IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 5. Cell Migration and Inflammation

MECHANISMS OF CELLULAR MIGRATION

The exit of leukocytes from the vascular bed through the endothelium occurs in several stages (the first three of which are shown in Fig. 5.10).

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Fig. 5.10. Three phases of leukocyte adhesion exemplified by a neutrophil (other leukocyte types and situations involve different sets of Cell-Cell Adhesion molecules). 1. Margination: binding of leukocyte CD15 to E-selectin causes the neutrophil to slow down and gradually stop as it rolls along the endothelium. 2. Activation: the detained neutrophil is activated through direct interaction with endothelial cell surface components or with chemokines and other chemotactic molecules present on the endothelium. 3. Arrest: activation mobilizes leukocyte Integrins (CR3 and LFA-1) to bind ICAM-1, the surface expression of which on the endothelium is induced upon activation.

✵ Margination: leukocyte movement along the venule slows down, and the Cells roll along the endothelium. This is primarily mediated by the interaction of selectins with carbohydrate ligands.

✵ Activation: arrested leukocytes are exposed to cytokines, chemotactic agents, endothelial surface components, and the Extracellular matrix. These factors are capable of activating The Cell and initiating its migration program.

✵ As a result of leukocyte arrest and integrin mobilization, the affinity of leukocyte integrins increases; these integrins interact with endothelial intercellular adhesion molecules and activate the cell for migration.

✵ Migration: assisted by a new set of adhesion molecules, leukocytes bind to the endothelial basement membrane and penetrate through it.

✵ Lysis: migrating cells release Enzymes that lyse Collagen and Other components of the endothelial basement membrane, enabling leukocytes to invade the tissue.

Cell adhesion molecules regulate leukocyte adhesion and transendothelial migration

Each type of cell movement is characterized by the involvement of a specific set of adhesion molecules and chemotactic agents.

Leukocyte migration to the site of inflammation. Neutrophils appear at the site of acute inflammation during its early stage, which is partly due to cytokine-Induced Expression of E-selectin on the endothelial surface in this region. Stimulation of endothelial cells in vitro by cytokines such as tumor necrosis factor alpha (TNFα) or interleukin-1 (IL-1) induces E-selectin expression within 4–12 hours, which subsides after 24 hours (Fig. 5.11); similarly, in vivo, this adhesion molecule appears during the early phase of the inflammatory response. Cells transfected with the E-selectin Gene express it in significant amounts, thereby acquiring a pronounced capacity to bind neutrophils. All these data indicate that E-selectin-mediated neutrophil arrest is the first necessary stage of their migration.

Fig. 5.11. Kinetics of the expression of various intercellular adhesion molecules on human umbilical vein endothelial cells following in vitro stimulation with TNFα.

Important roles in the migration of neutrophils, lymphocytes, and monocytes are also played by the leukocyte-expressed integrins LFA-1 and CR3, which bind to endothelial intercellular adhesion molecules belonging to the immunoglobulin superfamily. For instance, LFA-1 binds to ICAM-1 and ICAM-2 on the vascular endothelium. In culture, endothelial cells constitutively synthesize ICAM-2; it has therefore been suggested that this particular protein determines the baseline level of lymphocyte binding to various types of endothelium in vivo. For example, the level of ICAM-2 expression on cerebral vessel endothelium is normally relatively low, which corresponds to very modest transendothelial migration of lymphocytes. In contrast, the expression of ICAM-1, normally low on the endothelial surface, can be sharply upregulated by cytokines (TNFα, IL-1, or IFNγ, depending on the animal species). Under in vitro conditions, induced ICAM-1 expression is observed 8–96 hours post-stimulation (Fig. 5.11), which corresponds to the later arrival of lymphocytes and monocytes at the inflammatory site in vivo. The Role of CR3 in phagocyte recruitment has been demonstrated by in vivo experiments using anti-CR3 Antibodies, which were found to suppress the migration of these cells. Patients with leukocyte adhesion deficiency, who are prone to severe infections due to impaired phagocyte accumulation, exhibit a deficiency of all β₂-integrins (LFA-1, CR3, CR4). Notably, CR3 and LFA-1 bind to different regions of ICAM-1.

The expression of VCAM-1, like that of ICAM-1, is induced at sites of inflammation, and in vitro induction of these two molecules occurs synchronously (Fig. 5.11). (VCAM-1 binds to the α₄β₁ integrin expressed by certain lymphocyte subpopulations, and also mediates the selective adhesion of basophils and eosinophils, thus playing a crucial role in allergic reactions.) At the same time, the mechanisms governing the induction of E-selectin, ICAM-1, and VCAM-1 subtly differ among various lymphocyte populations and endothelial cells across different vascular beds. This ensures the precise fine-tuning of leukocyte transendothelial migration during inflammation and the sequential arrival of distinct cell populations at the inflammatory site. Lymphocyte adhesion to the endothelium can be inhibited by antibodies against lymphocyte or endothelial adhesion molecules, or by soluble preparations of these molecules themselves. This approach forms the basis for a novel therapeutic strategy against immunologically mediated diseases.

Normal leukocyte migration. The molecules described above that regulate cell migration to inflammatory sites must be distinguished from those responsible for normal lymphocyte homing. Naive lymphocytes express L-selectin, which mediates binding to carbohydrate ligands on high endothelial venules (HEV) endothelium (and participates in adhesion) in mucosal lymphoid Tissues and peripheral Lymph Nodes. Upon halting in a Peyer's patch, for instance, they can bind via the α₄β₇ integrin to MAdCAM-1 on the endothelial surface. Because the α₄β₇ integrin directs lymphocyte migration to mucosal lymphoid tissue, whereas the α₄β₁ integrin mediates binding to VCAM-1 on activated endothelium or to Fibronectin in the extracellular matrix, the expression of one or the other molecule dictates divergent migratory pathways—naive lymphocytes to normal lymphoid tissue versus activated T cells to sites of inflammation.

Leukocyte interactions with the extracellular matrix. Having crossed the vascular endothelium and entered the tissue, leukocytes inevitably interact with extracellular matrix Proteins (collagen, Laminin, fibronectin, etc.) as well as with tissue cells. Lymphocytes that have exited the Blood vessel immediately shed the now unnecessary L-selectin via enzymatic Cleavage. A functional phenotype switch occurs: the circulating cell adapts for locomotion within tissues.

Many leukocyte surface molecules that mediate interactions with the extracellular matrix belong to the group of β₁ integrins; they are termed very late Antigens (VLA) because they were first identified On the surface of T cells at a late stage of activation. Today, all β₁ integrins are referred to as VLA, although the majority of them are present on cells other than lymphocytes. This group includes receptors for collagen (VLA-2 and VLA-3), laminin (VLA-3 and VLA-6), and fibronectin (VLA-3, VLA-4, and VLA-5). The appearance of some of these molecules, representing a delayed consequence of lymphocyte activation, indicates that the cells are executing a differentiation program in which interaction with the extracellular matrix is one of the final stages.

Chemotactic molecules stimulate leukocyte migration and direct its course

The integrins that leukocytes use to penetrate the endothelium are present on the cell surface or stored in intracellular granules prior to deployment; most of them are inactive and require an activation signal originating from the endothelium to function. Signaling molecules may be products of the endothelium itself or Peptides released by the underlying tissue and deposited on the endothelium. Many of these signaling molecules also possess chemotactic properties; these include C5a, leukotriene B₄, and various low-molecular-weight cytokines collectively known as chemokines (Fig. 5.12).

Fig. 5.12. Molecules inducing chemotaxis of different leukocyte populations depending on the expression of specific receptors on their surface. Some of these chemokines exert a chemotactic effect on cells, while others act solely as activators.

Cell chemotactic activity should be distinguished from chemokinetic activity: chemotaxis is the directed migration of cells along a concentration gradient of chemotactic molecules, whereas chemokinesis represents random cell movement. Directed migration is driven by a cell's ability to detect a concentration gradient of a chemotactic mediator when the difference in concentration between its leading and trailing poles is at least 0.1%. Chemokinesis is associated with an overall increase in cell motility induced by a specific mediator, such as histamine.

Chemokines. These are a group of chemotactic, heparin-binding molecules comprising at least 25 low-molecular-weight cytokines, notably IL-8 and RANTES¹. Chemokines are released at the site of inflammation and can bind to the endothelial surface by interacting with the sulfate groups of local heparin. Many of these cytokines are also bound by the DARC antigen—the Duffy blood group antigen selectively expressed on venular endothelium. Endothelium-bound chemokines can increase the avidity of leukocyte integrins during the "margination" phase after the cells have been arrested by selectins. Although the majority of chemokines are synthesized by leukocytes, IL-8 and macrophage chemoattractant protein-1 (MCP-1) are produced, for example, by endothelial cell cultures, with pro-inflammatory cytokine activation further upregulating their synthesis. Chemokines and other chemotactic molecules discussed below act on cells via serpentine receptors characterized by seven transmembrane segments. Different serpentine receptors (with varying specificities) are selectively distributed among distinct leukocyte populations, which partly explains the selective effects of various chemokines—such as the fact that macrophage inflammatory protein-1β (MIP-1β) exclusively stimulates CD8⁺ T cells. Some chemokines merely activate cells, others primarily exhibit chemotactic properties, and still others combine both functions. The exact purpose of such a vast diversity of chemokines remains unclear, but it is presumed to allow the fine-tuned, selective regulation of leukocyte trafficking both at the endothelial surface and within tissues.

Other chemotactic molecules. A number of proteins induce chemotaxis in neutrophils and macrophages (Fig. 5.12). These cells possess receptors for N-terminally formylated peptides, notably the receptor binding the tripeptide f-Met-Leu-Phe (f-MLP). Since prokaryotic protein Translation uses formylmethionine as The initiating amino acid (unlike eukaryotic translation), it serves as a straightforward, specific signal indicating the presence of Bacteria toward which phagocytes must migrate. Neutrophils and macrophages also express receptors for C5a and leukotriene B₄. Both of these chemoattractants are generated at the site of inflammation: C5a through Complement activation, and leukotriene LTB₄ via the activation of various cells, most commonly macrophages and mast cells. Additionally, phagocyte chemotaxis can be triggered by molecules produced by the Blood Coagulation cascade, primarily fibrinopeptide B and Thrombin.

Upon activation, the cells that arrive first at the site of inflammation can trigger the next wave of leukocyte recruitment. For instance, activated monocytes secrete IL-8, which can stimulate the chemotaxis of neutrophils and basophils. Similarly, macrophage activation leads to the METABOLISM of arachidonic acid, resulting in the synthesis and release of leukotriene B₄.

¹RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted) is a chemokine secreted by activated non-immune T cells; molecular weight 7.8–8.7 kDa; a chemoattractant for monocytes and CD4⁺/CD45RO⁺ T cells. — Trans.