The cytokines tumor necrosis factor-α (TNFα), IL-1 and interferon-γ (IFNγ) are particularly important in this respect. TNFα is produced primarily by macrophages and other mononuclear phagocytes and has many functions in the development of inflammation and the activation of other leukocytes (Fig. 6.3). Notably, TNFα induces the adhesion molecules and chemokines on the endothelium, which are required for the accumulation of leukocytes. TNFα and the related cytokines, the lymphotoxins, act on a family of receptors causing the activation of the transcription factor NF-κB (Fig. 6.4), which has been described as a master-switch of the immune system. NF-κB is, in fact, a group of related transcription factors, which can also be activated by Toll-like receptors and IL-1. The activation of vascular endothelium by TNFα or IL-1 causes chemokine production and adhesion molecules to be expressed on the endothelial surface.

Fig. 6.3 TNFα is a cytokine with many functions

TNFα has several functions in inflammation. It is prothrombotic and promotes leukocyte adhesion and migration (top). It has an important role in the regulation of macrophage activation and immune responses in tissues (center), and it also modulates hematopoiesis and lymphocyte development (bottom).

Fig. 6.4 Intracellular signaling pathways induced by TNFα

TNFα induces the trimerization of the TNF receptor (TNFR), which causes adapter molecules to be recruited to the receptor complex. One pathway leads to the activation of caspase 8 and apoptosis. Other pathways lead to the activation of transcription factors AP1 and NF-κB, which activate many genes involved in adaptive and innate immune responses.

Once an immune reaction has developed in tissue, leukocytes generate their own cytokines (e.g. IFNγ, is produced by active Th1 cells), which also activate the endothelium and promote further leukocyte migration. The chemokines that are produced at the site depends on the type of immune response that is occurring within the tissue, and this in turn affects which leukocytes migrate into the tissue. This partly explains why different patterns of leukocyte migration and inflammation are seen in different diseases.

Leukocytes migrate across the endothelium of microvessels

The mechanisms that control leukocyte migration into inflamed tissues have been carefully studied because of their biological and medical importance. These mechanisms are also applicable in principle to the cell movement that occurs between lymphoid tissues during development and normal life.

The routes that leukocytes take as they move around the body are determined by interactions between:

• circulating cells; and

• the endothelium of blood vessels.

Leukocyte migration is controlled by signaling molecules, which are expressed on the surface of the endothelium, and occurs principally in venules (Fig. 6.5). There are three reasons for this:

• the signaling molecules and adhesion molecules that control migration are selectively expressed in venules;

• the hemodynamic shear force in the venules is relatively low, and this allows time for leukocytes to receive signals from the endothelium and allows adhesion molecules on the two cell types to interact effectively;

• the endothelial surface charge is lower in venules (Fig. 6.6).

Fig. 6.5 Leukocytes adhering to the wall of a venule

Scanning electron micrograph showing leukocytes adhering to the wall of a venule in inflamed tissue. × 16 000.

(Courtesy of Professor MJ Karnovsky.)

Fig. 6.6 Leukocyte migration across endothelium

Leukocytes circulating through a vascular bed may interact with venular endothelium via sets of surface adhesion molecules. In the venules, hemodynamic shear is low, surface charge on the endothelium is lower, and adhesion molecules are selectively expressed.

Although the patterns of leukocyte migration are complex, the basic mechanism appears to be universal. The initial interactions are set out in a three-step model (Fig. 6.7):

• Step-1 leukocytes are slowed as they pass through a venule and roll on the surface of the endothelium before being halted – this is mediated primarily by adhesion molecules called selectins interacting with carbohydrates on glycoproteins;

• Step-2 the slowed leukocytes now have the opportunity to respond to signaling molecules held at the endothelial surface – particularly important is the large group of cytokines called chemokines, which activate particular populations of leukocytes expressing the appropriate chemokine receptors;

• Step-3 activation upregulates the affinity of the leukocytes’ integrins, which now engage the cellular adhesion molecules on the endothelium to cause firm adhesion and initiate a program of migration.

Fig. 6.7 Three-step model of leukocyte adhesion

The three-step model of leukocyte adhesion and activation is illustrated by a neutrophil, though different sets of adhesion molecules would be used by other leukocytes in different situations. (1) Tethering – the neutrophil is slowed in the circulation by interactions between E-selectin and carbohydrate groups on CD15, causing it to roll along the endothelial surface. (2) Triggering – the neutrophil can now receive signals from chemokines bound to the endothelial surface or by direct signaling from endothelial surface molecules. The longer a cell rolls along the endothelium, the longer it has to receive sufficient signal to trigger migration. (3) Adhesion – the triggering upregulates integrins (CR3 and LFA-1) so that they bind to ICAM-1 induced on the endothelium by inflammatory cytokines.

Transendothelial migration is an active process involving both leukocytes and endothelial cells (Fig. 6.8). Generally leukocytes migrate through the junctions between cells, but in specialized tissues such as the brain and thymus, where the endothelium is connected by continuous tight junctions, lymphocytes migrate across the endothelium in vacuoles, near the intercellular junctions, which do not break apart.

Fig. 6.8 Lymphocyte migration

Electron micrograph showing a lymphocyte adhering to brain endothelium close to the interendothelial cell junction in an animal with experimental allergic encephalomyelitis. Adhesion precedes transendothelial migration into inflammatory sites.

(Courtesy of Dr C Hawkins.)

Migrating cells extend pseudopods down to the basement membrane and move beneath the endothelium using new sets of adhesion molecules. Enzymes are now released that digest the collagen and other components of the basement membrane, allowing cells to migrate into the tissue. Once there, the cells can respond to new sets of chemotactic stimuli, which allow them to position themselves appropriately in the tissue.

Leukocyte traffic into tissues is determined by adhesion molecules and signaling molecules

Intercellular adhesion molecules are membrane-bound proteins that allow one cell to interact with another. Often these molecules traverse the membrane and are linked to the cytoskeleton.

Q. Why is it important that cell adhesion molecules (CAMs) interact with the cytoskeleton?

A. By binding to the cytoskeleton the adhesion molecules allow a cell to gain traction on another cell or on the extracellular matrix, which allows the cell to move through tissues.

In many cases, a particular adhesion molecule can bind to more than one ligand, using different binding sites. Although the binding affinity of individual adhesion molecules to their ligands is usually low, clustering of the molecules in patches on the cell surface means that the avidity of the interaction can be high.

Cells can modulate their interactions with other cell types by increasing the numbers of adhesion molecules on the surface or altering their affinity and avidity. They can alter the level of expression of adhesion molecules in two ways:

• many cells retain large intracellular stores of these molecules in vesicles, which can be directed to the cell surface within minutes following cellular activation;

• alternatively, new molecules can be synthesized and transported to the cell surface, a process that usually takes several hours.

Selectins bind to carbohydrates to slow the circulating leukocytes

Selectins are involved in the first-step of transendothelial migration. The selectins include the molecules:

• E-selectin and P-selectin, which are expressed predominantly on endothelium and platelets; and

• L-selectin, which is expressed on some leukocytes (Fig. 6.9).

Fig. 6.9 Selectins

The structures of three selectins are shown. They have terminal lectin domains, which bind to carbohydrates on the cells listed. The EGF-R domain is homologous to a segment in the epidermal growth factor receptor. The CCP domains are homologous to domains found in complement control proteins, such as factor H, decay accelerating factor, and membrane cofactor protein.

Selectins are transmembrane molecules; their N terminal domain has lectin-like properties (i.e. it binds to carbohydrate residues), hence the name selectins. When tissue is damaged, TNFα or IL-1, induce synthesis and expression of E-selectin on endothelium. P-selectin acts similarly to E-selectin, but is held ready-made in the Weibel–Palade bodies of endothelium and released to the cell surface if the endothelium becomes activated or damaged. Both E-selectin and P-selectin can slow circulating platelets or leukocytes.

The carbohydrate ligands for the selectins may be associated with several different proteins:

• at sites of inflammation, E-selectin and P-selectin, which are induced on activated endothelium, bind to the sialyl Lewis-X carbohydrate associated with CD15, present on many leukocytes;

• some of the selectin ligands are selectively expressed on particular populations of leukocytes, for example the molecule PSGL-1 (P-selectin glycoprotein ligand) present on TH1 cells binds to E- and P-selectin, but a variant found on TH2 cells does not.

When selectins bind to their ligands the circulating cells are slowed within the venules. Video pictures of cell migration show that the cells stagger along the endothelium. During this time the leukocytes have the opportunity of receiving migration signals from the endothelium. This is a process of signal integration – the more time the cell spends in the venule, the longer it has to receive sufficient signals to activate migration. If a leukocyte is not activated it detaches from the endothelium and returns to the venous circulation. A leukocyte may therefore circulate many times before it finds an appropriate place to migrate into the tissues.

Chemokines and other chemotactic molecules trigger the tethered leukocytes

The chemokines are a group of at least 50 small cytokines involved in cell migration, activation, and chemotaxis. They determine which cells will cross the endothelium and where they will move within the tissue. Most chemokines have two binding sites:

• one for their specific receptors; and

• a second for carbohydrate groups on proteoglycans (such as heparan sulfate), which allows them to attach to the luminal surface of endothelium (blood side), ready to trigger any tethered leukocytes (Fig. 6.10).

Fig. 6.10 Chemokines

Chemokines bind to glycosaminoglycans on endothelium via one binding site while a second site interacts with chemokine receptors expressed on the surface of the leukocyte. Chemokines may be synthesized by the endothelial cell and stored in vesicles (Weibel–Palade bodies) to be released to the luminal surface (blood side) following activation (1). Alternatively chemokines may be produced by cells in the tissues and transported across the endothelium (2). Cells in the tissue can therefore signal either directly or indirectly to circulating leukocytes.

The chemokines may be produced by the endothelium itself. This depends on several factors including:

• the tissue;

• the presence of inflammatory cytokines; and

• hemodynamic forces.

In addition chemokines produced by cells in the tissues can be transported to the luminal side of the endothelium, by the process of transcytosis. Immune reactions or events occurring within the tissue can therefore induce the release of chemokines, which signal the inward migration of populations of leukocytes.

Q. What advantage is there in having different types of inflammation occurring in different tissues?

A. What constitutes an appropriate immune response depends on the pathogen, the amount of damage it is causing in a particular tissue, and the capacity of that tissue to repair and regenerate.

Chemokines fall into four different families, based on the spacing of two conserved cysteine (C) residues. For example:

• α-chemokines have a CXC structure, where ‘X’ is any amino acid residue; and

• β-chemokines a CC structure, where the cysteines are directly linked.

A single chemokine CX3CL1 is produced as a cell surface molecule, and it doubles-up as an adhesion molecule.

Chemokines receptors have promiscuous binding properties

All chemokines act via receptors that have seven transmembrane segments (7tm receptors) linked to GTP-binding proteins (G-proteins), which cause cell activation. There are also three non-signalling, scavenger receptors, which bind and clear chemokines, thereby helping to maintain chemotactic gradients.

Most chemokines act on more than one receptor, and most receptors will respond to several chemokines. Because of this complexity, it is easiest to understand what chemokines do by considering their receptors:

• the receptors for the CXC chemokines are called CXCR1, CXCR2, and so on; while

• the receptors for the CC chemokines are called CCR1, CCR2, etc.

Originally most chemokines had a descriptive name and acronym such as macrophage chemotactic protein-1 (MCP-1). The current nomenclature describes them according to their type, hence MCP-1 is CCL2, meaning that it is a ligand for the CC family of chemokine receptors (Fig. 6.11).

Fig. 6.11 Some chemokine receptors and their principal ligands

Some of the chemokine receptors found on particular leukocytes and the chemokines they respond to are listed. The cells are grouped according the principal types of effector response. Note that TH1 cells and mononuclear phagocytes both express chemokine receptor CCR5, which allows them to respond to chemokine CCL3, while TH2 cells, eosinophils, and basophils express CCR3, which allows them to respond to CCL11. This allows selective recruitment of sets of leukocytes into areas with particular types of immune/inflammatory response. Both groups of cells express chemokine receptors CCR1 and CCR2, which allow responses to macrophage chemotactic proteins (CCL2, CCL7, CCL8, and CCL13). Neutrophils express chemokine receptors CXCR1 and CXCR2, which allow them to respond to CXCL8 (IL-8) and CXCL1 and CXCL2. Bracketed entries indicate that only a subset of cells express that receptor.

The chemokine receptors are selectively expressed on particular populations of leukocytes (see Fig. 6.11) and this determines which cells can respond to signals coming from the tissues. The profile of chemokine receptors on a cell depends on its type and state of differentiation. For example:

• all T cells express CCR1;

• TH2 cells preferentially express CCR3; and

• TH1 cells preferentially express CCR5 and CXCR3. After activation in lymph nodes, the levels of CXCR3 on a T cell increase, so that it becomes more responsive for the IFNγ-inducible chemokines CXCL9, CXCL10, and CXCL11, which activate CXCR3. As a consequence, antigen-activated lymphocytes are more readily triggered to enter sites of inflammation where these chemokines are expressed.

Other molecules are also chemotactic for neutrophils and macrophages

Several other molecules are chemotactic for neutrophils and macrophages, both of which have an f.Met-Leu-Phe (f.MLP) receptor. This receptor binds to peptides blocked at the N terminus by formylated methionine – prokaryotes (i.e. bacteria) initiate all protein translation with this amino acid, whereas eukaryotes do not.

Neutrophils and macrophages also have receptors for:

• C5a, which is a fragment of a complement component generated at sites of inflammation following complement activation; and

• LTB4 (leukotriene-B4), a product of arachidonic acid, which is generated at sites of inflammation, particularly by macrophages and mast cells.

In addition, molecules generated by the blood clotting system, notably fibrin peptide B and thrombin, attract phagocytes, though many molecules such as these only act indirectly by inducing chemokines.

The first leukocytes to arrive at a site of inflammation, if activated, are able to release chemokines that attract others. For example:

• CXCL8 released by activated monocytes can induce neutrophil and basophil chemotaxis;

All of these chemotactic molecules act via 7tm receptors which activate trimeric G-proteins.

Q. Which of the chemotactic molecules described above has a microbial-associated molecular pattern (MAMP)?

A. fMLP. It is the only molecule that comes from bacteria; all other chemotactic molecules are produced by cells of the body.

Integrins on the leukocytes bind to CAMs on the endothelium

Activation of leukocytes via their chemokine receptors initiates the next stage of migration.

Leukocytes and many other cells in the body interact with other cells and components of the extracellular matrix using a group of adhesion molecules called integrins.

In the third step of leukocyte migration (see Fig. 6.7), the leukocytes use their integrins to bind firmly to cell-adhesion molecules (CAMs) on the endothelium. Leukocyte activation promotes this step in three ways:

• it can cause integrins to be released from intracellular stores;

• it can cause clustering of integrins on the cell surface into high-avidity patches;

• most importantly, the cell activation induced by the chemokines causes the integrins to become associated with the cytoskeleton and can switch them into a high-affinity form (Fig. 6.12) – this is referred to as ‘inside-out signaling’ because activation inside the cell causes a change in the position and affinity of the extracellular portion of the integrin.

Fig. 6.12 The affinity of integrins is controlled by inside-out signaling

Activation of the cell causes a change in the position of the two chains of the integrin, which become linked to the cytoskeleton via the adapter molecules vinculin and talin. The association produces an allosteric change in the extracellular portion of the molecule, causing the binding site to open and allowing the integrin to attach to its ligand.

Normally the binding affinity of integrins for the CAMs on the endothelium is relatively weak, but when sufficient interactions take place, the cells adhere firmly.

Many of the CAMs on the endothelium belong to the immunoglobulin superfamily. Some of them (e.g. ICAM-1 – intercellular adhesion molecule-1 and VCAM-1 – vascular cell adhesion molecule-1) are induced on endothelium at sites of inflammation, by inflammatory cytokines while others (e.g. ICAM-2) are constitutively expressed and not inducible (Fig. 6.13). Specific integrins bind to particular CAMs (Fig. 6.14), and since integrins vary between leukocytes and CAMs vary between endothelium in different tissues, the adhesion-step also affects which leukocytes will enter the tissue.

Fig. 6.13 Expression and induction of endothelial adhesion molecules

The graph shows the time course of induction of different endothelial molecules on human umbilical vein endothelium in vitro following stimulation by TNFα.

Fig. 6.14 Endothelial cell adhesion molecules

The molecules ICAM-1, ICAM-2, VCAM-1, and MAdCAM-1 are illustrated diagrammatically with their immunoglobulin-like domains. Their integrin ligands (see Fig. 6.w1) are listed above. MAdCAM-1 also has a heavily glycosylated segment, which binds L-selectin.

Integrins and CAMs – families of adhesion molecules

Integrins are present on many cells, including leukocytes. Each member of this large family of molecules consists of two non-covalently bound polypeptides (α and β), both of which traverse the membrane. In mammals, there are 18 alpha chains and 8 beta chains which can associate to produce approximately 24 different integrins. However, the integrins of interest to immunologists fall into three major families depending on which β chain they have; each β chain can associate with several α chains. Broadly speaking:

• the β₁-integrins are involved in binding of cells to extracellular matrix;

• the β₂-integrins are involved in leukocyte adhesion to endothelium or to other immune cells; and

• the β₃-integrins (cytoadhesins) are involved in the interactions of platelets and neutrophils at inflammatory sites or sites of vascular damage.

However, there are several exceptions to this rule, and some α chains associate with more than one β chain (Fig. 6.w1).

Fig. 6.w1 Integrins

The table gives the properties of some of the major integrins involved in leukocyte binding to endothelium or extracellular matrix.

The ability of integrins to bind to their ligands depends on divalent cations. For example, leukocyte-functional-antigen-1, LFA-1 (α_Lβ₂-integrin), has a Mg^2 + ion coordinated at the center of a binding site, which accommodates an aspartate residue (D) from the ligand. In many cases this aspartate residue is part of an amino acid sequence (RGD), or similar, which acts as a recognition sequence. Normally, integrins are expressed at the cell surface in a closed formation, where the binding site is not accessible by the ligand. Activation of the integrin by inside-out signaling causes the integrin to open like a jack-knife, so that its binding site faces outwards and can access its ligands.

The CAMs on the endothelium that interact with integrins are all members of the immunoglobulin supergene family. They include ICAM-1 and ICAM-2, VCAM-1, and mucosal addressin CAM (MAdCAM-1). All members of this family are expressed or inducible on vascular endothelium:

• ICAM-1 and VCAM-1 are both induced by inflammatory cytokines;

• ICAM-2 is constitutively expressed at low levels on some endothelia and is downregulated by inflammatory cytokines.

The two N-terminal domains of ICAM-1 are homologous to those of ICAM-2 and both molecules interact with LFA-1. (An additional ligand for LFA-1, ICAM-3, is expressed on lymphocytes and is important in leukocyte interactions.)

MAdCAM-1 is a composite molecule that includes:

• two immunoglobulin-like domains that interact with an integrin; and

• a glycosylated segment that can interact with selectins.

MAdCAM-1 was first identified on mucosal lymph node endothelium, but can also be induced at sites of chronic inflammation (Fig. 6.w2).

Fig. 6.w2 Mucosal addressin cell adhesion molecule of endothelium

The immunoelectron micrograph has been stained to show MAdCAM-1 as a dark border (arrow) on the luminal surface of endothelium. In this instance the molecule is expressed on brain endothelium in chronic relapsing experimental allergic encephalomyelitis induced by immunization of Biozzi AB/H mice with myelin basic protein.

Many of the integrins can bind to more than one ligand. For example:

• LFA-1 present on most lymphocytes binds to both intercellular CAM-1 (ICAM-1) and ICAM-2, which are expressed on endothelium;

• VLA-4 (α₄β₁-integrin) binds to vascular CAM-1 (VCAM-1) expressed on endothelium, or to fibronectin (an extracellular matrix component).

Notice that many of the β₁-integrins bind to extracellular matrix components. This group of integrins is also called very-late antigens (VLAs) because they were first identified on the T cell surface at a late stage after T cell activation. The whole group of β₁-integrins are now referred to as VLA molecules, although most of them are not just expressed on lymphocytes. They are present on many cell types in the body, allowing them to interact with extracellular matrix proteins. This group includes:

• receptors for collagen (VLA-1, VLA-2 and VLA-3);

• receptors for laminin (VLA-3 and VLA-6); and

• receptors for fibronectin (VLA-3, VLA-4, and VLA-5).

The fact that some of these molecules appear late after lymphocyte activation suggests that cells go through a program of differentiation, and that the ability to interact with extracellular matrix is one of the last functions to develop.

Leukocyte migration varies with the tissue and the inflammatory stimulus

Although the 3-stage mechanism described above applies to all leukocyte migration, distinct patterns of leukocyte accumulation are seen in different sites of inflammation, depending on:

• the state of activation of the lymphocytes or phagocytes – the expression of adhesion molecules and their functional affinity vary depending on the type of cell and whether it has been activated by antigen, cytokines, or cellular interactions. For example, activation of T cells induces both the chemokine receptor CXCR3, and the adhesion molecule LFA-1, which therefore promotes migration of activated cells into inflamed tissues.

• the types of adhesion molecule expressed by the vascular endothelium, which is related to its anatomical site and whether it has been activated by cytokines. For example: ICAM-1 is expressed at higher levels on brain endothelium than VCAM-1, whereas they are equally expressed on skin endothelium.

• the particular chemotactic molecules and cytokines present; receptors vary between leukocyte populations so that particular chemotactic agents act selectively.

Different chemokines cause different types of leukocyte to accumulate

In the second step of migration, neutrophils are triggered by chemokines such as CXCL8 synthesized by cells in the tissue, or by the endothelium itself. CXCL8 acts on two different chemokine receptors – CXCR1 and CXCR2 (see Fig. 6.11) – to initiate neutrophil migration.

In some tissues different sets of chemokines cause the local accumulation of other groups of leukocytes. For example:

• in the bronchi of individuals with asthma, CCL11 (eotaxin) is released, which causes the accumulation of eosinophils – CCL11 acts on CCR3, which is also present on TH2 cells and basophils, so by releasing one chemokine, the tissue can signal to three different kinds of cell to migrate into the tissue and this particular set of cells is characteristic of the cellular infiltrates in asthma;

• in sites of chronic inflammation, the chemokines CXCL10 and CCL2 are released by endothelium in response to IFNγ and TNFα – CXCL10 acts on activated TH1 cells (via CXCR3), while CCL2 acts on macrophages (via CCR5); consequently macrophages and TH1 cells tend to accumulate at sites of chronic inflammation.

Q. In what type of inflammatory reaction would you expect to see the production of CXCL10 and the accumulation of activated macrophages?

A. In chronic inflammatory reactions.

Preventing leukocyte adhesion can be used therapeutically

The importance of leukocyte adhesion has been pinpointed in a group of patients who have leukocyte adhesion deficiency (LAD) syndrome due to the absence of all β₂-integrins; they suffer from severe infections. The mechanism was confirmed by studies using antibodies to CR3 in experimental animals, which inhibit phagocyte migration into tissues.

A number of antibodies against adhesion molecules are now used therapeutically to treat inflammatory diseases. For example, antibodies against VLA-4 (the ligand for VCAM-1) are used to treat patients with multiple sclerosis, where the aim is to limit the migration of active T cells into the CNS.

Leukocyte migration to lymphoid tissues

Migration of leukocytes into lymphoid tissues is also controlled by chemokines and adhesion molecules on the endothelium.

Q. What is distinctive about the endothelium in secondary lymphoid tissues?

A. These tissues have high endothelial venules (HEVs) with columnar endothelial cells (see Figs. 2.53 & 2.54) that express high levels of sulfated glycoproteins and distinctive sets of adhesion molecules.

Up to 25% of lymphocytes that enter a lymph node via the blood may be diverted across the HEV. In contrast, only a tiny proportion of those circulating through other tissues will cross the regular venular endothelium at each transit. HEVs are therefore particularly important in controlling lymphocyte recirculation. Normally they are only present in the secondary lymphoid tissues, but they may be induced at sites of chronic inflammation.

In addition to their peculiar shape, HEV cells express distinct sets of heavily glycosylated sulfated adhesion molecules, which bind to circulating T cells and direct them to the lymphoid tissue.

The HEVs in different lymphoid tissues have different sets of adhesion molecules. In particular, there are separate molecules controlling migration to:

• Peyer’s patches;

• mucosal lymph nodes; and

• other lymph nodes.

These molecules were previously called vascular addressins, and their expression on different HEVs partly explains how lymphocytes relocalize to their own lymphoid tissue.

Naive lymphocytes express L-selectin, which contributes to their attachment to carbohydrate ligands on HEVs in mucosal and peripheral lymph nodes. Once they have stopped on the HEV, migrating lymphocytes may use the integrin α₄β₇ (LPAM-1, see Fig. 6.14) to bind to MAdCAM on the HEV of mucosal lymph nodes or Peyer’s patches.

Because the expression of α₄β₇ allows migration to mucosal lymphoid tissue, whereas α₄β₁ allows attachment to VCAM-1 on activated endothelium or fibronectin in tissues, expression of one or other of these molecules can alternately be used by naive lymphocytes migrating to lymphoid tissue or by activated T cells migrating to inflammatory sites.

Chemokines are important in controlling cell traffic to lymphoid tissues

Chemokines are also important in controlling cell traffic to lymphoid tissues. Naive T cells express CXCR4 and CCR7, which allows them to respond to chemokines expressed in lymphoid tissues. Initially, they recognize CCL21 on the endothelium and subsequently CCL19 produced by dendritic cells, which is thought to direct them to the appropriate T cell areas of the lymph node where dendritic cells can present antigen to them.

Once T cells have been activated they lose CXCR4 and CCR7, but gain new chemokine receptors (see Fig. 6.11), which allow them to respond to chemokines produced at sites of inflammation.

Naive B cells express CCR7 and CXCR5, a receptor for CXCL13, which is required for localization to lymphoid follicles within the lymph nodes. A subset of T cells, which are required to help B cell differentiation, also express CXCR5 causing them to colocalize with B cells in lymphoid follicles. Cells moving into lymphoid tissue therefore respond sequentially to signals on the endothelium and signals from the different areas within the tissue (Fig. 6.15).

Fig. 6.15 Chemokines and cell migration into lymphoid tissue

Cell migration occurs in stages. Naive T cells express CCR7, which allows them to respond to CCL21 expressed by secondary lymphoid tissues (1). Once the cells have migrated across the endothelium, the same receptor can respond to signals from CCL19 produced by dendritic cells (2), which promotes interactions with the T cells in the paracortex (T cell area) of the lymph node. B cells also express CCR7 and use similar mechanisms to migrate into the lymphoid tissues (1). However, they also express CXCR5, which allows them to respond to CXCL13, a chemokine produced in lymphoid follicles (2) – B cells are therefore directed to the B cell areas of the node. Mice lacking CXCR5 do not develop normal lymphoid follicles.

Mediators of inflammation

Increased vascular permeability is another important component of inflammation. However, whereas cell migration occurs across venules, serum exudation occurs primarily across capillaries where blood pressure is higher and the vessel wall is thinnest. This event is controlled in two ways:

• blood supply to the area increases;

• there is an increase in capillary permeability caused by retraction of the endothelial cells and increased vesicular transport across the endothelium – this permits larger molecules to traverse the endothelium than would ordinarily be capable of doing so, and so allows antibody and molecules of the plasma enzyme systems to reach the inflammatory site.

The four major plasma enzyme systems that have an important role in hemostasis and control of inflammation are the:

• clotting system;

• fibrinolytic (plasmin) system;

• kinin system; and

• complement system (see Chapter 4).

The kinin system generates powerful vasoactive mediators

The kinin system generates the mediators bradykinin and lysyl-bradykinin (kallidin). Bradykinin is a very powerful vasoactive nonapeptide that causes:

• venular dilation due to release of nitric oxide (NO);

• increased vascular permeability; and

• smooth muscle contraction.

Bradykinin is generated following the activation of Hageman factor (XII) of the blood clotting system, whereas tissue kallikrein is generated following activation of the plasmin system or by enzymes released from damaged tissues (Fig. 6.16).

Fig. 6.16 Activation of the kinin system

Activated Hageman factor (XIIa) acts on prekallikrein to generate kallikrein, which in turn releases bradykinin from high molecular weight kininogen (HMWK). Prekallikrein and HMWK circulate together in a complex. Various enzymes activate prokallikrein to tissue kallikrein, which releases lysyl-bradykinin from low molecular weight kininogen. Bradykinin and lysyl-bradykinin are both extremely powerful vasodilators.

The plasmin system is important in tissue remodeling and regeneration

The plasmin system can be activated by a soluble or a tissue-derived plasminogen activator, which leads to the enzymatic conversion of plasminogen into plasmin. Plasmin itself was originally identified by its ability to dissolve fibrin but it has several other activities, in particular it activates some matrix metalloproteases (MMPs), enzymes that are required for the breakdown and remodeling of collagen (Fig. 6.17). Additionally, it can promote angiogenesis (the formation of new blood vessels) by causing the release of cytokines that induce proliferation and migration of endothelial cells.

Fig. 6.17 The plasmin system

Plasmin is generated by the enzymatic activity of plasminogen activators. (MMP, matrix metalloproteinase; tPA, tissue plasminogen activator; uPA, urokinase plasminogen activator.)

Mast cells, basophils and platelets release a variety of inflammatory mediators

Auxiliary cells, including mast cells, basophils, and platelets, are also very important in the initiation and development of acute inflammation. They act as sources of the vasoactive mediators histamine and 5-hydroxytryptamine (serotonin), which produce vasodilation and increased vascular permeability.

Many of the proinflammatory effects of C3a and C5a result from their ability to trigger mast cell granule release, because they can be blocked by antihistamines. Mast cells and basophils are also a route by which the adaptive immune system can trigger inflammation – IgE sensitizes these cells by binding to their IgE receptors and the cells can then be activated by antigen. They are an important source of slow-reacting inflammatory mediators, including the leukotrienes and prostaglandins, which contribute to a delayed component of acute inflammation and are synthesized and act some hours after mediators like histamine, which are pre-formed and released immediately following mast cell activation.

Figure 6.18 lists the principal mediators of acute inflammation. The interaction of the immune system with complement and other inflammatory systems is shown in Figure 6.19.

Fig. 6.18 Inflammatory mediators

The major inflammatory mediators that control blood supply and vascular permeability or modulate cell movement.

Fig. 6.19 The immune system in acute inflammation

The adaptive immune system modulates inflammatory processes via the complement system. Antigens (e.g. from microorganisms) stimulate B cells to produce antibodies including IgE, which binds to mast cells, while IgG and IgM activate complement. Complement can also be activated directly via the alternative pathway (see Fig. 4.4). When triggered by antigen, sensitized mast cells release their granule-associated mediators and eicosanoids (products of arachidonic acid metabolism, including prostaglandins and leukotrienes). In association with complement (which can also trigger mast cells via C3a and C5a), the mediators induce local inflammation, facilitating the arrival of leukocytes and more plasma enzyme system molecules.

Platelets may be activated by:

• immune complexes; or

• platelet activating factor (PAF) from neutrophils, basophils, and macrophages.

Activated platelets release mediators which are important in type II and type III hypersensitivity reactions (see Chapters 24 and 25).

Pain is associated with mediators released from damaged or activated cells

The substances released from damaged cells, mast cells and basophils are also important in producing the sensation of pain. Platelet activating factor (PAF), histamine, serotonin, prostaglandins and leukotrienes act on C-fibers which are responsible for the poorly localized, dull-aching pain associated with inflammation. Substance-P released from activated nerve fibers further contributes to the feeling of pain. Various types of mechanical and physical damage can all also lead to the release of these mediators from the tissue cells, resulting in pain.

Lymphocytes and monocytes release mediators that control the accumulation and activation of other cells

Once lymphocytes and monocytes have arrived at a site of infection or inflammation, they can also release mediators, which control the later accumulation and activation of other cells. For example:

• activated macrophages release the chemokine CCL3 and leukotriene-B₄, both of which are chemotactic and encourage further monocyte migration;

• lymphocytes can modulate later lymphocyte traffic by the release of chemokines and inflammatory cytokines, particularly IFNγ.

Q. What determines whether an immune response is acute or chronic?

A. Ultimately the outcome of an acute inflammatory response is related to the fate of the antigen. If the initiating antigen or pathogen persists, then leukocyte accumulation continues and a chronic inflammatory reaction develops. If the antigen is cleared then no further leukocyte activation occurs and the inflammation resolves.

In recurrent inflammatory reactions and in chronic inflammation the patterns of cell migration are different from those seen in an acute response. We now know that the patterns of inflammatory cytokines and chemokines vary over the course of an inflammatory reaction and this can be related to the successive waves of migration of different types of leukocyte into the inflamed tissue.

Chronic inflammation is characteristic of sites of persistent infection and occurs in autoimmune reactions where the antigen cannot ultimately be eradicated (see Chapter 20).

Pathogen-associated molecular patterns

Before the evolutionary development of B cells and T cells, organisms still needed to recognize and react against microbial pathogens. Hence, a variety of soluble molecules and cell surface receptors developed which were capable of recognizing distinctive molecular structures on pathogens. Such structures are called pathogen-associated molecular patterns (PAMPs) and the proteins which recognize them are pattern recognition receptors (PRRs). Typical examples of PAMPs are carbohydrates, lipoproteins and lipopolysaccharide components of bacterial and fungal cell walls while some of the PRRs recognize the distinctive nucleic acids (e.g. dsRNA) formed during viral replication.

Strictly speaking, many of these molecules are found on non-pathogenic organisms, so some authors prefer to call them microbe-associated molecules patterns (MAMPs), and distinguish them from products of damaged cells, damage-associated molecular patterns (DAMPs).

Many of the PRRs that evolved in invertebrates have been retained in vertebrates and work alongside the adaptive immune system to recognize pathogens. However, the importance of different PRRs often differs between different species of mammals.

There are three main types of PRR:

• secreted molecules, present in serum and body fluids;

• receptors, present on the cell surface and on endocytic vesicles; and

• intra-cytoplasmic recognition molecules.

The intra-cytoplasmic recognition molecules are particularly important for macrophage-mediated recognition of internalized pathogens. The functions of the secreted molecules and the cell surface receptors are explained below. They are divided into families according to structure.

Some of the secreted molecules are acute phase proteins (i.e. they are present in the blood and their levels increase during infection). Indeed the first of these molecules to be recognized was C-reactive protein, which can increase by more than 1000-fold in serum, during infection or inflammation. This protein has been used as a clinical marker of inflammation for more than 70 years.

PRRs allow phagocytes to recognize pathogens

In many cases, the innate recognition mechanisms allow phagocytes to bind and internalize the pathogens and this is often associated with activation of the phagocytes, which enhances their microbicidal activity.

The binding of the pathogen to the phagocyte can be direct or indirect:

• direct recognition involves the surface receptors on the phagocyte directly recognizing surface molecules on the pathogen;

• indirect recognition involves the deposition of serum-derived molecules onto the pathogen surface and their subsequent binding to receptors on the phagocyte (i.e. the process of opsonization) (Fig. 6.20).

Fig. 6.20 The binding of pathogen to the macrophage can be direct or indirect

Macrophages can recognize PAMPs either directly or following opsonization with serum molecules.

Q. Give an example of an innate immune recognition system that allows macrophages to recognize and phagocytose bacteria

A. Complement C3b can become deposited on bacteria following activation of either the lectin pathway or the alternative pathway (see Fig. 4.4). The deposited C3b acts as an opsonin and is recognized by the receptors CR1, CR3, and CR4 on macrophages (see Fig. 4.12).

Opsonins come from a number of different families of proteins and include pentraxins, collectins and ficolins.

Soluble pattern recognition molecules

Pentraxins

The pentraxins are a family of molecules with a characteristic protein-fold, including C-reactive protein (CRP), serum amyloid-P (SAP), and pentraxin-3 (PTX3). In humans, CRP is the main acute-phase protein produced by liver hepatocytes in response to IL-6. It is a pentameric protein that binds to phosphorylcholine present on, for example, pneumococci and promotes their phagocytosis by binding to C1q and activating the complement classical pathway (see Fig. 4.4).

SAP also binds to bacteria and has a long evolutionary history (a homologue is present in horseshoe crabs). It is a major acute-phase protein in mice, although it appears to be less important in humans.

PTX3 is induced in leukocytes by inflammatory cytokines, and in vascular endothelium by IL-1. It binds to bacterial and fungal carbohydrates and to some viruses. Other members of the pentraxin family are produced in different tissues, and they appear to have important roles in recognizing and clearing apoptotic cells.

As well as activating the complement classical pathway PTX3, CRP and SAP may also directly act as opsonins, by binding to Fc-receptors.

Collectins and ficolins opsonize pathogens and inhibit invasiveness

Collectins are a family of PRMs that bind to carbohyrates. Each member of the group has subunits formed of a triple-helical collagenous tail and a lectin head (Fig. 6.w3). A number of subunits may be linked together in the complete molecule and the overall structure of these molecules is similar to that of C1q (see Fig. 4.5). Although C1q is not itself a lectin, it probably evolved from this group as an Fc receptor, at the time that antibodies developed. In addition to Fc-binding, C1q recognizes pentraxins and some bacterial components. There are several cell receptors for C1q, so it can directly promote phagocytosis. Indeed, since much of the C1q in serum is not complexed with C1r and C1s, its direct action may be as important as its role in the classical pathway of complement.

Fig. 6.w3 Structure of collectins and ficolins

Collectins and ficolins are oligomers of triple-helical molecules with C-type lectin domains. They recognize a variety of microbial PAMPs.

Mannan-binding lectin (MBL), which activates the complement lectin pathway (see Fig. 4.4) is an acute phase protein, produced by the liver, which varies structurally between individuals, affecting their susceptibility to a number of infectious and autoimmune diseases.

Surfactant proteins (SP-A and SP-D) are primarily produced by lung epithelium where they act as opsonins, but in the absence of infection, they may regulate the inflammatory actions of macrophages. All of these collectins can link to the C1q phagocytic receptor (C1qRp) via their collagenous tails. In addition, by binding to the surface of bacteria or viruses the collectins can inhibit their ability to invade the tissues. For example SP-A binds to the glycosylated hemagglutin molecule on the surface of influenza virus and reduces the ability of the virus to infect cells.

Ficolins are structurally similar to the collectins. They have a collagenous tail, but with fibrinogen-like domains at the C-terminus, that recognize microbes. Three members have been identified in humans two of which are present in serum. Because some collectins and ficolins promote activation of the complement alternative pathway, their opsonizing activity may ultimately be mediated by the interaction of C3b with complement receptors.

Phagocytes have receptors that recognize pathogens directly

Even in the absence of opsonins, phagocytes have a number of receptors that allow them to recognize PAMPs. These include:

• scavenger receptors;

• carbohydrate receptors;

• Toll-like receptors (TLRs).

The scavenger receptors and carbohydrate receptors are primarily expressed on mononuclear phagocytes, and will be described more fully in Chapter 7.

Q. Why do bacteria not just mutate their MAMPs so that they cannot be recognized by the innate immune system – after all, protein antigens of pathogens often mutate?

A. This is a difficult question because it requires deep insight into the evolutionary history of microbes, but many of the MAMPs are so fundamental to the structure of the bacterial or fungal cell walls that it is difficult to see how they could be altered without destroying the integrity of the microbe.

Toll-like receptors activate phagocytes and inflammatory reactions

The transmembrane protein Toll was first identified in the fruit fly Drosophila as a molecule required during embryogenesis. It was also noted that mutants lacking Toll were highly susceptible to infections with fungi and Gram-positive bacteria, suggesting that the molecule might be involved in immune defence. Subsequently a series of Toll-like receptors (TLRs) was identified in mammals that had very similar intracellular portions to the receptor in the flies.

The intracellular signaling pathways activated by the TLRs and the receptor for IL-1 are very similar and lead to activation of the transcription factor NK-κB.

The family of TLRs include ten different receptors, in humans, many of which are capable of recognizing different microbial components (Fig. 6.21). All of the TLRs are present on phagocytic cells, and some are also expressed on dendritic cells, mast cells, and B cells. Indeed, most tissues of the body express at least one TLR.

Q. TLR5 is present on the basal surface of epithelia in the gut, but not on the apical surface. What consequence might this have for immune reactions in the gut?

A. Bacteria within the gut would not stimulate the cell, whereas any microbes that have invaded the epithelial layer may do so. This may explain why non-pathogenic commensal bacteria in the gut do not elicit an inflammatory reaction, whereas pathogenic bacteria that have invaded the tissue do.

Expression of many of the TLRs is increased by inflammatory cytokines (e.g. TNFα, IFNγ) and elevated expression is seen in conditions such as inflammatory bowel disease. The functional importance of the TLRs has been demonstrated in mouse strains lacking individual receptors. Depending on the TLR involved, such animals fail to secrete inflammatory cytokines in response to pathogens and the microbicidal activity of phagocytes is not stimulated. These results show that the TLRs are primarily important in activating phagocytes in addition to any role they may have in endocytosis. Occasionally humans are deficient in individual TLRs. For example, TLR3 deficiency is associated with susceptibility to Herpes simplex, and a variant of TLR7 is associated with higher viral load and disease progression in HIV infection.

The TLRs make an important link between the innate and adaptive immune systems because their activation leads to the expression of co-stimulatory molecules on the phagocytes, which convert them into effective antigen-presenting cells. The binding of microbial components to the TLRs effectively acts as a danger signal to increase the microbicidal activity of the phagocytes and allows them to activate T cells.

Critical thinking: The role of adhesion molecules in T cell migration (see p. 434 for explanations)

An experiment has been carried out to determine which CAMs mediate the migration of antigen-activated T cells across brain endothelium using a monolayer of endothelium overlaid with lymphocytes in vitro. The endothelium is either unstimulated or has been stimulated for 24 hours before the experiment with IL-1. In some cases the co-cultures were treated with blocking antibodies to different adhesion molecules. The results shown in the table below indicate the percentage of T cells that migrate across the endothelium in a 2-hour period.

Blocking antibody	Percentage of T cells migrating in 2 hours
Blocking antibody	Unstimulated endothelium	IL-1-stimulated endothelium
none	18	48
anti-ICAM-1	3	16
anti-VCAM-1	19	28
anti-α_Lβ₂-integrin (LFA-1)	2	14
anti-α₄β_rintegrin (VLA-4)	17	32