Leukocyte Extravasation

Leukocyte Extravasation

Give a brief account of cellular events in acute inflammation.
Or
Describe exudation of leucocytes in acute inflammation.
Answer:

The cellular phase of inflammation consists of two processes:

• Exudation of leucocytes.
• Phagocytosis.

Cellular events in acute inflammation: Exudation of Leucocytes:

The escape of leucocytes from the lumen of the microvasculature to the interstitial tissue is the most important feature of inflammatory response. In acute inflammation, polymorphonuclear neutrophils (PMNs) comprise the first line of body defense, followed later by monocytes and macrophages. The changes leading to the migration of leucocytes are as follows:

1. Changes in the formed elements of blood: In the early stage of inflammation, the rate of flw of blood is increased due to vasodilatation. But subsequently, there is slowing or stasis of the bloodstream.

With stasis, changes in the normal axial fl0w of blood in the microcirculation take place. The normal axial flow consists of central stream of cells comprised by leucocytes and RBCs and a peripheral cell-free layer of plasma close to 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 cen- tral column come close to the vessel wall; this is known as pavements.

2. Rolling and adhesion: Peripherally marginated and pave mounted neutrophils slowly roll over the endothelial cells lining the vessel wall (rolling phase).

This is followed by the transient bond between the leucocytes and endothelial cells becoming firmer (adhesion phase). The following cell adhesion molecules (CAMs) bring about rolling and adhesion phases:

Leukocyte extravasation steps

• Selectins: These are a group of cell adhesion molecules 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 s-Lewis X molecule. Their role is to recognize and bind to glycoproteins and glycolipids on the cell surface of neutrophils. There are 3 types of selectins viz P-selectin, E-selectin and L-selectin.
• Integrins: These are a family of endothelial cell sur- face proteins having alpha (or CD11) and beta (CD18) subunits, which are activated during the process of loose and transient adhesions between endothelial cells and leucocytes. At the same time the receptors for integrins on the neutrophils are also stimulated. This process brings about fim adhesion between leucocyte and endothelium.
• Immunoglobulin gene superfamily adhesion molecules: This group consists of a variety of immuno- globulin molecules present on most cells of the body. These take part in cell-to-cell contact through various other cell adhesion molecules and cytokines. They have a major role in the recognition and binding of immunocompetent cells as under Intercellular adhesion molecule-l and vascular cell adhesion molecule-1 allow a tighter adhesion and stabilize the interaction between leucocytes and endothelial cells.
  Platelet-endothelial cell adhesion molecule-1 or CD31 is involved in leucocyte migration from the endothelial surface.

3. Emigration: After sticking of neutrophils to the endothelium, the former 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 colla geniuses and escape out into the extravascular space; this is known as emigration.

The damaged basement membrane is repaired almost immediately. 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, and escape of red cells through gaps between the endothelial cells, diapedesis takes place.

It is a passive phenomenon of RBCs being forced out either by raised hydrostatic pressure or may escape through the endothelial defects left after the emigration of leucocytes. Diapedesis gives her- a tragic appearance to the inflammatory exudate.

4. Chemotaxis: The transmigration of leucocytes after crossing several barriers to reach the interstitial tissues is a chemotactic factor-mediated process called chemotaxis. The following agents act as potent chemotactic substances for neutrophils:

Rolling adhesion transmigration

• Leukotriene B4 (LT-B4), a product of the lipooxygenase pathway of arachidonic acid metabolites
• Components of complement system (C5a and C3a in particular)
• Cytokines (interleukins, in particular, IL-8)
• Soluble bacterial products (such as formylated pep-tides).

Cellular events in acute inflammation Phagocytosis:

Phagocytosis is defined as the process of engulfment of solid particulate material by the cells (cell—eating). The cells perform- ing this function are called phagocytes. There are 2 main types of phagocytic cells:

• Polymorphonuclear neutrophils (PMNs) which appear early in acute inflammatory response, are sometimes called as macrophages.
• Circulating monocytes and fixed tissue mononuclear phagocytes, commonly called as macrophages. Neutrophils and macrophages on reaching the tissue spaces 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:

• Recognition and attachment
• Engulfment
• Killing and degradation.

Recognition and Attachment

Phagocytosis is initiated by the expression of cell surface receptors on macrophages which recognize micro organisms 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 opsonization.

Opsonins establish a bond between bacteria and the cell membrane of phagocytic cell. 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; it is the naturally occurring antibody in the serum that coats the bacteria while the PMNs possess receptors for the same.
• C3b opsonin is the fragment generated by the activation of the complement pathway. It is strongly chemotactic for attracting PMNs to bacteria.
• Lectins are carbohydrate-binding proteins in the plasma which bind to bacterial cell wall.

Engulfment

The opsonized 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 cell wall, enveloping it in a phagocytic vacuole.

Eventually, plasma membrane enclosing the particle breaks from the cell surface so that membrane—lined phagocytic vacuole or phagosome becomes internalized in the cell and lies free in the cell cytoplasm.

The phagosome fuses with one or more lysosomes of the cell and form a bigger vacuole called a phagolysosome.

Killing and Degradation

Next is the stage of killing and degradation of microorganisms to dispose it of which is the major function of phagocytes as scavenger cells. The microorganisms after being killed by antibacterial substances are degraded by hydrolytic enzymes.

However, this mechanism fails to kill and degrade some of bacteria like tubercle bacilli. The following are the mechanisms involved in the disposal of microorganisms:

1. Intracellular Mechanisms:

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

• Oxidative bactericidal mechanism by oxygen free radicals An important mechanism of microbicidal killing is by oxidative damage by the production of reactive oxygen metabolites. A phase of increased oxygen consumption 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. 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, HOl, HOBr). This is called H2O2 – MPO halide system and is more potent antibacterial system in polymorphs than H2O2 alone:
  • MPO independent killing: Mature macrophages lack the enzyme MPO and they carry out the bactericidal activity by producing OH- ions and superoxide singlet oxygen (O’) from H2O2 in the presence of O’ 2 or in the presence of Fe++ (Fenton reaction): Reactive oxygen metabolites are particularly useful in eliminating microbial organisms that grow within phagocytes For Example. Mycobacterium tuberculosis, Histoplasma capsulatum.
• The oxidative bactericidal mechanism by 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, and alkaline phosphatase. Progressive degranulation of neutrophils and macrophages along with oxygen free radicals degrades proteins, i.e. induces proteolysis.

• Non-oxidative bactericidal mechanism: Some agents released from the granules of phagocytic cells do not require oxygen for bactericidal activity. These include the following:
  • Granules: Some of the liberated lysosomal granules do not cause killing by oxidative damage but cause lysis of microbe within the phagosome. These are lysosomal hydrolases, permeability-increasing factors, cationic proteins (defensins), lipases, proteases, and DNAases.
  • Nitric oxide: Nitric oxide is reactive free radicals similar to oxygen free radicals which are formed by nitric oxide synthase. It is produced by endothelial cells as well as by activated macrophages. Nitric oxide is another potential mechanism of microbial killing.

2. Extracellular Mechanisms:

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

• Granules: Degranulation of macrophages and neutrophils explained above continues to exert its effects of proteolysis outside the cells as well.
• Immune mechanisms: Immune-mediated lysis of microbes takes place outside the cells by mechanisms of cytolysis, antibody-mediated lysis, and cell-mediated cytotoxicity.