Signaling Pathways In Increased Vascular Permeability Notes

The changes leading to the migration of leucocytes are as follows:

1. Changes In The Flow Of Blood:

• In the early stage of inflammation, the rate of flow of blood is increased due to vasodilatation. But subsequently, there is slowing or stasis of the bloodstream.

Read And Learn More: General Pathology Notes

• With stasis, changes in the normal axial flow of blood in the microcirculation take place. The normal axial flow consists of a central stream of cells comprised by leucocytes and RBCs and a peripheral cell-free layer of plasma close to the vessel wall.
• Due to slowing and stasis, the central stream of cells widens and the peripheral plasma zone becomes narrower because of loss of plasma by exudation.
• This phenomenon is known as margination. As a result of this redistribution, neutrophils of the central column come close to the vessel wall; this is known as pavement.

2. Rolling And Adhesion:

• Peripherally marginated and pavemented neutrophils slowly roll over the endothelial cells lining the vessel wall (rolling phase).
• This is followed by the transient sticking of leucocytes to endothelial cells (adhesion phase). The process of rolling is facilitated
  by following cell adhesion molecules (CAMs) or adhesion receptors (ARs).
• CAMs are expressed in response to cytokines (TNF, IL-1, chemokines) produced after exposure to microbial infection and cell damage

1. Selectins:  These are a group of CAMs expressed on the surface of activated endothelial cells and are structurally composed of lectins or lectin-like protein molecules, the most important of which is a sialyl-Lewis-X-modified protein (s-Lewis-X molecule or CD62). Their role is to recognise and bind to glycoproteins and glycolipids on the cell surface of neutrophils.
   • There are 3 types of selectins:
     • P-selectin (expressed on platelets and cytokine-activated endothelial cells, also called CD62P) is involved in rolling.
     • E-selectin (synthesised by cytokine-activated endothelial cells, also named ECAM or CD62E) is associated with both rolling and adhesion.’
     • L-selectin (expressed on the surface of lymphocytes, neutrophils and monocytes, also called LCAM or CD62L) is responsible for homing of circulating leucocytes to the endothelial cells in lymph nodes
2. Integrins:  These are a family of leucocyte surface proteins which mediate firm adhesion between endothelium and leucocytes. For each integrin, a corresponding ligand belonging to the immunoglobulin gene superfamily is present on endothelial cells. These ligands are stimulated by cytokines (TNF, IL-1). Important integrins on leucocytes and their corresponding ligands on endothelium participating in adhesion phase are as under:
   • β-1 integrin VLA-4 (CD49a/CD29) on monocytes and dendritic cells with corresponding ligand vascular cell-adhesion molecule-1 (VCAM-1, also named CD106) on endothelial cells.
   • β-2 integrins LFA-1 (CD11a/CD18) and MAC1 (CD11b/CD18) on leucocytes with corresponding ligand intercellular adhesion molecule-1 (ICAM-1, also called CD54) on endothelial cells

3. Transmigration:

After leucocyte-endothelium adhesion, neutrophils move along the endothelial surface till a suitable site between the endothelial cells is found where the neutrophils throw out cytoplasmic pseudopods.

• Subsequently, the neutrophils lodged between the endothelial cells and basement membrane cross the basement membrane by damaging it locally with secreted collagenases and escape out into the extravascular space; this is known as transmigration or diapedesis.
• The damaged basement membrane is repaired almost immediately. Transmigration of leucocytes across the endothelial wall is affected by platelet-endothelial cell adhesion molecule-1 (PECAM-1 or CD31) belonging to the immunoglobulin superfamily.
• As already mentioned, neutrophils are the dominant cells in acute inflammatory exudate in the first 24 hours, and monocyte-macrophages appear in the next 24-48 hours. However, neutrophils are short-lived (24-48 hours) while monocyte-macrophages survive much longer.
• Simultaneous to the emigration of leucocytes, the escape of red cells through gaps between the endothelial cells takes place.
• It is a passive phenomenon—RBCs being forced out either by raised hydrostatic pressure or may escape through the endothelial defects left after the migration of leucocytes. This gives a haemorrhagic appearance to the inflammatory exudate.

4. Chemotaxis Chemotaxis:

Is movement of leucocytes towards the direction of chemical molecules or factors is called chemoattractants.

• Transmigration of leucocytes after crossing several barriers (endothelium, basement membrane, perivascular myofibroblasts and matrix) to reach the interstitial tissues is a chemotactic factor-mediated process.

Chemo attractants for leucocytes may be of two types:

1. Exogenous agents Most important exogenous substances are soluble bacterial products such as formylated peptides.
2. Endogenous agents These include several chemical mediators

• • Leukotriene B4 (LT-B4), a product of the lipoxygenase pathway of arachidonic acid metabolites
  • Components of the complement system (C5a in particular)
  • Cytokines/chemokines (for example,IL-8, MCP-1, MIP-α, eotaxin etc)
  • Kallikerin, is the end-product of the kinin system.

Mechanism of chemotaxis The Process of chemotaxis proceeds in the following sequence:

• Chemoattractant binds to specific transmembrane G-protein-coupled receptors (GPCRs) present on the surface of leucocytes.
• GPCRs send signals for activation of effector molecules on neutrophils: phospholipase-C (PLC), phosphoinositol 3 kinase (PI3K), GTPase and tyrosine kinase.

• These effector molecules act on membrane phospholipids and increase cytosolic calcium.
• This results in cytoskeletal changes: there is polymerisation of actin at the front end of the cell while myosin gets localised at the back end of the cell.
• These changes result in the forward movement of the leucocyte towards the chemoattractant.

Phagocytosis:

After the site of infection has been infiltrated by leucocytes, the process of clearing off the microbial agent is set in action.

Phagocytosis is defined as the process of cellular engulfment of solid particulate material (for example, microbes, foreign particulate material); in other words, phagocytosis is cell-eating (on the other hand, cell-drinking is called pinocytosis).

The cells performing this function are called phagocytes. There are 2 main types of phagocytic cells:

1. Polymorphonuclear neutrophils (PMNs) which appear early in an acute inflammatory response, are sometimes called as macrophages.
2. Circulating monocytes and fixed tissue mononuclear phagocytes, commonly called macrophages.

Neutrophils and macrophages on reaching the tissue spaces at the site of infection produce several proteolytic enzymes—lysozyme, protease, collagenase, elastase, lipase, proteinase, gelatinase, and acid hydrolases.

These enzymes degrade collagen and extracellular matrix. Phagocytosis of the microbe by polymorphs and macrophages involves the following 3 steps

1. Recognition and attachment
2. Engulfment
3. Killing and degradation

1. Recognition and Attachment:

Phagocytosis is initiated by the expression of cell surface receptors on macrophages which recognise microorganisms: mannose receptor and scavenger receptor.

• The process of phagocytosis is further enhanced when the microorganisms are coated with specific proteins, opsonins, from the serum and the process is called opsonisation (meaning preparing for eating).
• Opsonins establish a bond between bacteria and the cell membrane of phagocytic cells.

The main opsonins present in the serum and their corresponding receptors on the surface of phagocytic cells (PMNs or macrophages) are as under:

• IgG opsonin is the Fc fragment of immunoglobulin G (IgG); it is the naturally occurring antibody in the serum that coats the bacteria. The corresponding receptor on PMNs is the Fc receptor for IgG called FcγRI.
• C3b opsonin is the breakdown product generated by the activation of the complement pathway. It is strongly chemotactic for attracting PMNs to bacteria. The corresponding receptor or C3b is complement receptors 1 and 3 (CR1 and 3).
• Collections are carbohydrate-binding lectins in the plasma which bind to the bacterial cell wall. The corresponding receptor for collectins is C1q.

2. Engulfment:

The opsonised particle or microbe bound to the surface of the phagocyte is ready to be engulfed.

• This is accomplished by the formation of cytoplasmic pseudopods around the particle due to activation of actin filaments beneath the cell wall, enveloping it in a phagocytic vacuole. ”
• Eventually, the plasma membrane enclosing the particle breaks from the cell surface so that membrane-lined phagocytic vacuole or phagosome becomes internalised in the cell and lies free in the cell cytoplasm.
• The phagosome fuses with one or more lysosomes of the cell and forms a bigger vacuole called a phagolysosome.

3. Killing and Degradation:

The major function of phagocytes as scavenger cells is killing and degrading microbes to dispose of. The microorganisms after being killed by antibacterial substances are degraded by hydrolytic enzymes. However, this mechanism fails to kill and degrade some bacteria like tubercle bacilli.

In general, the following mechanisms are involved in the disposal of microbes:

1. Intracellular mechanisms:
   • Reactive oxygen species
     • MPO-dependent
     •  MPO-independent
   • Nitric oxide
   • Lysosomal granules
2. Extracellular mechanisms:
   • Activated leucocytes
   • Neutrophil extracellular traps (NETs)
   • Immune mechanisms

These mechanisms are discussed below:

1. Intracellular Mechanisms:

Intracellular metabolic pathways are involved in killing microbes, more commonly by oxidative mechanism and less often by non-oxidative pathways:

• Reactive oxygen species:
  • An important mechanism of microbial killing is oxidative damage by the production of reactive oxygen species or intermediates (O^–₂, H₂O₂, OH’) and reactive species from halides (HOCl, HOI, HOBr).
    A phase of increased oxygen consumption (‘respiratory burst’) by activated phagocytic leucocytes requires the essential presence of NADPH oxidase.

NADPH-oxidase present in the cell membrane of phagosome reduces oxygen to superoxide ion (O^–₂):

Superoxide is subsequently converted into H₂O₂ which has bactericidal properties:

2O₂^– + 2H⁺  →  H₂O₂

• This type of bactericidal activity is carried out either via enzyme myeloperoxidase (MPO) present in the azurophilic granules of neutrophils and monocytes, or independent of enzyme MPO, as under:

• • MPO-dependent killing In this mechanism, the enzyme MPO acts on H₂O₂ in the presence of halides (chloride, iodide or bromide) to form hypohalous acid (HOCl, HOI, HOBr).
    1. This is called the H₂O_2^– MPO-halide system and is a more potent antibacterial system in polymorphs than H₂O₂ alone.
    2. Hereditary deficiency of MPO leads to susceptibility to infections.

• • MPO-independent killing Mature macrophages lack the enzyme MPO and they carry out the bactericidal activity by producing OH- ions from H₂O₂ by Fenton reaction or by Haber-Weiss reaction; the latter is superoxide-driven.
  • Fenton reaction:

Reactive oxygen species are particularly useful in eliminating microbial organisms that grow within phagocytes, for example, Tuberculosis, and Histoplasma capsulatum.

• Nitric oxide:
  • Nitric oxide is a reactive free radical similar to reactive oxygen species which is formed by nitric oxide synthase. It is produced by endothelial cells as well as by activated macrophages.
  • Nitric oxide is another potent agent of microbial killing by damage to lipids, proteins and nucleic acid of microbes and host cells.
• Lysosomal granules:
  • In this mechanism, the preformed granule-stored products of neutrophils and macrophages are discharged or secreted into the phagosome and the extracellular environment.
  • While the role of MPO is already highlighted above, other substances liberated by the degranulation of macrophages and neutrophils are protease, trypsinase, phospholipase, hydrolases, cationic proteins (defensins), DNAase and alkaline phosphatase.
  • Progressive degranulation of neutrophils and macrophages along with oxygen free radicals degrades proteins i.e. induces proteolysis.

2. Extracellular Mechanisms:

The following mechanisms explain the bactericidal activity at the extracellular level:

• Activated leucocytes: When leucocytes get activated on exposure to an offending agent, they perform their defensive antimicrobial role against invading organisms but at the same time they do not distinguish in causing harmful effects on the host cells too.
• Activated macrophages and neutrophils release granules into the extracellular environment causing proteolysis. Besides, they generate reactive oxygen species which may cause leucocyte-dependent tissue injury to the host.
• Neutrophil extracellular: traps (NETs) NETs are a fibrillary network of material released from nuclei of neutrophils which can trap microbes in their fibrils at the site of infection and prevent its spread. In other words, neutrophils commit beneficial suicide to liberate NETs.
• Immune mechanisms:  immune-mediated lysis of microbes takes place outside the cells by mechanisms of cytolysis, antibody-mediated lysis and cell-mediated cytotoxicity.

Acute Inflammatory Response:

• Acute inflammatory response by the host to an etiologic agent is a continuous process consisting of vascular and cellular events.
• Vascular changes include haemodynamic changes and altered vascular permeability.
• Haemodynamic changes in the microvasculature in sequence are: initial transient vasoconstriction, followed by persistent progressive vasodilatation, raised local hydrostatic pressure and transudation in extracellular space.
• At the same time, there is slowing and margination of the bloodstream.
• Increased vascular permeability and appearance of inflammatory oedema occur by different mechanisms: contraction of endothelial cells to produce gaps, mild to severe endothelial damage, direct or leucocyte-mediated injury to endothelial cells, transcytosis and excessive leakiness in neovascularization.
• The triple response is a demonstration of cardinal features of inflammation by firm stroking of the skin surface.
• A cellular phase of inflammation consists of the exudation of leucocytes and phagocytosis
• Leucocyte exudation begins from a change of normal axial blood flow to slowing and stasis, followed by margination, pavement, rolling, adhesion, and finally, transmigration of leucocytes across the endothelial wall directed to chemoattractants.
• Phagocytosis is cellular eating. The process of engulfment of foreign particulate material involves its initial recognition and opsonisation.
• The mechanisms of phagocytosis are largely intracellular (by reactive oxygen species, nitric oxide, and lysosomal granules) and a few extracellular mechanisms (activated leucocytes, NETs, immune).