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Leukocytes are the primary agents in the body's defence system acting against invading microorganisms (Weissman and Cooper, 1993). This system is divided into the non-specific (granulocytes and macrophages) and the specific (lymphocytes).

In the non-specific system, granulocytes (neutrophils, eosinophils, and basophils) destroy microorganisms by releasing cytotoxic compounds from their intracellular granules and by phagocytosing them (Lehrer et a/., 1993). Macrophages also destroy microorganisms in this way.

The specific lymphoid system involves the antigen-specific cellular immune defense. B lymphocytes produce antibodies which bind to foreign organisms. Antibodies act to destroy foreign organisms by the activation of either the complement system (which perforates the membrane of the microorganism) or phagocytosis. Phagocytosis is carried out by macrophages which have receptors for the antibodies coating the microorganism (opsonization).

T lymphocytes act primarily by cell to cell contact. There are two main populations of T lymphocytes. The first set destroys the cells (killer cells) which have foreign antigens. The second coordinates the haemopoietic cells in the immune response (helper cells) and causes the effector cells to multiply.

Leukocytes continuously circulate through the blood and lymphatic system. When there is tissue damage and inflammation, leukocytes are recruited to the site. This is highly specific (Butcher, 1991). This means that neutrophils selectively enter sites

General Introduction 52

of acute inflammation or tissue damage, while eosinophils enter sites of allergic reactions and parasitic infections.

To effectively survey and respond to infections, the cells of the immune system must circulate. They do this in a non-adherent state (in the blood and lymph). When activated they rapidly become adherent. Leukocyte migration and homing is a multi- step process involving tethering/rolling, triggering, strong adhesion, and migration. It also involves the interaction of leukocytes with the endothelium (Butcher, 1991; Springer, 1994).

Leukocytes have the lowest blood flow rates when in the postcapillary venules. The flow rate in these areas is reduced during inflammation because of vessel dilation. Tethering is a loose and transient adhesion mediated by the selectin family of adhesion molecules. (Carlos and Harlan, 1994; Kishimoto and Rothlein, 1994; Lawrence et al., 1994). This tethering is not strong enough to hold the cells permanently because of the shear forces generated by the blood flow. This gives rise to rolling.

There are three selectins, named because of their lectin-like affinity for carbohydrate bearing ligands. L-selectin is located on leukocytes, P-selectin and E-selectin on stimulated endothelium. Histamine and thrombin up-regulate P-selectin from intracellular stores. On the other hand, E-selectin upregulation requires do novo

protein synthesis. This is stimulated by interleukin 1 (IL-1) and tumour necrosis factor-a (TNF-a). More than one selectin can be expressed by cells, thus fine tuning the process.

The tethering leads to cell-cell interactions (leukocytes and endothelium). These interactions are a way of triggering the leukocytes.

Leukocytes respond to ligands on the endothelial cell surface activating strong adhesion (activating integrins) in less than a second. The integrins are present on circulating leukocytes but are not active. Chemokines and other chemotactic molecules can trigger integrin activation. Chemokines are heparin-binding molecules which can become associated with proteoglycans of endothelial cells. CD44 is a prominent proteoglycan and is known to bind P-chemokine MIP-1p (Tanaka etal.,

1993). Thus, a rolling T lymphocyte will encounter it and respond by activating the integrin a 4 p i causing tight adhesion. Rolling leukocytes can be triggered by the immunoglobulin superfamily adhesion molecule CD31. CD31 is found on endothelia, monocytes, neutrophils and some T lymphocyte populations. Occupancy of CD31 by ligand leads to signal transduction followed by integrin activation (Shimizu etal.,

1992).

Once integrins have been activated they bind to members of the immunoglobulin superfamily on the endothelial cells. The main integrins involved in this phase are the Pg CD11a/CD18, CD11b/CD18 and p^ integrin very late antigen-4 (VLA-4) (Carlos and Harlan, 1994; Kishimoto and Rothlein, 1994; Alon at a!., 1995). CD11a/CD18 and CD11b/CD18 interact with the intercellular adhesion molecule- 1

(ICAM-1) and other endothelium ligands. VLA-4 binds to vascular cell adhesion molecule-1 (VCAM-1) and to the matrix component fibronectin. There is an increase in the numbers of ICAM-1 and VCAM-1 due to IL-1 and TN F-a induction.

General Introduction 54

Once they have bound to the luminal side of the endothelium, leukocytes migrate through the endothelium within minutes. When they have reached the subendothelial basal membrane they are trapped by the extracellular matrix of the basal membrane (Hourihan etal., 1993). Transmigration begins with the locomotion of the adherent leukocyte to the endothelial cell-cell junction which it traverses by a mechanism that involves homotypic binding between CD31 on leukocytes and on endothelium (Muller etal., 1993; Berman and Muller, 1995; Bogen etal., 1994). The leukocyte is thought to be guided across the endothelium and through the tissues to its target by gradients of chemoattractants which are produced at the site of injury (such as N-formyl peptides, LTB4, and PAF). At the inflammatory lesion the

leukocyte becomes activated to carry out its functions in host defence.

For the cell to be mobile it must form new adhesion contacts at the 'front' while at the back' these must be reduced. At least two mechanisms are thought to be involved. First, the transient integrin activation. This is probably due to the rapid decrease in the CD31 and other chemokine signals (Muller et al., 1993). Secondly, shedding occurs. That is, L-selectin is shed from the cell surface of leukocyte after they are activated (Kishimoto et al., 1989). In addition, it is thought that large numbers of soluble adhesion molecules in the blood may reduce adhesion by blocking adhesion ligands and modulating migration (Schleiffenbaum etal., 1992; Gearing and Newman, 1993).