hallmark of acute inflammation. this results in reduction of the osmotic pressure in the vessels and its increase in interstitial space together with increase in the hydrostatic pressure due to vasodilatation leads marked out flow of liquids and formation of interstitial edema.

Cause of vascular leakage:

1. Formation of endothelial gaps in the venules which affects only venules of diameter of 26-60nm. It is due to histamine, bradykinin and is short lived

2. Cytoskeleton reorganization which manifests in endothelial retraction.

3. Increased transcytosis across the endothelial cyt, in the form of coated vesicles

4. Direct endothelial injury resulting in necrosis and detachment e.g. in burns.

5. Delayed prolonged leakage involving venules and capillaries caused by X rays

6. Leukocyte mediated endothelial injury due to active oxygen and proteolytic enzymes produced by leukocytes.

7. Leakage from new blood vessels.

III.Cellular events
These comprise leukocyte extravasation and phagocytosis
The critical function of the inflammation is delivery of leukocyte s to injured site. The ingest offending agents, kill bacteria, degradate necrotic tissue.
The leukocytes may prolong inflammation and induce tissue damage by releasing lysosomal enzymes, chemical mediators and active oxygen metabolites.



Extravasation

Can be divided into 3 phases:-
Margination, rolling and adhesion
Transmigration across the endothelial wall (diapedesis)
Migration in he interstitial space toward a chemiotatic stimuli.
Normally, RBC in the venule assume a central axial column displacing the leukocytes to the periphery. When the blood flow slows during the inflammation this phenomena amplifies and the endothelial surface become covered with leukocytes (margination.) Subsequently, individuals or rows of leukocytes tumble slowly along the surface transiently adhering (rolling) and finally come to rest at some point and adhere firmly on the surface. After firm adhesion, the leukocytes insert pseudo bodies into the junction of the endothelial cells and the rest of the cell squeezes into the junction and enter the space between endothelial and basal lamina and finally into the extra vascular space.




Extravasation and transmigration
Is determined by the binding of complementary adhesive molecules on the leukocytes and endothelial cell surface. These adhesive molecules belong to 4 families: selectins, immunoglobulins, integrins and mucin like glycoproteins.





1. Selectins

Are characterized by an extra cellular N-terminal domain related to sugar binding
-E selectin (CD62E) is for endothelial cells
-P selectin (CD62P) is for platelets and endothelial cells
-L selectin (CD62L) is for leukocytes.

Selectins bind through their lectin domain to the sialylated oligosaccharides which themselves covalently bound with mucin like glycoprotein on the leukocytes.

2. Immunoglobulin
Of the endothelial cells are ICAM (intracellular AM) and VCAM (vascular cell AM)
Both of them interact with integrins on the leukocytes.

3. Integrins

Are transmembrane AM hetrodimeric glycoproteins they have Alfa and beta chains. The receptors of ICAM are beta integrin LFA-1 and MAC-1. the VCAM receptors are integrin alfa4, beta1. They also function as ECM receptors.

4. Mucin like glycoproteins e.g. PSGL1, GLYCAM1

Induction of adhesion mechanism
Redistribution of AM on the surface of the cell .
P selectins are normally present in the membrane of intracytoplasma granules. On stimulation due to histamine, thrombin and PAF rapidly redistribute on the cell surface where it binds with leukocytes.
Synthesis of adhesive molecules.
Some inflammatory mediators like IL1 and TNF induce the synthesis and surface expression of the EAM e.g. E selectin is not present in the normal endothelium. Some cytokines also increase the expression of ICAM and VCAM which are present at low level in normal endothelium.
Increased avidity of the binding
LFA-1of the leukocytes do not adhere on its ligand ICAM on the endothelial cell. To firmly adhere the neutrophil needs to be activated by a chemotatioc agents with conformational changes in of the integrins.
During inflammation, increased affinity of the LFA-1 on the activated leukocyte coupled with increased ICAM-1 expression on the endothelium induced by cytokines gives strong LFA-1/ICAM-1 binding causing firm adhesion between leukocyte and endothelium which appears necessary for subsequent trans migration across the endothelium.

The sequence of events of the adhesion mechanism

1. Endothelial activation by inflammatory mediators in the site of injury increases the expression of E-selectins.

2. Rolling initially rapid with relatively loose adhesion due to interaction of selectins and their ligands.

3. Firm adhesion: leukocytes activated by chemokines increase the avidity of their integrins.

4. Transmigration mediated by interaction between ICAM/ integrins and further enhanced by PECAM-1(platelet endothelial cell adhesive molecules) on both the leukocytes and endothelium. The PECAM-1 are homophilic adhesive molecules which bind themselves.

· The proof of the importance of the adhesive molecules lies in the existence of certain genetic deficiencies of these molecules which predispose recurrent bacterial infections due to lack of adhesion of leukocytes. E.g. leukocyte adhesion deficiency type I from defect in the synthesis of β2 chain integrin; in type II there is absence of sialyl-Lewis X, the ligand of E-selectin. Ab anti AM also abrogate leukocyte extravasations in experimental acute inflammation.

Chemo taxis
Chemo taxis is locomotion orientated along chemical gradient where the leukocytes emigrate toward the site of injury through the tissue.

Chemical attractants can be exogenous or endogenous
-Exogenous are bacterial products especially peptides with N-formyl methionin terminal amino acids; others are lipids
-Endogenous include:

a) Components of the complement e.g. C5a.

b) products of lipooxygenase pathway e.g. leukotrienes B4

c) cytokines especially the chemokine family e.g. IL-8

The leukocyte movement
The binding of the chemotatic agent on the receptor on the leukocyte stimulate the activation of the phospholipase C mediated by protein G leading to the hydrolysis of PIP2 (phosphatidyl inositol 4-5 biphosphate) into inositol triphosphate (IP2) and diacyl glycerol (DAG ) and the release of calcium ions from the ER and mitochondria and Ca influx from the extra cellular space.
The increase of Ca in the cytosol triggers the assembly of the contractile elements responsible for the movement of the leukocytes. They move by extending pseudopodia through the endoth, junction and this pulls the rest of the cell.
Locomotion involves rapid assembly of the actin monomers into linear polymers at pseudopod’s leading edge followed by their disassembly away from the leading edge. These complex events are controlled by effect of the Ca ions and phosphoinositol on a number of actin regulating proteins e.g. filamin, profilin. These proteins interact with actin and myosin to produce contraction.
Leukocytes migrate in the tissue in a step-by-step manner in response to one chemioattractant after other. The position of the leukocytes is determined by the pattern of attractant receptor they express and the sequence of chemokine gradient they encounter.



Leukocyte activation
Chemotatic agents stimulate locomotion and in high concentration induce other responses of the leukocytes. These responses are can also be induced by phagocytosis of Ag- Ab complexes.

The responses are:

a) Production of arachidonic acid and its metabolites from membrane phospholipids through the activation of phospholipase A2 by DAG and increase in Ca ions concentration in the leukocyte.

b) Degranulation and release of lysosomal enzymes and activation of oxidative burst due to DAC mediated activation of protein G kinase.

c) Modulation of leukocyte adhesive molecule’s function.

Phagocytosis
The phagocytosis and the release of lysosomal enzymes by neutrophiles and macrophages constitute the 2 major benefits derived from the accumulation of leukocyte at the site of inflammation.

Phagocytosis occurs in 3 steps:

a) Recognition and attachment of the particle to be ingested.

b) Engulfment and formation of phagocytic vacuole.

c) Degradation of the ingested material

I. Recognition and attachment
Microorganisms are not recognized until they coated by opsonins which bind specific receptor on the leukocyte. The opsonization enhance the efficiency of phagocytosis.

Major opsonins are:

1)The FC of the IgG

2) C3b called opsonazing fragment of the complement

3) Carbohydrate binding protein (lectins) of the plasma called collectins which binds to the microbial cell wall.

II. Engulfment
The binding between FC and its receptor on leukocyte is enough to trigger engulfment but is markedly enhanced by C3b/ receptor binding. The C3b alone can not stimulate engulfment.

III. Degradation.
Phagocytosis stimulate oxidative burst, oxygen consumption, glycogenolysis and increase of glucose oxidation via hexose-mono phosphate shunt and production of reactive oxygen metabolites due to rapid oxidation of the NADPH by an oxidase (NADPH oxidase) with the resultant reduction of oxygen molecule into super oxide which is then converted into hydrogen peroxide by spontaneous dismutation.

NADPH oxidase
Is a complex enzyme protein formed by at least 7 proteins. In a resting leukocyte, the component proteins are in different locations in the membrane and cytoplasm. On leukocyte activation by chemotatic agents, the proteins assembly and translocate to plasma membrane or phagosome membrane where the oxidative burst is to occur and become functionally active

Accomplishment of the oxidative burst
The hydrogen peroxide formed is not enough in killing microbes and in the presence of a halide such as chlorine ions the hydrogen peroxide is converted into hypochlorus acid (HOCl) by the action of MOP (myeloperoxidase) present in azurophile granules of neutrophiles.
The HOCl acid is a potent microbcid destroying microbes by halogenations or by oxidation of the constituent proteins or lipids e.g. lipid per oxidation.
Other method of killing microbes by leukocytes are bacterial permeability increase protein (BPI), by lysosomal enzymes like hydrolases and ect.



Release of leukocyte products and leukocyte induced tissue damage

· The changes that occur in leukocytes during chemo taxis activation and phagocytosis result in release of their products not only in phagosomes but also in extra cellular space. These products which are oxygen active metabolites, lysosomal enzymes and products of arachidonic acid metabolites can have powerful mediators of endothelial and tissue injury which amplifies the effects of initial inflammation stimuli.

· Thus if persistent and unchecked, the leukocyte infiltrate itself becomes the offender and leukocyte dependent tissue injury underlies many acute and chronic diseases.

Mechanism of release of the leukocyte products

1) Regurgitation during ingestion: some of the products come out spontaneously during feeding.

2) Frustrated phagocytosis: potentially ingestible materials like Ab/Ag, when on flat surface like glomerulus's BM their attachment by leukocyte triggers membrane movement and phagocytosis doesn't occur but the products are released in place

3) During surface phagocytosis, leukocyte release their products to facilitate ingestion of microbes in trapping them against a resistant surface.

4) Phagocytosis of potentially membranolytic materials like urates

5) Exocytose of specific granules of neutrophiles during phagocytosis



· After phagocytosis, neutrophiles undergo apoptotic cell death and are ingested by macrophages or cleared by lymphatic.

Defects in leukocyte function

· Leukocytes are the cardinal elements in the defense against infection and impairment of these function from adhesion to the oxidative burst, leads increased vulnerability to infections. These defects can genetic or acquired:

1)Defect in adhesion: e.g. genetic deficiency of β2 integrins gives recurrent bacterial infection and impaired wound healing.

2) Defects in phagocytosis: e.g. Chediak Higash syndrome; there is a reduced transfer of lysosome enz, to phagocytic vacuoles and hence susceptibility to infection and other signs.

Defect in microbicidal activity: some genetic defects in NADPH oxidase activities predispose recurrent bacterial infection and chronic granulomatose diseases.