Migration of cells from the blood into tissue, and movement of cells in tissues.

The process by which cells leave the bloodstream and cross the endothelium to enter into various tissues is called extravasation. Although the particular molecules involved may differ in different situations, the fundamental process is the same. Extravasation can be divided into three stages – rolling, activation and firm attachment, and trans-endothelial migration. Once cells have left the bloodstream they must be guided to the right location within the tissue. The entrance of neutrophils into a site of inflammation is the best understood example and will be described to illustrate the basic steps involved in these processes.
1. Rolling. Neutrophils, like other leukocytes, normally travel in the centre of the blood flow away from the endothelium. At a site of inflammation asodilation occurs, slowing down and disturbing the blood flow so that the neutrophils can ‘bump’ along the endothelium, a process known as rolling. Due to the action of inflammatory mediators, especially TNFα, the endothelial cells are activated to express P-selectin and E-selectin on their surface. These selectins can bind to sialyl-Lewisx on the surface of the neutrophil, slowing down the neutrophil so that it rolls along the endothelium.
2. Activation and firm attachment. The binding of the selectins to the sialyl-Lewisx is not strong enough for the neutrophil to adhere strongly to the endothelium. Strong attachment requires the binding of the integrin LFA-1 on the neutrophil to ICAM-1 on the endothelium. Before it can bind to ICAM-1, the LFA-1 must change conformation. One of the factors produced in an inflammatory response is interleukin- 8 (IL-8), which is a chemokine. Chemokines are a group of cytokines with chemotactic and other functions. Some of the IL-8 produced is held in the extracellular matrix on the endothelial cell surface and can bind to IL-8 receptors on the neutrophil surface. The binding of IL-8 to the neutrophil activates the neutrophil and LFA-1 changes conformation and binds firmly to ICAM-1 on the endothelium.
3. Transendothelial migration. Once the neutrophil is firmly attached to the endothelium it squeezes between the endothelial cells, making contact with the basement membrane underneath. This process is poorly understood but involves additional adhesion molecules. Finally enzymes digest the basement membrane, allowing the leucocyte to pass through into the tissue space.
4. Movement in the site of inflammation. In the inflamed tissue there will be a gradient of IL-8, with maximum levels at the centre of infection. Neutrophils that have left the bloodstream and entered the tissue will travel along the IL-8 gradient, moving towards increasing concentration of the chemokine so that they will accumulate at the centre of infection.

The way in which other leukocytes cross endothelia, leave the bloodstream and migrate through tissues is essentially the same as for neutrophils, although the adhesion molecules and chemokines may be different for different cell types. Many adhesion molecules and chemokines exist to control adhesion, integrin activation and movement of different types of cells in various tissues. In sites of inflammation other factors such as complement components and prostaglandins can also act as chemoattractants.

Adhesion molecules.

There are four families of adhesion molecules called selectins, integrins, mucin-like vascular addressins and members of the immunoglobulin superfamily and each family contains many members. Different adhesion molecules bind to each other in a specific manner and enable cells to interact with each other. Cell–cell adhesion is controlled both by the expression of particular adhesion molecules and in some cases by the activation status, or actual binding capacity, of the adhesion molecules.

Adhesion molecules.

Different adhesion molecules are expressed on different cell types; some are expressed constantly on the cell surface and others are induced by cell activation, e.g. by cytokines.
By altering cell-adhesion molecule expression or activity on endothelial cells or leukocytes, it is possible to control whether particular leukocytes bind to endothelium at a particular tissue site and, hence, the entry of the leukocytes into the tissue.
Selectins are glycoproteins that are lectins, i.e. sugar-binding molecules, some of which are expressed on leukocytes and some on endothelial cells.
Mucin-like vascular addressins are heavily glycosylated proteins and therefore can bind to the selectins. Some are expressed on leukocytes and some on endothelial cells.
Integrins are heterodimeric proteins consisting of an α-chain and a β- chain and are expressed on leukocytes. There are many α- and β-chains and they can pair to give many combinations of integrins with different expression and binding specificity. Some integrins will bind to target molecules only following activation of the leukocyte by various factors.
Immunoglobulin superfamily: these molecules contain immunoglobulin (Ig)-like domains (110 amino acids flanked by an intra-chain disulphide bond) and are the binding target for the integrins. They are expressed on endothelial cells.

Cell migration.

The movement of cells around the body must be carefully controlled so that the cells go only to where they are required. This control is at two levels: one level is controlling where leukocytes leave the bloodstream; the second level is controlling where the cells go within tissues and organs once they have left the bloodstream. For a single cell, most organs are pretty big places and the cell must go to the right location within the organ or tissue.
Two important factors play an important role in controlling the movement of cells to and within specific tissue sites. Adhesion molecules are present on leucocytes and endothelial cells, and interactions between adhesion molecules allow leucocytes to bind to endothelium as part of the process of migrating across the endothelium. Chemotactic agents, especially the chemokines, are also important in controlling cell migration. They can act directly on cells and cause them to move in a particular direction or they can act indirectly by altering the expression or binding activity of adhesion molecules.

The inflammatory response and cell migration.

If a pathogen has successfully invaded a tissue, the macrophages in the tissue may recognise the pathogens with one of the receptors and attempt to phagocytose and kill the pathogens. Often there are not enough macrophages present in a tissue to phagocytose and remove all the pathogens and therefore the tissue macrophages initiate a response that will bring additional phagocytes, together with a variety of proteins, to the site of infection from the blood. These cells and proteins then help to remove the pathogen. This response is known as the inflammatory response. The aim of the inflammatory response is to recruit cells and other factors from the bloodstream into tissues to aid in the remove of pathogens and dead cells or tissue. Leukocytes (white blood cells) are unique in their ability to move throughout the body. They travel through the bloodstream and also have the ability to leave the bloodstream and enter tissue or organs. This ability to move around the body is also referred to as ‘cell migration’.

Cytokines.

The term cytokine covers a large number of smallish proteins (usually less than 20 kDa) that serve a hormone-like function in enabling cells to communicate with each other. Most people are familiar with hormones such as insulin and growth hormone, which are produced in one organ or tissue and travel through the bloodstream to other organs where they bind to receptors on the cells of that organ and stimulate a particular response. Hormones that are produced in one organ and act on a distant tissue are said to be acting in an endocrine manner. Cytokines do not usually act in an endocrine manner; rather, they act locally. They are produced by cells in a particular tissue and act on ‘cells’ in that tissue. Cytokines therefore act in a paracrine or autocrine manner. Paracrine action means that the cytokine binds to receptors on cells close to those producing the cytokine; by ‘close’ we are probably talking about a microenvironment of microns to 1 mm. Autocrine means that the cytokine actually binds to receptors on the cell that produced the cytokine. Thus the role of cytokines is to enable cells to communicate with each other in a local environment. A few cytokines can also act in an endocrine manner.
There are many cytokines and they can be divided into families. The main families of cytokines are the interleukins, colony-stimulating G-CSF, granulocyte-CSF; M-CSF, macrophage-CSF; GM-CSF, granulocyte/monocyte-CSF; MCP, macrophage chemotactic protein; TGF, transforming growth factor; IGF, insulin-like growth factor.

Action of hormones. Endocrine: the hormone is secreted at one site of the body and travels through the bloodstream. The hormone will bind to receptors on cells at a distant site (blue cells) and cause a response in those cells. Paracrine: hormones produced by cells in a tissue bind to receptors on other cells in the immediate vicinity (blue cells). Cells in other parts of the same tissue are not affected by the hormone (white cells). Autocrine: the secreted hormone binds to receptors on the cell that produced the hormone (blue cell).

The functions of cytokines will be described in detail at the appropriate times when particular mechanisms are being explained. It is important to realise that in the body, cells are never exposed to a single cytokine – they will be exposed to a number of different cytokines, probably produced by a number of different cell types. Different cytokines can either act cooperatively in promoting a response or act antagonistically in inhibiting each other’s actions. It is the combination of cytokines to which a cell is exposed that determines the behaviour of the cell.

The cellular response to infection – production of new factors.

When cells of the innate immune system encounter pathogenic products they can respond in ways other than phagocytosis. Cells can be stimulated to synthesise and/or secrete an enormous variety of new products. Some of these products may be directly involved in killing pathogens. Other products are involved indirectly in recruiting other cell types to try and eliminate the pathogen. An important group of proteins that can be secreted in response to pathogenic stimuli are known as cytokines.

Recognition by phagocytes. Phagocytes must distinguish microbes and dead host cells from healthy host cells so that healthy host cells are not phagocytosed. Phagocytes have receptors on their surface that recognise sugars present on microbes or sugars that are newly expressed on dead or damaged host cells. These sugars are not present on healthy host cells and therefore the host cells are not phagocytosed.

The cellular response to infection – phagocytosis.

Phagocytosis is the ingestion and destruction of microbes by cells called phagocytes. The two main types of phagocytes are the macrophages and neutrophils described above. The way in which macrophages and neutrophils phagocytose particles is essentially the same and can be divided into four stages.

1. Attachment of the phagocyte to the particle being phagocytosed, which may be a pathogen, a dead or damaged host cell or a piece of tissue.
2. Ingestion. By extending membrane protrusions called pseudopodia around the particle, the phagocyte is able to engulf the particle, which is taken into the cell in a phagocytic vacuole.
3. Killing. If the ingested particle is a live cell of a pathogen (e.g. a bacterium) the phagocyte will normally kill the cell by one of a number of mechanisms.
4. Degradation. The phagocytosed particle, whether it is a dead cell or a piece of tissue, is broken down by enzymes in the phagocytic vacuole.

 Phagocytosis. Phagocytes can take up and remove bacteria and dead host cells or tissue debris. The figure shows phagocytosis of a bacterium: ➀ The phagocyte binds to the bacterium. ➁ The phagocyte extends projections around the bacterium and engulfs it in a phagocytic vacuole. ➂ The phagocyte kills the engulfed bacterium. ➃ The bacterium is degraded by proteolytic enzymes.

Although the basic process of phagocytosis is similar in neutrophils and macrophages, there is an important difference. While neutrophils are only able to phagocytose small organisms such as bacteria and viruses, macrophages are able to phagocytose larger particles such as dead cells and tissue debris in addition to microorganisms. Therefore macrophages are involved in eliminating pathogens from tissues and also in cleaning up damaged tissue by removing dead or damaged host cells. Macrophages are able to distinguish between healthy host cells and dead/damaged cells because the receptors they have for recognising sugars on microbes also recognise sugars that are exposed by dead or damaged host cells.