Infectious diseases Part 2: Immunology

Regulatory T cells (Tregs)

A functionally-defined subset of T cells that express high levels of the IL-2 receptor (CD25) and that are important in controlling immune and inflammatory reactions mediated by T helper cells.

Major histocompatibility complex

A gene locus, referred to as HLA in humans, which encodes a large number of genes, including MHC class-I and class-II genes. These highly polymorphic loci encode cell surface molecules, which present antigenic peptides to T cells.

Pentraxins

A group of acute-phase pentameric molecules present in serum that recognize PAMPs and opsonise bacteria for phagocytosis.

Collectins

A group of large polymeric proteins, including conglutinin and mannan-binding lectin (MBL), which can opsonise microbial pathogens.

Large granular lymphocytes (LGLs)

A group of morphologically defined lymphocytes containing the majority of NK cell activity. They have both lymphocyte and monocyte or macrophage markers.

Scavenger receptors

A group of receptors that recognise cell debris and are involved in the phagocytosis of apoptotic cells and some pathogens.

Complement system

A group of serum molecules involved in the control of inflammation, removal of immune complexes and lysis of pathogens or cells sensitised with antibody. The complement system is important in mediating adaptive and innate immunity and in controlling inflammation. Complement system molecules interact with each other in a series of enzyme cascades, where the product of one reaction acts as the enzyme which catalyses the next. This is conceptually similar to the blood clotting system - a small initial trigger can precipitate a fast-growing chain of reactions. Complement system molecules are produced constitutively by the liver, although they can also be produced locally at sites of inflammation by macrophages. The principal functions of the complement system are: • attraction of phagocytes to a site of inflammation (chemotaxis) • opsonisation of pathogens for phagocytosis, i.e. acting as an adapter between pathogen and phagocyte • opsonisation of immune complexes for uptake by phagocytes • activation of the phagocytes to destroy material they have taken up • triggering of mast cells to release their inflammatory mediators • lysis (osmotic rupture) of cells and some bacteria and viruses which have been recognised as foreign. The complement system can be activated via three different pathways (Figure 2.11), all of which converge on the central reaction of the pathway, the activation of C3. When C3 is activated it is cleaved enzymatically into two fragments: the larger, C3b, is involved in opsonisation and the smaller, C3a, can activate mast cells (Figure 2.12). The pathways all generate enzymes (C3 convertases), which cleave the C3, but the triggers for each pathway are different. When C3 is cleaved it breaks an internal bond (thioester) exposing a very reactive group on the larger fragment, C3b, which can bind covalently to hydroxyl groups (-OH) or amine groups (-NH2) present on nearby proteins or carbohydrates. This tags the molecule/pathogen, so that it can be recognised by cells that have receptors for C3b. However, because the reactive group is very transient, if it fails to combine quickly with a protein or carbohydrate then it decays and becomes inactive. This means that C3b will only bind in the immediate vicinity of whatever caused the activation. This is usually an antibody combined with an antigen, or the surface of a microorganism. The deposition of C3b on the surface of pathogens targets them for phagocytosis by cells that have C3b receptors. Receptors for C3b are present on mononuclear phagocytes, neutrophils and NK (natural killer) cells. As these cells also have Fc receptors for IgG, this means that antibody and C3b can act together to opsonise antigens and immune complexes. A different kind of C3b receptor is found on B cells and follicular dendritic cells and this receptor has an important role in the development of the antibody response. It allows these cells to accumulate antigen as immune complexes early in an immune reaction, something which is required for development of the secondary antibody response. • Occasionally, children are born with a genetic deficiency of C3. What effect do you think this would have? • These children are extremely susceptible to infection with Gram-positive bacteria, e.g. staphylococci and streptococci, because their macrophages do not phagocytose them efficiently. When antibodies bind to antigens they form immune complexes - multimolecular complexes containing a number of molecules of antigen bound to specific antibody. If the complexes activate complement with the formation of C3b, it too may become bound to the complex. One of the functions of complement is to keep immune complexes in solution (i.e. to prevent the formation of huge insoluble aggregations of antigen and antibody), so that they can be taken up by phagocytes. If C3b has become bound on the surface of a pathogen, it can recruit additional complement proteins, belonging to the lytic pathway, which assemble a multimolecular complex called the membrane attack complex (MAC). On plasma membranes, this complex forms a doughnut-shaped pore through which the contents of the cell can leak. It also allows other components of the immune system access to the inside of a pathogen. • What kinds of pathogens have a lipid bilayer exposed on their external surface, and might therefore be susceptible to damage by the membrane attack complex? • Gram-negative bacteria have an outer membrane, and enveloped viruses have a plasma membrane derived from the infected cell. Note, however, that the inner plasma membrane of Gram-positive and Gram-negative bacteria is beneath the bacterial cell wall and is not normally exposed to this kind of attack. Eukaryotic pathogens, including worms and protists, also have an external plasma membrane, so are susceptible to the lytic pathway of complement. The smaller fragments generated by complement activation, specifically C3a and C5a, have a central role in the development of inflammation, including the triggering of mast cells to release inflammatory mediators and in attracting phagocytes to a site of infection.

Defensins

A group of small antibacterial proteins produced primarily by neutrophils.

Lectin pathway

A pathway of complement activation, initiated by mannan-binding lectin (MBL), which intersects the classical pathway. The lectin pathway is activated by carbohydrate groups that are typically found on the surfaces of bacteria and some fungi. The initiation of the pathway is different from the classical pathway, but the result is the same, i.e. the formation of a C3 convertase.

Kinin system

A plasma enzyme system that generates mediators of inflammation.

Basophils

A population of polymorphonuclear leukocytes that stain with basic dyes and that have important roles in the control of inflammation. Secrete histamine (and in some species serotonin) to help mediate inflammatory reactions.

Affinity maturation

A second event that occurs during the development of an immune response is a progressive rise in the affinity of the antibodies, which is called affinity maturation. A closer look at events in the lymphoid tissue will help you to understand how this process occurs at a cellular level. Interestingly, the processes that occur at the cellular level involve mutation and diversification of B cells, followed by selection of the fittest B cells. Does this sequence look familiar? In fact, it is wholly analogous to the processes underlying evolution on the grand scale. However, in this case, the 'natural selection' of the antibody response takes place within our bodies over the period of a few days. As noted above, class switching happens in the germinal centres of the spleen, lymph nodes, Peyer's patches and other lymphoid tissues. As an immune response develops, individual B cells enter the germinal centre and undergo very rapid cell division. As they do this, a mutation mechanism is activated that affects just the region around the immunoglobulin heavy chain genes. The mechanism for the increased localised mutation is not known, but it is so high that, if it occurred throughout the whole of the genome, many of the cell's essential enzymes would be rendered non-functional due to damaging mutations. Each of the B cell progeny from a single precursor will develop different mutations in their heavy chain genes. In other words, the B cells from the same clone will diversify slightly with respect to the antibodies they produce. • What effect will the mutations have on the affinity of the antibodies produced by a clone of B cells derived from a single precursor? • Most antibodies will be unaffected. A few antibodies will have mutations in their hypervariable regions. In many cases this will mean that the antibody binds less well to the antigen (lower affinity). In the worst case, the antibody will be completely unable to bind to its epitope. Just occasionally, however, the mutated antibody will have a better fit for the epitope than the original did, i.e. the mutated antibody will have a higher affinity for the antigen. At this stage a selection process comes into play. Remember that B cells only receive help from T cells if they present antigen to the T cells. The B cells in the scenario presented here have diversified, some with high and some with low affinity antibodies as their antigen receptors, but any cells that have lost their ability to bind the antigen cannot take it up. Consequently they have no antigen to present to the T cells and they do not receive T cell help. The situation is actually more critical than this. In the lymphoid tissues the quantity of antigen available is limiting, so only B cells with the highest affinity antibodies are able to take up sufficient antigen to present to the T cells and get help (Figure 3.7). In other words, only B cells that have developed high affinity antibodies get T cell help. The way this works is as follows. • When the B cells enter the germinal centre of the follicular dendritic cell network (where the stores of antigen are held) and divide, they become programmed to die (by apoptosis) unless they are rescued by interaction with Th cells. • The molecular signals that are involved in the rescue process include the costimulatory molecule CD40, and the result is that only high affinity B cells survive passage through the germinal centre and go on to divide and differentiate. The overall effect of the selection process is that high affinity B cells develop during an immune response so that the affinity of the antibodies produced, increases as the immune response progresses (see Figure 2.6). The processes of class switching and affinity maturation happen synchronously, and the molecular events underlying class switching are interrelated. As T cells promote class switching, you can now see why T-independent antigens do not induce IgG responses, while T-dependent antigens do. Also, as class switching is associated with the increase in affinity, it is clear why IgG antibodies tend to be of higher affinity than IgM. While class switching can be understood in broad outline, many important details are still being investigated. In particular, although it is known, for example, that B cells in mucosal lymphoid tissues tend to switch to IgA production and cytokines can affect the class switching, this is not the whole story.

Phagosome

A vesicle formed by phagocytosis. Diameter of a phagosome is determined by the size of the particle being ingested, and they can be almost as large as the phagocytic cell itself.

C-reactive protein

An acute phase protein and opsonin that binds to phosphoryl choline.

Lactoferrin

An iron-binding protein.

Antigens

Antibodies, or immunoglobulins, can be subdivided into different classes according to their structure. There are five classes, called IgG, IgE, IgD, IgA and IgM each having different functions. The class of an antibody is determined by its heavy chains and each antibody can be produced either as a cell surface antigen receptor on B cells or as a secreted molecule. The secreted form of IgA is a dimer of the basic four chain structure and IgM is a pentamer. Naive B cells which have not encountered their antigen initially express IgM as their surface receptor. The heavy chain of IgM is called a m chain and a B cell makes a m chain by transcribing its recombined VDJ gene together with exons for the constant domains of the IgM heavy chain. These are called C genes. C genes for the other immunoglobulin classes lie downstream of those for IgM. An individual B cell can switch the class of antibody it produces. This process occurs in the germinal centres of the lymph nodes, spleen and other secondary lymphoid tissues. Class switching is accompanied by a second process, somatic mutation, which increases the overall affinity of the antibodies produced. A primed B cell enters a germinal centre and undergoes rapid division. At the same time a mechanism is activated which introduces mutations into the DNA around the recombined VDJ gene, so each B cell has a slightly different antibody. Occasionally a mutation will generate antibody with a higher affinity for its antigen. Follicular dendritic cells in the germinal centre hold stores of antigen on their surface. B cells compete with each other for the antigen, and only those B cells whose mutations have produced higher affinity antibodies will compete effectively for the limiting amount of antigen. This is now processed and presented to T cells, which signal division and differentiation. But B cells with low affinity antibodies have no antigen to present to the T cells and die by apoptosis. The interaction with T cells also promotes immunoglobulin class switching. Look now at the heavy chain gene locus. The IgM C gene is preceded by a switching region which corresponds to switching regions preceding the C genes for all the other immunoglobulin classes except IgD. Here we see the event which occurs as a B cell switches from making IgM to IgG1. The C gene for IgG1 replaces the C gene for IgM placing it next to the VDJ gene. Now the transcript from this encodes an IgG1 heavy chain but with the same specificity as the IgM since it uses the same VDJ gene which encodes the variable (antigen-binding) part of the antibody. Exactly which switch will take place, depends on the T cells and the cytokines they release, so the T cells determine what type of antibody response will be produced. B cells leaving the germinal centre may become antibody secreting plasma cells or memory cells. As a consequence of these processes, the affinity of the antibodies tends to increase during an immune response and this is associated with a switch to production of IgG, IgA and IgE. Secreted antibodies have many functions. The IgG antibody can act as an adaptor. The two Fab arms of antibody have identical antigen binding sites. These form multiple non-covalent bonds with the specific antigen. The Fc portion of the antibody can link to Fc receptors on cells, including phagocytes and natural killer cells. This allows these cells to recognise their targets. For example IgG can opsonise bacteria, for phagocytosis by neutrophils. It can also activate the complement system, causing deposition of C3b. IgG is also transferred across the placenta from the mother to protect a child in the uterus and during its first few months of life. Meanwhile other B cells may have switched to the production of IgE. This type of antibody binds to receptors on mast cells and basophils so that if they should contact antigen they release inflammatory mediators. Other B cells, particularly in the Peyer's patches may have switched to the production of IgA. IgA is produced as a dimer of the basic 4-chain immunoglobulin unit, linked by a joining or J chain. Cells of mucosal epithelia can bind IgA and transport it across to protect the mucosal surface. Some antigens, particularly polymeric carbohydrates are not recognised by T cells and are called T-independent antigens. So the antibody response to these antigens shows neither increasing affinity nor class switching. The antibody against such antigens is predominately secreted IgM, which is also the principle antibody in the earliest stages of an immune response to conventional T-dependent antigens. Because secreted IgM is a pentamer of the basic 4 chain structure, it is effective at cross-linking antigens and also fixes complement. Here we see a blood vessel lined with endothelial cells with a normal flow of blood containing lymphocytes. We are going to follow one particular lymphocyte to see how it migrates through the endothelium and out into the surrounding tissues. As the vessel widens into what is known as a venule, the blood flow decreases and the lymphocytes slow down. In the presence of an infection such as bacteria, an immune response takes place and cytokines such as interleukin 1 and tumour necrosis factor are released from tissue cells and resident macrophages. In addition, peptides of the complement system cause mast cells to degranulate. Cytokines such as interleukin 1 and tumour necrosis factor act on the endothelium to induce adhesion molecules, while chemokines attach to the endothelium to act as stimulators of migration. The cytokines induce the expression of adhesion molecules on the surface of endothelial cells complementary to those on the lymphocytes. Molecules on the endothelial cells such as E-selectin interact with carbohydrate molecules on the surface of the lymphocyte such as CD15. These interactions cause the lymphocytes to slow down and roll along the endothelium enabling them to sample other adhesion molecules which have been induced onto its surface. Amongst these are the chemokines which bind to the lymphocyte and trigger an activation mechanism causing an influx of calcium ions into the cell and the activation of another group of adhesion molecules on its surface. These adhesion molecule proteins called LFA1 and VLA4 interact with other adhesion molecule proteins on the surface of the endothelium called ICAM1 and VCAM1 respectively. These latter molecules can also be induced on the endothelial surface by cytokines such as tumour necrosis factor thereby enhancing binding, just at this site. This multi-point adhesion onto the endothelium is called pavementing. Both the adhesion molecules on the lymphocyte and those on the endothelium are each attached to their respective cell's cytoskeleton. This can be thought of as a molecular motor which acts like a tractor enabling the lymphocyte to pull itself down and out through the endothelium. With a firm anchoring point to move against, the lymphocyte proceeds to extend its pseudopod down into the intercellular junction between the endothelial cells which retract as they are pushed aside. When the pseudopod makes contact with the basement membrane, the cell moves new adhesion molecules down to its leading edge enabling it to form a new anchoring point. The lymphocyte now releases granules from this leading edge. These contain destructive enzymes such as collagenase and elastase, which puncture the basement membrane allowing the cell to squeeze down through the damaged area and migrate out into the tissue. This migratory process is called diapedesis. The damaged endothelium then seals over again and reforms. The lymphocyte then migrates towards the site of infection under chemical stimuli in a process known as chemotaxis. Another feature of inflammation is an increase in capillary permeability. Mediators from activated mast cells such as histamine cause the endothelial cells to retract. This allows larger serum molecules such as antibody and complement to leak into the inflamed area. These mediators can also act on the smooth muscle in the blood vessel wall, increasing the diameter of arterioles and producing enhanced blood flow. This causes increased supply of serum molecules and cells to the inflammatory site. Lymphocytes must generate diverse receptors in order to recognise the many different antigens which the individual might encounter during their lifetime. B cells mature in the bone marrow, and generate cell surface immunoglobulin as their receptor. Ultimately, if they become activated following contact with their specific antigen, they develop into plasma cells and will produce a secreted form of this immunoglobulin. These secreted immunoglobulins are the serum antibodies. T cells mature in the thymus where they generate diverse T cell receptors, or TCRs. The process by which immunoglobulin diversity is generated is analogous, although the two molecules are quite distinct. Inside each pre B cell nucleus there are sets of genes which encode the protein chains which combine to form immunoglobulin. A molecule of immunoglobulin consists of two identical heavy chains and two identical light chains, joined by disulphide bonds and non-covalent interactions. Each of the two chains is encoded by its own DNA. There is one gene locus for the heavy chain, and two loci for light chains, termed kappa and lambda, although a B cell will only use one or other of the sets of light chain genes. Heavy and light chain genes are unusual because they undergo a process of recombination during B cell development. The gene for the heavy chain or light chain is assembled from different gene segments by the recombination. The heavy chain gene locus has numerous V gene segments and several J gene segments, but also includes additional D (diversity) segments, so that a heavy chain variable domain is encoded by a recombined VDJ gene. Each light chain gene locus has numerous V gene segments and several J gene segments which can recombine at random to make a recombined VJ gene, which will encode the variable, antigen-binding domain of the light chain. The DNA base sequences flanking the V, D, and J gene segments cause them to become aligned during B cell development, and enzymes break and rejoin the DNA strands to make the final recombined VJ or VDJ gene. This random process of gene segment recombination provides an enormous amount of diversity in the antigen binding domains of the antibodies in different B cells. In order to synthesise immunoglobulin, the cell must transcribe the immunoglobulin gene. The DNA strands separate and RNA polymerase makes a faithful transcription of the immunoglobulin gene. The transcript includes the recombined gene for the variable domain, the genes for the constant domains and the intervening stretches of DNA which do not encode protein. These non-coding sections are termed introns, and the coding portions are exons. This process is called transcription. In a second stage, the RNA transcript is processed to make messenger RNA by removal of the introns. The messenger RNA migrates out of the nucleus into the cytoplasm. In the cytoplasm, ribosomes translate the information contained in the mRNA. Each triplet of bases in the mRNA encodes one amino acid in the protein. Initially a short signal sequence is translated. This binds to a signal-recognition protein which blocks further translation until the mRNA is docked on the endoplasmic reticulum. Translation of the protein chain now occurs across the endoplasmic reticulum. The signal sequence is removed by cleavage. In the endoplasmic reticulum, the new heavy and light chains combine to form immunoglobulin. They then associate with polypeptides termed CD79, which will transduce signals to the B cell. Vesicles separate from the endoplasmic reticulum to join the Golgi apparatus, where the immunoglobulin is glycosylated. Finally, vesicles containing the immunglobulin bud from the Golgi body and migrate to the plasma membrane. At the cell surface, the immunoglobulin remains in the membrane, presenting its antigen-binding site to the outside. However, if the cell has produced secreted immunoglobulin, this is released as a soluble molecule into the lymph. Antibodies are folded into domains which are named according to whether they are variable, V, or constant, C, and according to whether they belong to the light chain, L , or heavy chain, H. T cells replicate in the sub capsular region of the thymus. At this stage they start to recombine genes for the T cell receptor, in an analogous way to the B cell's recombination of antibody genes. Within the nuclei of all pre T cells are genes for T cell receptor chains termed alpha, beta, gamma, and delta. Most T cells use the alpha and beta genes so the TCR is formed of an alpha chain and a beta chain. The beta gene locus contains V, D, and J gene segments, while the alpha gene locus contains V and J gene segments. These are analogous to those of antibodies, but completely distinct. During T cell development, the beta gene recombines a particular combination of V, D, and J gene segments to make a recombined VDJ gene. Similarly, one of the alpha V gene segments recombines with one of the J gene segments at random, to make a recombined VJ gene, which will encode the variable, antigen-binding domain of the alpha chain. The recombined genes together with the genes for the C domain and the intervening introns are transcribed into RNA. This RNA is then processed to make messenger RNA for the alpha and beta chains. Once in the cytoplasm, ribosomes synthesise alpha and beta chains across the membrane of the endoplasmic reticulum. The T cell receptor is now formed by assembly of one alpha chain with one beta chain. The T cell receptor now associates with other components of the T cell receptor complex, termed CD3 and the whole receptor complex is transported to the Golgi body. The receptor is processed and vesicles pinch off from the Golgi carrying the assembled receptor complex to the cell surface, where it remains anchored, to become the T cell's receptor for antigenic peptides. The alpha and beta chains are folded into variable, V, and constant, C, domains, similar to those in antibody. Here we have a population of mature B cells expressing surface antibody, and educated T cells with T cell antigen receptors on their surfaces. At any one time in a population of lymphocytes there will be millions of different clones of cells each carrying a different receptor, and therefore only able to recognise a particular type of antigen. This means that in a normal immune system there is always a small sub-population of cells which are able to recognise a specific antigen. If this were not the case, we would be completely unable to protect ourselves from pathogens. Such cells are said to share the same specificity. This is one of the reasons why lymphocytes circulate around the body to ensure that the small number of cells which are able to recognise a specific antigen will actually come into contact with it. Consequently, most of the cells in the human body are never called upon to fight infection. They are simply there just in case they are needed to combat any new strains of pathogen that might enter the host. We are going to look at how B cells and T cells differentiate and the mechanisms which bring this about. Here we have a virgin lymphocyte pool. In the presence of an antigen only one of these many different B cells is able to bind to that antigen successfully. This process known as clonal selection initiates a primary immune response which causes the cell to undergo a series of irreversible changes. The antigen is taken up from solution by the B cell's surface immunoglobulin. It is internalised and processed. Antigen fragments are subsequently re-expressed on the B cell surface associated with MHC class II molecules for presentation to Th2 cells. Receptors for interleukin 4 are also induced on the B cell. Presentation of the antigen to the Th2 cell causes vesicles within the T cell to fuse with the cell surface and release cytokines, including interleukin 4, which then binds to the interleukin 4 receptors on the B cells. Together with costimulatory signals, the IL-4 activates the B cell triggering its division. Other cytokines released from the Th2 cell including interleukin 2, interleukin 4, interleukin 5, and interleukin 6 then bind to the appropriate receptors on the B cells, causing them to differentiate. Some of the cells become plasma cells which produce a secreted form of the original antibody. Consequently they are also known as AFCs or antibody forming cells. Plasma cells are a different shape from B cells and have lost all their original cell surface antibody. They will go on dividing several times but within days or weeks they will die. Other cells become memory cells. They have undergone a series of phenotypic changes making them more efficient at reacting to the same antigen, if it is ever presented again in the future. This means that there is always a ready supply of cells with the appropriate specificity to fight infection. These cells can live for many years, and this increased efficiency of the memory cells to fight infection underlies the enhanced secondary immune response. Memory cells confer lasting immunity against a pathogen, which is the basic principle behind vaccination. Now we shall look at how T cells differentiate. The process is quite similar. Again we have a pool of virgin lymphocytes. There are millions of different clones of T cells, each carrying a different T cell antigen receptor. In the presence of an antigen-presenting cell such as a macrophage, only one of these many different T cells is able to bind to that antigen successfully. As we have seen, this is called clonal selection. The antigen-presenting cell interacts with the T cell and releases cytokines such as interleukin 1, which cause the expression of interleukin 2 receptors on the surface of the T cell. There are also crucial interactions between pairs of costimulatory molecules on the surfaces of each of the two cells. In addition to being able to bind interleukin 2 using its new receptor, the T cell is now also able to produce its own interleukin 2 enabling it to stimulate itself. This ability to produce cytokines which can act on cells of the type that produced them is called an autocrine action, whereas producing cytokines which stimulate another cell type is a paracrine action. The interleukin 2 binds to the receptors on the surface of the T cell triggering division. This causes the induction of new costimulatory molecules onto the surface of the T cell. The T cell divides, and after five or six cycles there is a gradual loss of the interleukin 2 receptor. This prevents the cells from receiving any positive signals to continue dividing. All of the T cells eventually lose their interleukin 2 receptors but retain their T cell antigen receptor. However, some of them also retain the signalling costimulatory molecules and those become the memory T cells. As before, these cells are able to mount an effective attack against the specific pathogen in the event of future infection. Memory cells remain in the lymphatic system where they can live for many years. Other cells lose their costimulatory molecules and become effector cells, which migrate out into the surrounding tissue to participate in effector responses to eliminate infection. These cells die within a few weeks. Both B cell and T cell clonal proliferation is driven by the presence of antigen. As soon as the antigen has been destroyed, the whole immune response switches off, leaving more cells in the system sharing the same specificity than existed prior to the original infection.

Antigen presenting cells

Antigen-presenting cells (APCs) are a group of functionally defined cells that are capable of taking up antigens and presenting them to lymphocytes in a form they can recognise. Dendritic cells, macrophages, B cells and sometimes even tissue cells can present antigen to Th cells. The description of macrophages above reintroduced the idea of antigen presentation. Macrophages are just one of a group of cells which can perform this function. For example, B cells can act as antigen-presenting cells for Th2 cells. So the definition of an antigen-presenting cell is a functional one; it is not the description of any particular cell lineage. Effectively, it means any cell that can present antigen to a T helper cell. The term is so often used in immunology that it is very frequently abbreviated to APC. The basis of two types of cellular interaction can be seen in Figure 1.11: 1. Macrophages present antigen to Th1 cells. They respond to cytokines released by Th1 cells by enhanced microbicidal activity so that the Th1 cells help macrophages to destroy the pathogens they have phagocytosed. 2. B cells present antigen to Th2 cells, and respond to cytokines released by these cells by dividing and differentiating into antibody-producing plasma cells so that the Th2 cells help the B cells to make antibody. This division is not absolute - cytokines from Th1 cells affect B cell development and those from Th2 cells affect macrophage activation, i.e. most immune responses display both types of response although they are often polarised to a Th1 or a Th2 response. Moreover, there are important interactions between the two sets of cells and the responses they mediate. Nevertheless, it is useful to distinguish these two basic types of immune response, while acknowledging that, in many cases, the immune response is not completely polarised to one type or the other.

Soluble mediators of antibacterial immunity

Anyone who has a genetic deficiency that reduces their ability to make antibodies or complement molecules becomes more susceptible to infection with bacteria. This tells us that antibodies and complement are important in defence against such infections. Antibody provides a specific way of recognising bacterial molecules, and most importantly, it can direct complement activation onto the bacterial surface via the classical pathway. This section introduces some ways in which antibodies and complement can protect against bacterial infection. The simplest mechanism is neutralisation of toxic molecules. Many bacteria produce their damage by releasing toxins that move through the blood. Examples are the toxins from tetanus and diphtheria or the toxic molecules produced by some organisms which cause food poisoning (e.g. botulism). If an antibody binds to these molecules, it can often neutralise them completely and high affinity antibodies are most effective at neutralisation. • What class of antibody is likely to be most effective in countering such toxins? • IgG, the main serum antibody, prevents toxins spreading through the blood. Also, IgG antibodies are generally of higher affinity than IgM. Many bacteria produce their damage by invading tissues, while producing enzymes and toxins that allow spreading and dispersal. Antibodies against these components are also important in limiting spread. An antibody that is bound to the active site of an enzyme will prevent it from working properly. Even antibodies that bind to other sites of the enzyme may affect its function and will promote the formation of immune complexes that can be phagocytosed and broken down by neutrophils and macrophages. Antibodies against flagella or related components often reduce bacterial motility and thus limit spread of the bacteria through the body.

Clonal selection

As the numbers of lymphocytes which recognise an infectious agent are relatively small, the first thing that the immune system must do when an infection occurs is to expand the number of cells available which are capable of responding to those antigens. This process is called clonal selection, because the antigen selects and activates those clones of cells that recognise it. When an antigen binds to the antigen receptors on a lymphocyte, provided that the lymphocyte receives appropriate additional signals, it will be driven to divide. Lymphocyte division within a lymph node can occur every 6 hours, an extraordinarily high rate of cell division. And it needs to be, because a race is now on between the lymphocytes and the infection. Some bacteria can divide every 20 minutes while eukaryotic cells typically divide every 20 hours. It is essential therefore for the immune system to generate as many specific lymphocytes as possible in the shortest time, in order to mount an adequate immune response. One consequence of the considerations above is that most lymphocytes cannot recognise the ongoing infection. Indeed most lymphocytes do not recognise any particular infectious agent, and will never be called upon during a lifetime to engage in an immune response. • What is the point of having millions of lymphocytes present in the body which, although they can recognise antigens, cannot recognise antigens from any known infectious agent? • The immune system does not know what it will encounter during a lifetime. We must have lymphocytes available which can recognise any foreign molecule. If we only had cells available that could recognise extant pathogens, when pathogens mutate we would have no cells available to recognise them and would thus be wide open to infection.

reactive nitrogen intermediates (RNIs)

Bactericidal metabolites produced by phagocytic cells, including nitric oxide and peroxynitrites.

Mycobacterium leprae

Causes leprosy and are usually transmitted by nasal secretions. As they are very slow-growing, the incubation period is up to ten years. The bacteria typically grow in the skin, but in some cases affect other organs including nerves, and they may survive in macrophages in some individuals. The unique characteristic of leprosy is that the type of immune response that develops in different individuals varies enormously. Some patients develop a very strong cell-mediated immune response, which eliminates virtually all of the bacteria. Indeed the response can be so powerful that it causes a lot of damage in the host. Such patients are said to have a 'tuberculoid' type of immune response. In contrast, other individuals produce no cell-mediated responses at all, although they do produce a good antibody response against the bacteria. These patients are said to have a 'lepromatous' type of response. Unfortunately, the antibody response is not effective against leprosy and the skin and other tissues of these patients are teeming with millions of bacteria. In these cases, it is the bacteria that produce damage throughout the body. Tuberculoid leprosy reflects a Th1-type immune response, while lepromatous leprosy reflects the activation of B cells and antibody production directed by Th2 cells. Interestingly, patients can swing between having one type of immune response and another. As they do so, the external appearance of the lesions on the skin changes. The problem with leprosy is that the bacteria are extremely resistant to destruction by macrophages. Once it has gained a hold in the individual, neither a strong cell-mediated response (Th1) nor a strong antibody response (Th2) is sufficient to eliminate it. In this disease, drug therapy is the best option.

Salmonella typhi

Causes typhoid which is contracted from contaminated water and has a high mortality rate if not treated. The bacteria enter the body from the gut by crossing the cells that are above the Peyer's patches. This gives them direct access to cells of the immune system, and from there they can spread throughout the body, to infect mononuclear phagocytes in many different tissues. The symptoms, which develop after 1-3 weeks, include fever, severe abdominal cramps, diarrhoea, muscle pains and dizziness. Serious developments such as intestinal ulceration and bleeding produce the mortality. Because S. typhi does not produce disease in other animals, much of our knowledge comes from the related mouse disease caused by Salmonella typhimurium. Control of this infection depends on macrophages and the bacteria are susceptible to cationic proteins and the oxygen-dependent killing mechanisms (ROIs). However, they have a mechanism to delay lysosomes and granules from fusing with the phagosome that therefore limits their exposure to the toxic molecules. Also, if the bacteria survive for a few hours inside the macrophage, they adapt to the intracellular environment, expressing a range of proteins that limit the damage caused by the macrophage and are then able to survive and replicate at a low level. Moreover, the bacteria produce proteins that activate the intrinsic pathways of apoptosis in the host cells. Survival of the bacteria is therefore partly dependent on whether the macrophages are activated at an early stage of infection, and this depends on IFNγ and TNFα. Treatment of macrophages with IFNγ in vitro increases the rate at which lysosomes fuse with phagosomes containing Salmonella. Conversely, inhibiting these cytokines makes the animals much more susceptible to infection. The cell-mediated response against Salmonella is clearly important, but antibodies are also required, to allow the bacteria to be opsonised. For example, it is possible to protect mice against infection by transferring into them both antigen-specific T cells and antibacterial antibody - the T cells activate the macrophages and the antibodies allow the macrophages to recognise the bacteria. One more important feature of typhoid should be noted: a proportion of individuals who recover from typhoid, and a small number who never exhibit the disease, act as chronic carriers of the bacteria for many years. Their macrophages have a permanent low level of infection. The carriers have high levels of immunity, including specific antibodies (against Salmonella), but they continue to shed the bacteria in faeces. From the viewpoint of the bacteria, this situation is ideal: a carrier who does not appear to be ill, continues life as normal and can continuously contaminate food or water with the bacteria.

Acidic phase

Following this defensin release, the lysosomes fuse with the phagosome to produce a phagolysosome. The lysosomes of activated macrophages contain an antimicrobial peptide, cathelicidin, which is toxic for many microbes. In the final phase, the phagolysosome becomes acidic and degradative enzymes from the lysosome break down the pathogen, which by this stage is (we hope) dead. Just the process of acidification is toxic for some pathogens, but the enzymes, which are active at low pH, are also damaging. The phagolysosome can also contain growth inhibitors. For example, neutrophils produce a molecule, lactoferrin, which binds avidly to iron. Since bacteria and parasites need iron to grow, its removal by binding to lactoferrin prevents them getting the supply that they need to divide. Evidently a phagolysosome is not a comfortable place for a microorganism, and all persistent pathogens have evolved countermeasures that allow them to deflect some of the phagocytes' defences. • If a pathogen had managed to enter the cytoplasm of the macrophage, either because it had infected the cell directly or because it had escaped from the phagosome, do you think that the macrophage would still be able to kill it? Explain your answer. • No, the only way the macrophage can let loose its chemical arsenal is by confining everything to the phagolysosome. If a pathogen escapes into the cytoplasm, the macrophage is now an infected cell and the pathogen can replicate inside it. This is what occurs in tuberculosis and with a number of other pathogens that live inside macrophages.

Control of cell migration

How does the tissue signal to the endothelium to promote leukocyte migration into an area of infection? At such sites, inflammatory cytokines, such as interleukin-1 (IL-1) and tumour necrosis factor-α (TNFα), are produced which activate the endothelium locally. The inflammatory cytokines induce both the selectins that slow the leukocytes and the cell adhesion molecules that bind to the leukocyte integrins (Figure 3.9). Mononuclear phagocytes, which are present in most tissues, produce IL-1 and TNFα in response to infection or cell damage. Another important cytokine is interferon-γ (IFNγ) which is produced by activated Th1 cells. This means that a T cell that has entered the tissue and encountered its antigen is able to signal to the endothelium to display adhesion molecules that are required for the migration of more leukocytes. Although the adhesion molecules are essential for migration, it is the chemokines that principally determine which cells will migrate where. There are a large number of different chemokines, each of which promotes the migration of a particular group of leukocytes. For example, monocyte chemotactic protein-1 (CCL2) promotes the migration of monocytes. Chemokines can be synthesised by cells in the tissues, or by the endothelium itself in response to inflammatory cytokines. The chemokines that are produced in inflammation vary from one tissue to another, and also depend on the type of infection, damage and immune response that is taking place. • You have learned that different types of leukocytes are characteristic of different types of inflammation. How can chemokines selectively attract one population of cells into an area? • Only a cell that has receptors for that specific chemokine will be able to respond to it. As noted above, different populations of leukocytes express distinct sets of chemokine receptors. In addition to the chemokines, there is a variety of other chemotactic molecules (Table 3.2). You have already encountered the complement fragment C5a, and this works in a very similar way to the chemokines, attracting monocytes and neutrophils to a site of inflammation. Other important chemotactic molecules are bacterial peptides. Bacteria initiate their protein synthesis using a special amino acid called formylmethionine (fMet) and there are receptors on phagocytes for peptides that have this amino acid. It means that if a bacterial infection is present, phagocytes will automatically be attracted to it.

Immunological synapse

In addition to the costimulatory molecules, a number of other cell surface adhesion molecules contribute to the interaction between the antigen-presenting cell and the T cell. It is important that the T cells and the antigen-presenting cells stay bound to each other for long enough for the T cell to receive appropriate stimulation and costimulation. Several molecule pairs can contribute to this adhesion, and may enhance the effectiveness of the signalling between the two cells. The structure that is formed between the two cells may last for hours and is called an 'immunological synapse'. In this structure, TCRs and MHC molecules cluster together at the centre of the synapse, while adhesion molecules interact in a ring around the periphery. The diagrams in Figures 2.21 and 2.22 show just one MHC molecule, and one T cell receptor. However, a cell could have more than 100 000 MHC molecules present on its surface. Of these, perhaps only 100-200 are required to bind to receptors on the T cell, in order to activate it, i.e. a T cell only needs to recognise about 0.1% of the MHC/antigen complexes on the surface of a cell in order to respond to it. This is appropriate, because the cell may express hundreds of different MHC/antigen molecules from all the different antigens that it is processing. Therefore, only a tiny proportion of the total can be recognised by any one T cell.

Mediators of inflammation

In addition to their role in promoting leukocyte traffic, inflammatory mediators control other aspects of inflammation, too. Mast cells are very important sources of mediators, since they have granules containing chemotactic molecules and histamine, which acts on blood vessels to increase blood flow and capillary permeability. In addition, if they are activated, mast cells synthesise a group of molecules called eicosanoids (a group of small molecules derived from fatty acids via arachidonic acid), some of which modulate the local blood supply, some are chemotactic (e.g. LTB4) and others can mediate sensations of pain. Another important group of mediators is produced by the kinin system. Like complement, this is a plasma enzyme system, but its main function is in controlling blood supply and capillary permeability. The kinin system is activated when damage occurs in the tissue and not only when there is infection. It can also be activated in association with other plasma enzyme systems. One of the mediators of this system, bradykinin, is extremely potent at causing dilation of blood vessels and enhancing permeability. The ways in which the systems interact with each other in inflammation are outlined in Figure 3.10 and its caption.

Cell migration

In order to understand how different types of leukocyte traffic develop in different conditions, we need to look at the underlying cellular and molecular processes that direct cell migration from blood vessels into the tissues. The key to this process is the interaction of the circulating leukocytes with signalling molecules and adhesion molecules that appear on the surface of the endothelial cells at sites of inflammation. Adhesion molecules include several families of molecules that allow cells to adhere to other cells or to components of the extracellular matrix (e.g. collagen). On the endothelium these include selectins, which slow circulating cells, and cell adhesion molecules (CAMs), which mediate leukocyte migration across the endothelium. Cell migration takes place across venules (small veins), partly because the shear force pushing cells through the circulation is lowest in venules, and partly because this is where the signalling molecules are located. The initial adhesion of leukocytes to the endothelium takes place in three phases (Figure 3.9). 1. Initially, circulating leukocytes are slowed by binding to selectins induced on the endothelium by inflammatory cytokines such as TNFα and IL-1 produced in the tissue. The slowed leukocytes roll along the endothelium. 2. The leukocytes may now receive signals from molecules attached to the endothelial surface called chemokines (a family of cytokines that are involved in chemotaxis and cell activation). The chemokines may have come from cells in the tissue, or may be produced by the endothelium itself. Receptors for chemokines are distributed on different populations of leukocytes, and each population has a different profile of receptors. In addition, activation of a cell often changes the number and types of chemokine receptors that it expresses. 3. A leukocyte that has been activated by a chemokine will reorganise and activate molecules on its surface that belong to the family called integrins. (This is a group of adhesion molecules, which are expressed on leukocytes, and which can bind to different CAMs on the endothelium; examples that are mentioned in the animation on inflammation at the end of this unit (Video 3.4) are LFA-1 and VLA-4.) The integrins can now interact with cell adhesion molecules that have been induced on the endothelium by inflammatory cytokines. Once a leukocyte has attached to the endothelium, it can pull itself down through the endothelium, a process called diapedesis and make its way into the tissue where it can participate in the immune reaction.

Immunity

In the presence of infection, chemotactic molecules are released from the complement system, mast cells and leukocytes. These in turn stimulate macrophages to bind to endothelial surfaces in blood vessels near the site of the infection facilitating their passage through the endothelial wall.Once the macrophage is within the tissue fluid, it migrates towards the site of infection across the surface of the extra-cellular matrix.This migratory process is termed chemotaxis. Its direction is controlled by the concentration gradient of free chemotactic molecules such as C5a.The macrophage achieves this migration by means of surface integrin molecules, which are able to bind to proteins within the extra-cellular layer as long as the cell moves up a chemotactic gradient.Each molecule of integrin attaches to the extra-cellular surface, then is moved by the cyto-skeleton towards the rear of the macrophage. On reaching the trailing end of the macrophage it detaches and is reabsorbed within the cell. Other integrin molecules emerge from the leading surface to continue the process.Once the chemokine concentration ceases to increase, receptors are no longer stimulated and the integrin stops binding to the extra-cellular surface. The macrophage therefore comes to rest as it is now at the intended destination.The macrophage is equipped with CR3 receptors for the C3b residues deposited on the surface of the target bacterium by the complement system. When this binding takes place, the anti-microbial mechanism is triggered. The bacterium is held and enveloped by the macrophage, and is endocytosed, a process called phagocytosis.The macrophage attacks the enclosed bacterium in a variety of ways. Enzymes assemble in the phagosome membrane which can reduce oxygen by the addition of an electron to form super-oxide anions. These anions further react to form a variety of reactive oxygen intermediates which can directly damage the bacterial membrane. Within the body of the macrophage, there are small vesicles called lysosomes, containing enzymes. When a bacterium is endocytosed, the lysosomes may fuse with the vacuole and release an enzyme called myeloperoxidase. This can act further on the peroxides to form even more chemically reactive species. These processes constitute the oxygen dependent mechanism of bacterial killing.In the first fifteen minutes after the fusion of a lysosome with the vacuole, the pH rises. During this period cationic peptides called defensins are able to penetrate bacterial membranes and create channels for ions to enter the bacterium, disrupting its metabolism. Additionally, an iron-chelating agent, lactoferrin, is released. This binds free iron, denying it to the bacteria, which need it to reproduce.Subsequently, pH falls as hydrogen ions are pumped into the phagosome. This tends to inactivate the defensins, but creates a suitable environment for other lysosomal enzymes which attack bacterial membranes. The lactoferrin is also active under acidic conditions to inhibit bacterial division.When destruction of the engulfed organism is complete, debris is ejected.The preceding process will occur even if the immune system does not specifically recognise the bacterium.If, however, Th1 cells exist with receptors for the bacterial proteins, they recognise bacterial products presented by the macrophage and release cytokines, notably interferon gamma.These cytokines stimulate the macrophage's killing mechanism, increasing its ability to destroy bacteria with oxygen radicals. Also, another pathway which is not oxygen dependent is activated. This pathway generates reactive nitrogen intermediates, such as nitric oxide, which attacks bacterial membranes.The other mechanisms seen in non-activated macrophages are still found but occur more intensely. Lysosomes release cationic proteins which may penetrate bacterial coatings during the high pH alkaline phase followed by lytic enzymes which are active in the acid environment which follows.The activated macrophage destroys engulfed bacteria rapidly, and through the use of reactive oxygen and nitrogen intermediates will deal with some varieties of bacteria not susceptible to attack by non-activated cells. Tuberculosis is a disease caused by mycobacteria which gain access to the body in aerosol droplets and then infect lung macrophages. They normally act as intracellular pathogens that can survive and may divide inside the phagosomes of macrophages which are usually present within the lung or local lymphoid tissues. Occasionally however the infection does spread to other sites. The mycobacteria which produce this disease including Mycobacterium tuberculosis are very widespread infecting at least 1.5 billion people worldwide. However, infection is usually contained by the immune system so that only about one per cent of infected people actually have the disease. However, if the effectiveness of the host's immune defence is compromised by malnutrition or immunodeficiency, for example AIDS, then the disease is much more likely to develop. This partly explains why the highest rates of tuberculosis occur in poorer countries as can be seen in this map which shows notification rates in 1998. Even within wealthy countries people belonging to poor communities are usually more frequently affected. The cell wall of mycobacteria is extremely resistant to degradation. This is crucial since the bacterium has to survive inside the cell type which is chiefly responsible for its destruction. If you look at the diagram of the structure of the mycobacterial cell wall and compare it with that of a bacterium such as Staphylococcus you can see the additional complexity of the mycobacteria. In addition to the inner lipid bilayer and cell wall the compound structure includes an outer layer containing glycolipids and mycolic acids bound to a phosphorylated polysaccharide backbone. Notice also the lipoarabinomannan, a glycolipid which spans the cell wall. In addition to being very tough the components of the mycobacterial cell wall are very effective in activating macrophages, since macrophages have developed receptors which can specifically recognise mycobacterial components. On the other hand mycobacteria have evolved a group of strategies which allow them to evade or suppress immune responses. The main line of defence against tuberculosis is provided by the macrophages. In an infected individual they present bacterial antigens by both the Class II pathway to CD4 positive T cells and by the Class I pathway to CD8 positive T cells. T cells which recognise the bacterial antigens are induced to release interferon gamma which activates the macrophages to destroy the mycobacteria which they have phagocytosed. We know that interferon gamma is important in protection against tuberculosis because of children with rare genetic defects affecting either interferon gamma or its receptor. These children are susceptible to a progressive disseminating form of disease even when they have been infected with harmless strains of mycobacteria which are normally used as vaccines. It is clear that Th 1 cells are central to the protective response against mycobacteria. Production and activation of Th 1 cells is promoted by interleukin-12 released by macrophages. Mycobacteria are strong inducers of interleukin-12 and it is notable that a deficiency of IL-12 also predisposes to severe disease produced by normally innocuous mycobacteria. We know that macrophages and mycobacteria go back together a long way. Macrophages have receptors which belong to the evolutionary ancient Toll family and some of these transduce signals from mycobacterial components to provoke the synthesis of interlelukin-12. The mycobacterial components are also important in inducing costimulatory molecules on the macrophages so that they can present antigens to the T cells effectively. In addition to these cytokines tumour necrosis factor produced by macrophages also plays an important role in host defence. It promotes accumulation of cells at the site of infection and it acts in concert with interferon gamma to activate the macrophages antimicrobial defences. We have known for a long time that macrophages use their MHC Class 1 and Class 2 molecules to present mycobacterial antigens to T cells. More recently it emerged that another group of MHC Class 1 like molecules could specifically present components of mycobacterial cell walls to T cells. The molecules which present these glycolipid antigens belong to the CD1 family. One of these is shown here. You can see it is indeed very similar to an MHC Class 1 molecule. However, the antigen-binding groove is particularly deep and hydrophobic so that it can accommodate long lipid groups. Some of the mycobacterial antigens are shown here. They all have long hydrophobic chains, which can fit into the deep groove on the CD1 molecule. And hydrophilic sugar and phosphate groups which would be exposed to the T cell receptor. It's fascinating that our macrophages appear to have developed specific systems to present the antigens of mycobacteria. The killing of phagocytosed mycobacteria only takes place effectively within activated macrophages. Within the membrane of the phagosome an enzyme is assembled that generates reactive oxygen intermediates or ROIs. And these can go on to produce hydrogen peroxide. In the presence of chloride or iodide ions and myloperoxidase hypohalites are formed. Hypochloride for example is that noted antibacterial agent bleach. But all of these compounds are potentially toxic for the mycobacteria. Even more important are reactive nitrogen intermediates such as nitric oxide and peroxynitrites. Nitric oxide is formed by the action of nitric-oxide synthase on arginine. Production of the enzyme inducible nitric oxide synthase occurs in macrophages, which have been activated by interferon gamma. The cocktail of highly reactive oxygen and nitrogen intermediates is critical in killing the phagocytose mycobacteria. In addition macrophages can limit the availability of iron which the bacteria need for intracellular survival. However, this is a bit of a double-edged sword because the macrophages also need iron for the generation of their bactericidal reactive oxygen and nitrogen intermediates. Also important are the lysosomes. These organelles contain a collection of enzymes which degrade macromolecules. The enzymes work best at low pH and the lysosome is maintained at an acid pH by proton pumps in its membrane. Fusion of the lysosome with the phagosome allows the enzymes to digest the bacteria. All of the macrophages' defences work most effectively if the cells have been activated by interferon gamma. Although interferon gamma is critical the process also involves other inflammatory cytokines such as tumour necrosis factor, interleukin 1 and interleukin 6. In addition to their role in immune response some of these cytokines are also responsible for the wasting and fever which is seen in patients with tuberculosis. In people who develop tuberculosis the balance between the immune system and the bacteria is tilted in favour of the pathogen. And bacteria escape from the phagosome into the cytoplasm of the macrophage and start to divide, eventually killing the cell. Within the lung of an infected individual areas of cell death or necrosis start to form. Around these areas palisades of activated macrophages can be seen which are referred to as epithelioid cells. Sometimes the macrophages fuse to form giant cells each having several nucleii. The appearance of this type of lesion is called a granulomatous reaction and it shows the co-lateral damage which may develop when the immune system generates a very strong cell-mediated immune reaction against a persistent antigenic stimulus. In patients who develop tuberculosis the immune reaction does not control the infection. The bacteria proliferate and in this picture they can clearly be seen stained by the fluorescent dye or-amine in the sputum of an infected individual. The damage created by cell death and the spreading infection produces the lung shadows, which were often used to diagnose tuberculosis. In this X-ray picture the patient has streaking on the upper part of both lungs and there is also some enlargement of the heart. However, in most people who become infected with tuberculosis the immune response limits the infection to a small granuloma and the infection progresses no further. It is possible to tell whether a person is sensitised to the bacteria because they have a positive skin reaction if antigens from the mycobacteria are pricked into the skin in the so-called Heaf test. The result can be seen here after one week This person has been previously sensitised to the bacteria and shows a raised area of inflammation caused by cellular infiltration. One might well ask why only a small proportion of people develop tuberculosis even though most have been infected. Variation in disease susceptibility is partly due to genetic differences. For example specific MHC molecules have been identified which are associated with either susceptibility or resistance. In addition a gene called Nramp1 which determines resistance to infection in mice appears to affect susceptibility in humans too. This gene produces a protein called natural resistance associated macrophage protein, which acts as an ion pump to divalent metal ions from the phagosome. Interestingly bacteria have a related gene, which they use to concentrate divalent metal ions. This means that there is a competition between the phagocyte and the mycobacteria for essential ions such as iron, zinc and copper. Another protein associated with disease resistance is the vitamin D receptor expressed in macrophages and activated lymphocytes. Apart from its role in regulating calcium metabolism vitamin D is known to modulate immune responses. Although a few of the relevant genes have been identified there are clearly many more which contribute to susceptibility or resistance to tuberculosis. One must not forget however that tuberculosis is a disease where it is not just the bacterium and the immune response which determines susceptibility. Environmental conditions also play a large part in determining whether an infected person will succumb to the disease. Before a bacterium can infect body tissues, it must first gain entry. There are several non-specific defence mechanisms which help prevent this. The skin forms a physical barrier, impermeable to many bacteria. This is further protected by acidic secretions which are toxic to some varieties. The epithelial surfaces of the airways and digestive tract are another possible route of entry. They are washed by mucosal secretions, and low pH contributes to the protection of some mucosal surfaces. Throughout the body, non-pathogenic commensal organisms help block establishment of pathogens. There are three ways in which bacteria can affect the body adversely. They may invade the tissues and multiply, colonise the tissue surface and cause damage by releasing toxins into the tissues, or thirdly, they may do both; invade and release toxins. If these general defences prove inadequate, other more specific mechanisms come into play. Take as an example of the first type, N. gonhorroeae in the genitourinary tract. Normally bacteria are flushed out by the passage of urine. In order to attach to the epithelial surface, the bacillus must bind using some receptor. The body's cells attempt to pre-empt this by blocking those receptors. Immunoglobulin A is produced by B-cells which may be transported across the epithelium from the abluminal to the luminal surface. To do this, it first becomes bound to the poly-Ig receptor on the abluminal surface of the epithelial cell. The cell draws in the IgA bound to its receptor, and forms a vesicle which migrates towards the external, luminal surface. At the luminal surface, the IgA is released by cleavage of the receptor and is secreted into the genitourinary tract. When the IgA encounters a bacterium, it can bind to its surface receptors. This prevents the bacterium from attaching to the epithelium, and it is washed away by the passing fluid. Another possible route of entry for bacteria is through broken skin, for example C. tetani, the causative agent of tetanus. A bacterial colony could become established following a wound. The bacteria multiply and release toxins into the surrounding tissue. If the individual has been immunised against tetanus, tetanus specific toxin immunoglobulin exists in serum. When it encounters the bacterial toxins, it binds and blocks the deleterious effect this toxin has on neuronal transmission. The bacterial colony will be phagocytosed in the same way as any bacillus within the body. Should the protective effects of IgA be inadequate, and bacteria succeed in attaching to the tissue wall, such as the epithelium in the gut, they will divide on the luminal surface and invade the underlying tissue. An example of this is seen with S. typhi, the causative agent of typhoid fever. The bacteria can invade macrophages and survive within them. However, if there are specific T cells which recognise bacterial peptides, presented by the macrophage, then the T cells release interferon gamma which activates the macrophages to destroy the bacteria they have taken up. Antibodies and complement in the serum also provide their own immune response to these bacteria, by damaging their membranes and limiting their invasiveness. As the immune system has evolved these mechanisms, bacteria have also evolved counter measures to avoid destruction. Some attempt to evade the macrophages by intercepting the chemotactic messengers sent out by the complement system. Without the presence of C3a and C5a, the macrophages cannot determine the site of infection. If some bacteria are engulfed by macrophages, they release substances which inhibit the fusing of lysosomes with the vacuole, thus preventing contact with the macrophage's arsenal of chemicals. Those that do allow the lysosome to join with the vacuole can have defences against the reactive species they bring in. Others are adapted to survive in the hostile environment of the vacuole through the presence of extremely resistant coatings. Some follow the premise that the best defence is offence. They are able to escape the vacuole into the safety of the cytoplasm of the macrophage, where they divide, and can kill the host cell.

Inflammation

Inflammation is an essential element of immune defence since it brings cells and molecules of the immune system into an area of infection. The outward features of inflammation include the pain, redness and swelling that often accompany an infection. These signs can be related to the underlying cellular processes that are taking place in the infected tissue. There are three principal components of inflammation. • an increased blood supply to the area • an increase in the leakiness of the local capillary blood vessels • migration of leukocytes out of the blood vessel and into the tissue. Each of these functions serves to bring cells and molecules of the immune system to the site of infection. Smooth muscle in arterioles determines the diameter of the vessels and controls the blood flow through a site of inflammation. The increase in blood supply means that there are more cells and serum molecules (including antibodies and complement molecules) arriving in the area. It is also responsible for the redness seen at a site of inflammation. The increase in leakiness of the blood vessels is more usually called increased capillary permeability. Normally most of the large sera molecules are retained within the serum, since the endothelial cells that line the internal wall of the blood vessels prevent large molecules from crossing. But at sites of inflammation, the endothelial cells retract in response to inflammatory mediators and this allows the larger molecules to leak out in greater amounts. This is very desirable, since it is important to get antibodies and complement into the tissue to combat infection. Leakage of serum shows up as the swelling. The migration of leukocytes into the tissue is equally important, since they are needed at the site to phagocytose pathogens, or to attack infected cells in the tissue. So, the function of inflammation is to get appropriate immune defences to a site of infection. The process of inflammation is very carefully choreographed by signalling molecules (cytokines) released in the tissue. The type of inflammation that develops does in fact depend on the type of infection and on the tissue involved. Remember that an appropriate immune defence depends on what infectious agent is involved. So, for example, an acute local infection of the skin with Staphylococcus (a boil) will induce many neutrophils and macrophages to enter the tissue, whereas a chronic infection of the lung with Mycobacterium tuberculosis will cause the inward migration of T cells and macrophages.

Lymph nodes

Lymph nodes are small organs varying in size from a few millimetres to 1-2 cm. They contain collections of leukocytes and are distributed in different areas of the body. Lymph nodes are linked in chains by lymphatic ducts, so that the lymph flowing out of one lymph node via the efferent lymphatic vessel becomes the inflow to the next in line, via the afferent lymphatics. Lymph is a fluid derived from the tissues that carries cells and foreign material to the lymph nodes. Eventually, lymph returns to the bloodstream via one of the body's two major lymphatic ducts. Secondary lymphoid organs can be thought of as guard posts that are strategically placed to intercept any infectious agent that enters an area of the body. So, for example, the lymph nodes in the axilla of the arm (the armpit) will intercept infections which enter that part of the body. In this analogy, leukocytes are the troops of the immune system and they are able to move between the guard posts and into the hinterland of the tissues. Lymph nodes have a well defined structure (Figure 1.5), with different sub-regions. Different types of leukocyte are localised within the regions, so that they can interact with each other appropriately to initiate and develop the immune response. Antigens and cells enter the node through afferent lymphatics, and cells and fluid leave through the efferent lymphatic. Cells (lymphocytes) can also enter the node from the blood by migrating across the specialised high endothelial venules (as was indicated in Figure 1.2). Within the node, cells distribute to distinct zones. B cells proliferate and develop within the follicles of the cortex, while T cells are primarily located in the paracortex. The capsule, medullary cords and hilus are fixed structural elements of the tissue. Lymphocytes located in the local lymph nodes are responsible for the initial recognition of the infection and the development of the immune response. Once the immune response has developed, the cells will migrate out from the lymph node to the blood and, ultimately, cells will move to the site of infection to combat the pathogen there.

Auxiliary cells

Many cells of the body take part in immune reactions by producing or responding to cytokines or by interacting with lymphocytes. There is, however, a smaller group of cells that contributes to the control of inflammation and these cells are often called auxiliary cells. Of these, the mast cell is particularly important. Mast cells lie close to the blood vessels in virtually all tissues of the body and contain numerous granules, full of mediators that promote inflammation, such as histamine that causes increased blood flow to an area. When they are activated, mast cells release the contents of their granules, thereby inducing inflammation (Figure 1.12). They can also synthesise mediators (signalling molecules) which cause leukocytes to be attracted to a site of inflammation. This process is called chemotaxis and the mediators are chemoattractants. Mast cells are related to basophils that also contain inflammatory mediators. Basophils can be triggered in a similar way to the mast cells, although basophils are generally confined to the blood. Blood platelets also act as auxiliary cells, although these are not strictly cells since they do not contain a nucleus. Platelets are produced by the fragmentation of a large cell in the bone marrow called a megakaryocyte, and they then circulate in the blood. Their principal role is in promoting blood clotting, but they also contain a number of inflammatory mediators and chemoattractants that are released as the platelets aggregate during blood clotting.

Opsonisation

Mononuclear phagocytes and neutrophils have receptors on their surface that bind to the Fc region of IgG (Fcγ receptors). Antibody bound to the Fc receptor and to the pathogen (via the antigen-binding site) acts as a cross-bridge between the phagocyte and pathogen and so facilitates phagocytosis (Figure 2.8). Immune complexes (soluble antigens bound to antibody) can also be taken up by this mechanism. There are three main receptors for IgG (FcγRI, FcγRII and FcγRIII) distributed on different leukocyte populations. All mononuclear phagocytes express FcγRI to FcγRIII - these are the receptors that the macrophage uses for binding to opsonised antigens. FcγRI (CD64) is a specific marker of mononuclear phagocytes, but FcγRIII is also present on most large granular lymphocytes, and they can use it to recognise antibody bound to infected cells. FcγRI and FcγRIII are present on neutrophils that also use the receptors for recognising opsonised antigens. In effect, when antibody binds to an antigen, it labels it for phagocytosis. Another receptor, FcγRII, is present on B cells but this has quite a different function. It allows a B cell to respond to the level of antibodies in solution and adjust its own production of antibody accordingly. The structures of some of the different Fc receptors. Receptors for other classes of antibody (IgA, IgM, IgE) can also allow leukocytes to recognise pathogens, for example, IgE receptors (Fcε) on eosinophils allow them to engage helminths (flatworms). A process by which phagocytosis is facilitated by the deposition of opsonins (e.g. antibody and C3b) on the antigen. 2.2 Opsonisation of bacteria If bacteria enter the body, clearance is dependent on phagocytosis, and the efficiency of the phagocytes often depends on both antibody and complement, which are needed to opsonise the bacteria Many bacteria use countermeasures to evade the immune response of the host. For example, they can release molecules calledimmunorepellents that prevent macrophages and neutrophils from moving to the site of infection. In addition, the capsule of many bacteria resists phagocytosis. In each case, antibodies neutralise the countermeasure and allow the phagocytes to engage the bacteria.

T-dependent antibody responses

Most antibody production by B cells requires help from T cells. However, there are a few antigens that can stimulate B cells directly, causing them to divide and differentiate into antibody-forming plasma cells. These antigens are typically large polymeric molecules, with arrays of repeated epitopes, e.g. bacterial carbohydrates. They activate the B cells by cross-linking their surface receptors (Figure 3.1, left). Such antigens typically induce the formation of IgM antibodies that are of low affinity. Other antigens (polyclonal activators, Figure 3.1, right) are intrinsically biologically active and also act on other receptors on the B cell surface (see a) to cause cell activation. At high doses, such molecules activate many clones of B cells, regardless of their antigen specificity. They are thus called polyclonal activators. At lower doses, B cells that have a B cell receptor for such antigens will concentrate the polyclonal activator to their cell surface and will then be triggered by it. Unlike the response to most antigens, the B cells do not switch to IgG production. The idea that B cells need help from T cells to make antibody goes back to the 1960s. Indeed, the term T helper (Th) cell came from experiments that demonstrated just this. The processes involved are now much better understood and T cell help for antibody production is considered a Th2-type of response. However, this is somewhat simplified, since the exact nature of an antibody response depends on cytokines from both Th2 and Th1 cells.

Natural killer cells

NK cells. A group of lymphocytes that have the intrinsic ability to recognise and destroy some virally infected cells and some tumour cells.

PAMPS

Pathogen-associated molecular patterns (PAMPs) are distinctive structures found on the surface of microbes, which can be recognised by pattern recognition receptors (PRRs), part of the innate immune system. For example, lipopolysaccharide present on Gram-negative bacteria is recognised by TLR4, a receptor present on macrophages. TLR4 is toll-like receptor-4, one of a family of such receptors found on various cells of the body. The archetypal molecule 'toll', from which this family has evolved, was originally identified in Drosophila melanogaster (the fruit fly). More information on this sort of receptor is provided in Section 2.2. The PRRs fall into two basic types: soluble molecules, which act as adapters between pathogens and cells of the body, and cell surface receptors, such as TLR4, which allow a cell to directly bind and recognise the pathogen. The following two sections give an overview of these systems. Even in the absence of opsonins, cells have a number of receptors, which allow them to directly recognise pathogens. They are: • lectin receptors, which bind to carbohydrates in bacterial and fungal cell walls • scavenger receptors which recognise cell debris; in addition to their role in recognising pathogens, they also help to clear dead cells • toll-like receptors (TLRs). The lectin receptors and scavenger receptors are expressed primarily on macrophages, where they promote recognition and phagocytosis of microorganisms. The TLRs are more widely distributed and some of the TLRs help protect against viral infections by recognising viral nucleic acids. For example, TLR3 recognises dsRNA and TLR7 and 8 recognise ssRNA. These receptors for nucleic acids are normally found in the membrane of endocytic vesicles, rather than on the plasma membrane. • Why would TLR3 recognise viral RNA but not host RNA? Why would it be advantageous to have TLR7 expressed on an endosome? • Long dsRNA molecules are formed during the replication of RNA viruses, and are not a normal component of cellular metabolism, so TLR3 discriminates when a virus is present. When an RNA virus is endocytosed, it will be taken up in an endosome where it uncoats and the genetic material is free to escape into the host cell; hence TLR7 will be well placed to detect the viral nucleic acid as it is released. In addition to their role in recognising the pathogen, the TLRs signal to the cell, causing it to become activated and increase its microbicidal defences. In doing this, they use some of the same pathways as inflammatory cytokines (TNFα and IL-1). A number of bacteria and many viruses replicate within the cytoplasm of the host cell. Cells have a number of cytoplasmic proteins that can detect such internal pathogens and activate the cell's defences, or cause the cell to die, which will limit the capacity of the pathogen to replicate and spread. Table 2.1 summarises some of the main PAMP receptors and their targets. Biological macromolecules produced by microbial pathogens that are recognised by receptors on mononuclear phagocytes and some opsonins in serum and tissue fluids.

Neutrophils

Polymorphonuclear granulocytes, which form the major population of blood leukocytes. There are two major types of phagocyte: mononuclear phagocytes and neutrophils. The mononuclear phagocytes are long-lived cells that are distributed throughout the body, while the neutrophils are the self-sacrificing cells of the immune system. Neutrophils are produced in huge numbers in the bone marrow, migrate from there to the tissues, phagocytose material and die, all within about 48 hours. Neutrophils belong to a group of cells called 'polymorphonuclear leukocytes' (Figure 1.6) or polymorphs for short, so called because the nucleus has several lobes. There are three types of cell in this category that are distinguished according to the way they stain with different dyes. The three types are: • neutrophils that stain with neutral dyes and are mostly involved in antibacterial defence • eosinophils that stain with acidic dyes such as eosin, and which are important in defence against parasitic worms • basophils that are important in the control and development of inflammation. Of these three cell types, only the neutrophils are phagocytes and they are by far the most abundant. Nearly 70% of the leukocytes in the blood are neutrophils. Unlike most other cells of the immune system that can move between the lymphoid tissues and the other tissues, neutrophils make a one-way trip from the bone marrow to the different tissues of the body. However, they tend to accumulate particularly at sites of bacterial infection. For example, the pus that is produced in an infected wound contains huge numbers of neutrophils, which are attracted to the area by products released by the bacteria, and by cytokines released by cells of the immune system to control the spread of infection. This process is called chemotaxis and will be examined in detail later, together with the mechanism by which phagocytes destroy microorganisms.

Macrophage activation

Process by which macrophages acquire enhanced microbicidal properties.

CD system

Since many of the different types of leukocytes look similar, immunologists had to find reliable ways of identifying them. The CD system of markers developed from this work. It uses antibodies that specifically bind to surface molecules on lymphocytes. If the antibodies are tagged with a fluorescent dye then cells that have the marker will be stained. Originally, the researchers would submit antibodies that they had produced to scientific workshops for evaluation. If an antibody bound to one particular lineage of cells it was said to identify a 'Cluster of Differentiation', hence the CD naming system. For example, antibodies of type CD3 bind to T lymphocytes and not to other leukocytes. CD3 is therefore a T lymphocyte marker. In time, the term CD3 came to be used for the molecule on the cell surface which was recognised by the antibodies. So the molecule CD3 is now known to be part of the T lymphocyte's receptor for antigen. Over the years, the number of defined CD molecules has expanded enormously and the system now runs from CD1 to CD339, with several of the numbers being subdivided, e.g. CD62E, CD62L, CD62P. All cells have many different CD molecules on their surface at any one time. Markers are not always lineage specific. Some may be present on several different lineages. All the cells of the immune system are initially derived from stem cells in the bone marrow, which differentiate into the various cell lineages (Figure 1.6). Lymphoid stem cells give rise to all lymphocytes, while myeloid stem cells give rise to all the other leukocytes. (A third stem cell type in bone marrow gives rise to red blood cells and platelets, which are not part of the immune system.) Even cells of the thymus, the other primary lymphoid organ, migrate there initially from bone marrow. Each cell type is distinguished primarily on the basis of the molecules that it expresses on the cell surface. These are called markers, and they are organised using a nomenclature called the 'CD' system (see Box 1.1). More than 300 different cell surface markers have been identified. Fortunately, you only need to know a few of these, which act as key markers of particular cell lineages; others can always be looked up if needed from resources like the Science Gateway (2012) website.

Development of the antigen response

So far, you have seen that T cells help B cells to make antibodies, that they direct antibody class switching and that the events which underlie the development of the antibody response take place in lymphoid tissues. The following sections will give you some insight into these events and an understanding of the molecular and cellular processes that occur as an antibody response develops. • At the level of an individual: antibodies in serum switch from low affinity IgM to high affinity IgG during the response to a T-dependent antigen as in Figure 2.6. • At the cellular level: individual B cells switch from producing IgM to IgG, IgA or IgE. • At the molecular level: the immunoglobulin genes that a B cell uses to make its antibodies are switched as the B cell matures.

PRRs

Soluble PRRs include three families of molecules: the pentraxins, the ficolins and the collectins. Many of these proteins are produced by the liver, particularly in response to inflammatory cytokines such as IL-6 and TNFα. The first of these proteins to be recognised was the pentraxin C-reactive protein, which recognises a group (phosphoryl choline; also known as phosphocholine) present on the surface of pneumococci. It also binds to a receptor on macrophages, so it can act as an adapter which allows macrophages to recognise pneumococci. This process, by which an adapter molecule promotes phagocytosis, is referred to as opsonisation and any molecule that performs this function is an opsonin. C-reactive protein also activates the complement system, a group of proteins that are themselves important in opsonisation and the control of inflammation (Figure 2.1). C-reactive protein is also an acute-phase protein, meaning that its level increases during infection or inflammation (CRP itself can increase up to 1000-fold). Such molecules are very useful clinical markers of infection and/or inflammation. Although many of these proteins are produced by the liver, some can be induced in other tissues, for example pentraxin-3 is synthesised by leukocytes and vascular endothelium and the surfactant proteins are produced by epithelium in the lung. Groups of molecules that can detect PAMP, for example the Toll-like receptors and the collectins.

Cross-reactivity

Some antisera are not totally specific for their inducing antigen but bind related (cross-reacting) antigens, either due to their sharing a common epitope, or because the molecular shapes of the cross-reacting antigens are similar. One more idea needs to be introduced here - cross-reactivity (Figure 2.6). Up to this point you have learned that each antibody binds to one antigen and only one antigen, and in most cases this is effectively true. However, you now know that an antibody recognises a molecular shape. It is just possible that the same molecular shape (epitope) could appear on two different antigens (a shared epitope). In this case the antibody would not be specific for the one antigen. It would bind to both, and such antibodies are described as cross-reactive. This is not generally a problem: indeed it could be quite useful to have antibodies which recognise several different strains of the same pathogen. However, there is one circumstance where cross-reactivity can lead to major problems in immune defence. (Cross-reactivity can also occur when an antibody binds an epitope because it has sufficient similarity to the epitope that the antibody normally recognises.)

Immune defences against intracellular bacteria

Some bacteria can survive and proliferate inside host cells, particularly macrophages (e.g. mycobacteria, Salmonella, Listeria, Legionella and Shigella). In each of these infections, macrophage-mediated immunity is an important component of protection, but the macrophage is also the cell that becomes infected by the bacteria. In order to do this and survive, these groups of bacteria deploy a variety of measures that allow them to evade the macrophages' defence mechanisms. In this case, the effectiveness of the immune response often depends on whether the macrophage has been activated by a pathogen-specific T cell. If the macrophage cannot eliminate the pathogen, then a chronic infection develops.

Functions of antibodies

Some functions of antibodies depend solely on their ability to bind to antigen. For example, if an antibody binds to a receptor on the surface of a virus, then it may prevent the virus from attaching to a host cell. More often however, it is necessary to recruit other immune defences. You have already seen that different antibody classes have different functions, related to the type of heavy chain they possess, and this is dependent on the Fc region of the molecule (see Figure 2.3). All cells of the immune system have receptors for the Fc region of antibodies (Fc receptors), but there are many types of Fc receptor, each one being specific for a particular class of antibody. Each cell type has its own set of Fc receptors, which means that it can bind to a particular set of immunoglobulin classes which mediate a range of functions. Basophils and mast cells both have a high affinity receptor for IgE (FcεRI) and most of the IgE in the body is bound to the surface of these cells. The cells are said to be sensitised by the IgE, because if antigen cross-links the surface-bound IgE it causes the cells to release their granules, which contain inflammatory mediators (Figure 2.10), and to synthesise new mediators. Inflammation and the role of mast cells and basophils will be looked at later in this unit. At this stage, it is sufficient to know that IgE antibodies are involved in inflammation. One important component of immune defence is the complement system, a group of about 20 proteins, present in blood and tissue fluids, which interact with each other and with many cells of the body, carrying out a number of protective functions. IgG and IgM antibodies are able to activate complement functions by binding to the first component of the system, C1q, which is structurally related to the collectins of the innate immune system. In this case it acts as a receptor for antibody Fc regions, rather than a PAMP. IgM and some IgG molecules activate complement by binding to C1q, and this constitutes the 'classical pathway' of complement activation (see Section 4.1). • In immune responses against helminths, the body often produces high levels of specific IgE. In responses against enteric bacteria, a strong IgA response occurs, and in responses to malaria, IgG is the major antibody. What reason or advantage (if any) is there in producing different classes of antibodies, in different infections? • The appropriate type of immune response depends on the pathogen. A response that is effective against one pathogen can be useless or even detrimental against another. Each of the antibody classes mediates a different type of immune defence. The body tends to synthesise the class of antibody that is most appropriate for dealing with each type of infection.

T cells

T cells are absolutely central to immune defence. In fact, there are several types of T cell, each having different functions. The cells described above, which are responsible for recognising and destroying infected cells, are called cytotoxic T cells or Tc cells. The term 'cytotoxicity' means being able to kill cells, and it is usually very desirable to kill a cell which has become infected. • Under what any circumstances (if any) might it not be desirable to kill an infected cell? • Some cells of the body, such as nerve cells, are irreplaceable. If the immune system destroys a nerve cell, it has gone for good. For this reason, the immune response in the brain and spinal cord is limited. Nerve cells may remain chronically infected for years, as in a herpes virus infection (cold sores), for example. In this case the damage caused by the virus is moderate, and the alternative would be the destruction of the sensory nerve in which the virus has established its infection. Tc cells are distinguished by the marker CD8 and about one-third of the T lymphocytes belong to this population. The remaining two-thirds express the marker CD4 and are called helper T cells (Th cells). Note that the markers are mutually exclusive: a mature lymphocyte has either CD4 or CD8 but not both. The idea of 'help' is very pervasive in immunology. It relates to the fact that leukocytes usually require signals from other cell types before they will divide, or become active in an immune response. Such signals constitute the help and this is provided by the helper T cells. Helper T cells have a number of functions. Principally: • Th1 cells help phagocytes to destroy pathogens that they have taken up • Th2 cells help B cells to make antibodies. Additional subsets of T cells are also identifiable based on function. For example, regulatory T cells (Tregs) are a subset of the CD4+ T cells, which are important in controlling autoimmune reactions, although their role in response to infection is less certain. There are markers that distinguish these populations, but it has often been more useful to distinguish them according to the molecules they secrete.

Alternative pathway

The activation pathways of the complement system involving C3 and factors B, D, P, H and I, which interact in the vicinity of an activator surface to form an alternative pathway C3 convertase. Activation of the alternative pathway is more complex; it is continuously being activated, but only at a very low level, to form another C3 convertase (C3bBb). Cells of the body have a group of molecules on their surfaces that limit the activation of complement. They are referred to as complement control proteins. Bacteria and fungi lack the control proteins, so the alternative pathway is rapidly activated in their presence - they are said to have 'activator surfaces'. So the alternative and lectin pathways act as a recognition system for microorganisms, while the classical pathway is directed to attack whatever the antibodies recognise.

Adaptive

The adaptive immune defence refers to the tailoring of an immune response to the particular foreign invader. It involves differentiating self from non self and involves B cells and T cells (lymphocytes). A key feature of the adaptive immune system is memory. Repeat infections by the same virus are met immediately with a strong and specific response. Lymphocytes are said to be part of the adaptive immune system, because the response of lymphocytes retains a 'memory' of previous infections that improves or adapts, on subsequent encounters with antigen. However, even before the evolution of lymphocytes, organisms had primitive immune systems with phagocytes and had developed a number of soluble molecules and cell surface receptors to recognise generic microbial molecules. Many of these evolutionarily early recognition systems have been retained within the present-day mammalian immune system and they are said to be part of the innate immune system, which provides an immediate, but non-specific, defence. In practice, the two systems interact extensively and work together so it is impossible to make an absolute distinction between them. The process is very efficient. For example, the concentration of IgA in blood is 2 mg/ml while the concentration in tears is 16 mg/ml. However, you should note that serum IgA and secretory IgA are slightly different: the latter has the secretory piece attached as it is transported across the epithelium and this polypeptide tends to prevent the IgA from being degraded by enzymes in the secretions (e.g. digestive enzymes). When devising vaccines to combat infections that enter through mucous membranes it is usually desirable to induce good IgA responses. This is done by promoting a local immune response at the site of entry, rather than a general immune response. Antibodies to fimbriae, which some bacteria use to attach initially to a host cell, are particularly important targets for this type of defence.

Anitgen presentation

The breakdown of the pathogen is not the end of the story. Remember that the macrophage is also an antigen-presenting cell. Peptide fragments can be transported through the cell to become combined with MHC molecules, which are then presented to Th1 cells. • What two signals are required to activate a Th1 cell? • A signal from the T cell antigen receptor (TCR) and a costimulatory signal from CD28 induced by binding to B7 on the antigen-presenting cell. The expression of B7 is induced on macrophages following recognition of bacterial products by toll-like receptors (TLRs). Consequently, when a macrophage detects bacterial components, it becomes a more effective antigen-presenting cell.

Antibody affinity

The discussion above leads on to the ideas of antibody affinity and avidity (Figure 2.5). These terms have precise meanings and can be measured experimentally, but put simply, an antibody which makes a strong bond with its antigen - many non-covalent bonds, and a good complementary shape - is said to have a high affinity. Generally speaking, high affinity antibodies are better at combating infection than low affinity antibodies. Also, it is generally found that IgA, IgG and IgE have higher affinity than IgM antibodies. However, we now introduce the idea of avidity. This concept takes into account not just affinity (the strength of a single antigen/antibody bond) but also the fact that antibodies have several combining sites. Thus IgM can have a high avidity for an antigen, because it has ten combining sites, even though the individual binding sites are of low affinity. This is called the bonus effect of multi-valency. So IgM antibodies can be very useful in circumstances where a pathogen has a cluster of identical epitopes on its surface. For example, the surface of a bacterium may have millions of identical carbohydrate epitopes and an IgM molecule can make several bonds with it. This is where the segmental flexibility of the hinge region is important, allowing the IgM to bend so that several epitopes can be bound simultaneously. The binding strength of a single antigen-combining site for its epitope.

C3 convertases

The enzyme complexes C3b, Bb and C4b2a that cleave complement C3.

Avidity

The functional combining strength of an antibody with its antigen, which depends on both the affinity of the reaction of the epitope with the antigencombining site, and the valencies of the antibody and antigen.

IgG

The main antibody in blood and tissue fluid. It has a large number of functions, including neutralising many toxic molecules, preventing viruses from attaching to cells, allowing phagocytes to recognise and internalise pathogens, and protecting the fetus and newborn babies. (It is the only antibody class that can cross the placenta.)

Epitopes

The parts of an antigen that contact the antigen-binding site of an antibody or the T cell receptor.

Antigen presentation

The process by which antigen is presented to lymphocytes in a form they can recognise. Most CD4+ T cells must be presented with antigen on MHC class II molecules, while CD8+ Tc cells only recognise antigen on MHC class I molecules. Antigen must be processed into peptide fragments before it can associate with MHC molecules. Before leaving the subject of antigen presentation, it is worth reviewing some of the ways in which infection can affect the process - remember that antigen presentation goes on all the time, regardless of whether infection is present. You should note that PAMPs can enhance antigen presentation by increasing the expression of costimulatory molecules. It has long been known that components of bacteria can enhance immune responses, and when they are included in vaccines, such components are called adjuvants, and a molecule that enhances a response is said to have adjuvant activity. We can now relate the actions of these bacterial adjuvants to their ability to activate macrophages and promote costimulation and antigen presentation. The simple pattern recognition systems used by macrophages pre-date the highly specific recognition systems of lymphocytes by many millions of years. Nevertheless, the original recognition systems now work in concert with the more recently evolved adaptive immune system to provide both stimulation and costimulation as joint elements of antigen presentation. The breakdown of the pathogen is not the end of the story. Remember that the macrophage is also an antigen-presenting cell. Peptide fragments can be transported through the cell to become combined with MHC molecules, which are then presented to Th1 cells. • What two signals are required to activate a Th1 cell? • A signal from the T cell antigen receptor (TCR) and a costimulatory signal from CD28 induced by binding to B7 on the antigen-presenting cell. The expression of B7 is induced on macrophages following recognition of bacterial products by toll-like receptors (TLRs). Consequently, when a macrophage detects bacterial components, it becomes a more effective antigen-presenting cell.

BALT

The respiratory tract has its own collections of lymphoid cells called bronchus-associated lymphoid tissue (BALT) and there are other specialised lymphoid tissues in the gut (Peyer's patches), the genitourinary tract and the eye. This is the first line of defence of the immune system - the place where pathogens first come into contact with lymphocytes which can recognise them. Later, in the effector phase of the immune response, lymphocytes in the sub-mucosal tissues produce a particular type of antibody called immunoglobulin A (IgA) that is released into the mucous secretions to combat the infection. Most potential pathogens never get beyond these first lines of defence. Lymphoid tissue associated with the respiratory tract, i.e. a subset of the mucosa-associated lymphoid tissues.

Spleen

The spleen is a large organ which lies just behind the stomach. It has a number of functions, one of which is as a lymphoid organ. In fact, the spleen contains two types of tissue called red pulp and white pulp and it is the white pulp that is a lymphoid tissue (the principal function of red pulp is to destroy ineffective red blood cells). The white pulp is distributed around the arterioles in the spleen and is often called the peri-arteriolar lymphatic sheath (PALS). The key immunological function of the spleen is to take up infectious agents that may be circulating in the blood. The role of the spleen is made clear in people who have had their spleen removed surgically (to prevent internal bleeding after injury to the spleen). These people are much more susceptible to sepsis (septicaemia), i.e. bacterial infection of the blood. You are now aware that the cells of the immune system move between lymphoid organs and the other tissues of the body. The patterns of cell migration are complex and depend on the type of cell involved and whether it has been activated. In the rest of this unit you will look at the cells of the immune system in detail and examine their functions in an immune response.

Lytic pathway

The three complement activation pathways are clearly essential for opsonising bacteria. However, the lytic pathway - in which all three pathways converge to generate the membrane attack complex (MAC) - is also important for defence against some groups, particularly Neisseria meningitidis (a causative agent of meningitis). These Gram-negative bacteria have an outer cell membrane susceptible to MACs, so people who are deficient in components of the lytic pathway are more susceptible to this disease.

Alkaline phase

This chemical barrage that the pathogen is subjected to is just the first phase of the body's attack. Immediately after the phagosome has formed, the pH inside it starts to rise. This can partly be attributed to hydroxyl ions produced as part of the process described so far. (Later on, the pH will fall and the phagosome becomes acidic.) During the alkaline phase, a new defence comes into action. Neutrophils (but not macrophages) have a group of cationic proteins called defensins in their granules that are released into the phagosome. They have a wide spectrum of activity against Gram-positive and Gram-negative bacteria, spirochaetes and yeast, including the ability to damage the outer membrane of the pathogens by assembling ion channels in them. • State any other instances that you have read about when pathogens are damaged by the assembly of channels in their outer membranes. • The membrane attack complex (MAC) of the complement lytic pathway produces channels through viral envelopes and the outer membranes of some bacteria (see Unit 2 Section 4.2 of this block).

Immune response

Two principal phases of an immune response can be distinguished: • the recognition of the infectious agent • the effector phase of the response, when the immune system aims to eliminate the infection, or at least contain it and minimise the damage it causes. Exactly how the body responds depends on which infectious agent is involved, and numerous other factors, such as the genetic make-up of the individual, the route of entry into the body and whether this is the first time the pathogen has been encountered. You have already come across the enormous range of pathogens, which vary from sub-microscopic viruses a few nanometres in diameter which replicate inside cells, to tapeworms located in the gut that can be more than a metre long. As shown in Figure 1.3, the immune system has to combat a great diversity of pathogens in different locations. • Viruses have an intracellular and extracellular phase to their life cycle and different immune defences are relevant to each phase. • Bacteria may live in the blood (septicaemia or sepsis) or within cells (e.g. tuberculosis) or within tissues (e.g. staphylococci) or on surfaces (e.g. cholera in the gut). • Parasites also may live inside or outside cells, and many have complex life cycles involving both phases. Parasitic worms can live in tissues (e.g. schistosomes) while large parasitic worms can occupy spaces such as the gut. • Fungi are broadly divisible into superficial (surface) infections and deep mycoses that occupy tissues. The type of immune defence required to combat a virus, for example, is completely different from that needed to deal with a tapeworm. So let us consider some of the key features of an infection as far as the immune system is concerned. One major distinction is between intracellular and extracellular infections. Many bacteria and parasites live and divide outside the cells of the host and these pathogens come into direct contact with the cells and molecules of the immune system. By contrast, all viruses, many bacteria and some parasites live and replicate inside cells of the host. The type of response that develops against an intracellular pathogen is quite different from the response to an extracellular pathogen because the cells of the immune system recognise, not the infectious agent per se, but the infected cell of the body. Of course, to reach the host cell, intracellular pathogens have to travel through the blood or tissue fluids, and at this time they are susceptible to the immune defences which recognise extracellular pathogens. This characteristic, where different stages of infection are controlled by different immune defences, is typical of very many infections. • From your knowledge of malaria, what stages of the life cycle would you expect to be recognised by antibodies that protect against extracellular infections and which stages would be susceptible to lymphocytes that recognise infected host cells? • Antibodies can recognise the sporozoite as it passes through the blood to the liver cell following first infection and later on different antibodies act against the merozoites as they move between red blood cells. Lymphocytes recognise infected liver cells and, in some circumstances, the infected red blood cells.

Phagolysosome

Vesicle formed by the fusion of lysosomes with a phagosome. Here the ingested material is degraded, indigestible substations remain as residual bodies.

Antigen processing and presentation

We now look at how the antigenic polypeptides come to be associated with MHC molecules - a process called antigen processing - and starting with the class I pathway. All nucleated cells express MHC class I molecules and present antigens via this pathway. • Why is it advantageous for all nucleated cells to present antigen via the class I pathway? • Any nucleated cell of the body may become infected with a virus. In order to be surveyed by Tc cells every cell needs to present antigen. The MHC class I molecules carry out this function, so every cell has them. Within cells, an organelle called the proteasome takes protein molecules that the cell has tagged for breakdown and cuts them into polypeptide fragments. All cells turn over and renew their proteins, so this is a perfectly normal cellular function. Transporter molecules take some of these fragments and carry them into the endoplasmic reticulum, where MHC class I molecules are synthesised. Each peptide is loaded into the cleft of one MHC molecule, where it binds specifically, but non-covalently to amino acid residues around the cleft. Like antigen-antibody binding, the antigenic peptide and the MHC molecule form multiple non-covalent bonds, and the shape of the peptide is complementary to the shape of the antigen-binding cleft. The MHC class I molecules with their bound antigenic peptides are now transported to the cell surface (Figure 2.18). Once they have arrived at the cell surface, the antigen-loaded MHC molecules can interact with the T cell receptor on Tc cells. Once again the interaction is non-covalent. A trimolecular complex is formed in which the antigenic peptide occupies the groove on the MHC molecule and the TCR interacts with residues on both the antigen and the MHC molecule. • If a mutation in a gene encoding an antigen causes an amino acid change in an antigenic peptide, do you think that the change in the peptide would affect its ability to form the trimolecular complex of MHC molecule/antigen/TCR? If so, what would be the effect, and how would it be mediated at a molecular level? • Changes in the antigenic peptide often, but not always, affect the formation of the trimolecular complex. Mutation of residues involved in the binding to the MHC molecule may prevent the antigen from binding to the groove on that particular MHC molecule. Likewise, mutation of residues sticking out of the groove can prevent the TCR from binding to the MHC/antigen complex. Put simply, if an antigenic peptide has mutated it is likely that a T cell that recognised the original peptide is no longer able to do so, or will do so less efficiently. The CD8 molecule (a characteristic marker of Tc cells) is also involved in the interaction between the T cell and a cell expressing the class I molecules. It binds to a site on the MHC class I molecule, in one of the domains adjoining the membrane, that is, distant from the antigen-binding cleft (Figure 2.19). There are now four molecules interacting at the cell surface. CD8 is important in initiating the intracellular signals that activate the T cell. It does this by bringing an enzyme into proximity with the TCR that phosphorylates CD3 (which you may remember is part of the T cell receptor). A complex series of interactions follows that leads to the activation of the T cell. If a cytotoxic T cell recognises the antigen presented by the MHC molecule as foreign then it is able to kill the target cell. The way in which it does this is rather subtle. In fact, it instructs the target cell to commit suicide via a process called apoptosis. This process will be examined later in the module, when you learn about antiviral defences. We will continue here by looking at the class II pathway, and comparing it with the class I pathway. Antigen presentation via the class II pathway is carried out by dendritic cells, mononuclear phagocytes and B cells, the principal cells in the body which express MHC class II molecules. However, in some circumstances, particularly at sites of inflammation, and in response to IFNγ, cells from the tissues (not leukocytes) may also be induced to express class II molecules. However, such tissue cells are often less effective at presenting antigen. The macrophage will be used here as an example of an antigen-presenting cell. Macrophages that have degraded microbial antigens in their phagosomes can return the antigenic peptides to the cell surface for presentation to Th cells. The MHC class II molecules are synthesised, like the class I molecules, on the endoplasmic reticulum with a chain (Ii) blocking their combining site. From here, vesicles bud off, carrying the class II molecules to an acidic endosome (vesicle) compartment within the cell called the MIIC compartment. Vesicles containing degraded antigenic peptides from the phagosome fuse with the MIIC compartment. Within the MIIC compartment the antigenic peptides are loaded onto the class II molecules with the aid of another MHC-encoded molecule called DM. At this stage, the Ii chain is cleaved into a fragment called CLIP and replaced by the antigenic peptide. Class II molecules are synthesised with a polypeptide occupying the antigen-binding cleft, and this is displaced and degraded as the antigenic polypeptides are loaded. The MHC class II molecule loaded with antigenic peptide is now transported to the cell surface to present antigen to Th cells. The process of loading both class I and class II molecules with antigen is complex and involves a number of other molecules, but this goes beyond the scope of the module. The peptides that have been loaded onto the class II molecules are trimmed to size by proteases. Then the MHC molecule, loaded with its antigenic peptide, moves towards the cell surface to be expressed on the plasma membrane. In practice, a single cell will express thousands of MHC molecules loaded with a variety of different peptides. The process is illustrated diagrammatically in Figure 2.20. Once they have arrived on the plasma membrane, the MHC/antigen complex can be recognised by receptors on helper T cells. In this case the CD4 molecule, which is a characteristic marker of the Th cell, binds to part of the MHC class II molecule in an analogous way to the binding of CD8 to MHC class I. Indeed, the way in which Th cells and Tc cells recognise antigen is very similar, although the ensuing reactions are quite different. At this point you should review your understanding of MHC molecules and their role in antigen presentation by working through the self-assessment questions below. Although recognition of an antigenic peptide is the first requirement to activate a Th cell, other interactions are also required before it will divide or release its cytokines. The most important additional interaction is 'costimulation'. Put simply: • if a T cell recognises antigen/MHC and receives a costimulatory signal, the cell will be activated • if the cell sees antigen but does not get a costimulatory signal, it will not be activated. Important costimulatory molecules which are expressed on antigen-presenting cells are B7-1 and B7-2 (CD80 and CD86 respectively), which interact with CD28 present on the T helper cell (Figure 2.21). The costimulatory molecules are typically expressed on mononuclear phagocytes, and are increased by stimulation with microbial components, such as lipopolysaccharide or by cytokines which are produced in inflammation. The T cell integrates a signal from its antigen receptor (TCR) and CD28 within the cell in order to become activated. • What advantage is there (if any) in the T cell requiring two signals to become activated, i.e. correctly presented antigen and then costimulation? • Antigen-presenting cells present antigen all the time and mostly the antigens are harmless molecules, so an immune response is not appropriate; it could even be damaging. However, if infection or inflammation occurs, an immune response is appropriate, and this is when the costimulatory molecules are most highly expressed. Having two signals is a fail-safe device, so immune responses are only promoted when they are appropriate. The association of the TCR and CD28 on the T cell causes phosphorylation of the TCR, which initiates a signalling cascade, causing the activation of transcription factors and potentially stimulating cytokine synthesis and cell division (Figure 2.22). While this discussion has concentrated on the interactions which occur between Th1 cells and mononuclear phagocytes, an analogous set of costimulatory actions takes place when Th2 cells interact with cells, and we shall return to this later when we look at how T cells help B cells to make antibodies.

Antibody responses

When an individual encounters an antigen that has already been contacted previously, the production of antibody is greater than on the first encounter. These are called primary and secondary antibody responses (Figure 2.7). The secondary response differs from the primary in several ways: • the lag time is reduced • the overall level of the antibodies is much higher • the antibodies persist for longer • there is a switch from IgM production to IgG • the affinity of the antibodies is generally higher. All of these effects mean that the secondary response is more efficient than the primary one, and this is the basis of vaccination. Immunisation aims to induce a primary antibody response using a harmless agent (e.g. an attenuated strain of a microorganism which is not pathogenic), so that if the real pathogen is encountered, a strong secondary immune response will develop immediately which can attack the pathogen before disease develops.

Recognition of intracellular bacteria

You have already encountered some of the pattern recognition molecules (Section 2.1), including the toll-like receptors which allow macrophages to recognise products of extracellular bacteria, but how can a cell recognise an intracellular pathogen? There are a number of cytoplasmic molecules that recognise pathogen products and activate the cell's defences. These receptors belong to the NOD family (nucleotide oligomerisation domain), as they share a common domain. For example, the protein NOD-1 recognises peptidoglycans from Shigella flexneri and some strains of E. coli. When the peptidoglycan binds, it activates transcription factors that induce a number of genes, including those for MHC class II, the costimulatory B7 molecules (CD80/CD86) and cytokines including TNFα and IL-1β. Consequently it enhances the ability of macrophages to present antigen to T cells (Figure 4.7). NOD proteins are also expressed in epithelial cells of the gut and this allows them to sense whether the epithelial layer has been invaded by bacteria. Defects in NOD-2, which recognises products of bacterial cell walls, are associated with Crohn's disease, a chronic inflammatory condition which affects the intestine. Antigen presentation by MHC class I molecules is also very important in allowing a T cell to recognise whether a cell has become infected. Figure 2.18 in Unit 2 of this block illustrates how the cell samples the internal proteins and presents them at the cell surface. In addition to MHC class I molecules, many cells express structurally related molecules that directly present microbial products. For example, CD1b is one member of a family of four molecules which have a similar overall shape to MHC class I molecules, but with a deep antigen-binding pocket that binds mycolic acid, a component of the mycobacterial wall. Another family member (CD1d) presents lipid antigens from malaria and trypanosomes.

Mycobacteria

cause the diseases TB and leprosy and survive in macrophages. This group of pathogens has particularly tough cell walls that allow them to survive indefinitely within macrophages, where they can divide slowly. Immunity mediated by Th1 cells is again particularly important, and one defence used by the bacteria is to interfere with the signalling systems inside the macrophage, so that they cannot respond to IFNγ. The countermeasures that a bacterium uses are selectively directed at just those immune defences which are directed against that bacterium.

Routes of infection

Despite the differences between pathogens, there are only a limited number of routes by which infection can enter the body. Skin: Some infections remain superficial (e.g. the fungi which cause athlete's foot) and normal intact skin is a very effective barrier against infection, since very few organisms are able to penetrate it. If, however, the skin is breached by a wound or a burn, the underlying tissues may become infected. Bacterial infections of wounds can be very serious, for example, hospital-acquired infections following surgery. Viruses too can enter the body following damage to the skin. For example, the rabies virus carried in saliva enters the body at wounds caused by the bite of a rabid animal. It does not require a major wound to allow infection to enter the body across the skin. Splinters can cause infection with the bacteria that cause tetanus, and the bite of an infected insect is sufficient to cause the transfer of diseases such as malaria or yellow fever (a viral infection). Mucous membrane: A major route for infection is across the mucous membranes of the respiratory tract, the gut and the genitourinary tracts. These membranes have a variety of unspecialised mechanisms that reduce the chances of pathogens attaching to them. These include the production of mucus, which traps many pathogens, and cilia on the surface of the cells that move the mucus up the bronchial tree to where it can be swallowed. Acid in the stomach and enzymes in the gut also have antimicrobial actions. The local bacterial fauna affords another important method of non-specific protection for these sites. The gut and vagina have their own normal collection of harmless microbes, which can compete with pathogens, so in some cases disease may develop if the normal balance of bacteria and yeasts at these sites is disturbed. Taken together, the non-specific barriers to infection, illustrated in Figure 1.4, are essential adjuncts to specific immune responses. Gut cavities: Many pathogens that enter the body do not cross the mucous membranes into the tissues. For example, the parasitic worms that infest the gut, enter the body by ingestion, and live in the intestine but, as they are lying within the gut, they are still effectively outside the tissues of the body. Bacteria such as cholera produce devastating damage, but they too do not enter the tissues, and they produce their damage while attached to the cells lining the gut. Many pathogens, however, do get into the body. The infection may be limited to the immediate point of entry. For example, Neisseria gonorrhoeae, which cause the sexually transmitted infection gonorrhoea, first attach to the cells which line the urethra and then enter and infect just these cells (i.e. there is a single, specific site of infection here). Other pathogens distribute more widely, and may move through the blood and tissue fluids, either in suspension or within cells they have infected and these are called systemic infections. Once inside the body such infections will usually provoke a strong immune response, which develops within the lymphoid tissues. • If you think about the major routes of infection, where in the body would you expect most lymphocytes to be located? • Most of the body's lymphocytes are located in lymphoid organs that are associated with the mucous membranes of the respiratory, gastro-intestinal and genitourinary tracts.

IgD

A class of antibody that is involved in the activation of B cells, but does not have a direct role in protecting the body against pathogens.

IgE

A class of antibody that is involved in the development and control of inflammation.

IgA

A class of antibody that is prevalent in mucous secretions, and protects against infections in the gut, respiratory tree and genitourinary tract. When considering how bacteria invade the body, you have learned that IgA protects the mucous membranes and prevents bacteria from attaching to these surfaces. B cells that produce IgA tend to localise in mucosa-associated lymphoid tissues (MALT), but the antibody they produce needs to be transported across the epithelium to reach the mucosal surface (Figure 4.1). IgA becomes bound to the poly-Ig receptor on the tissue side of epithelial cells. The bound IgA is transported across the epithelium in vesicles and is released at the mucosal surface, by cleavage of the poly-Ig receptor. The segment of the receptor that remains bound to the secreted IgA is the secretory component, which helps prevent degradation of the antibody by enzymes.

IgM

A class of antibody that is the first to be produced in an immune response.

Cathelicidin

A family of antimicrobial peptides found in lysosomes in macrophages and polymorphonuclear leukocytes (PMNs).

Helper T cells (Th cells)

A functional subclass of T cells, which can help to generate cytotoxic T cells, cooperate with B cells in the production of antibody responses or can activate macrophages. Helper cells express CD4 and recognise antigen presented on MHC class II molecules. They can be subdivided into Th1 cells, which activate macrophages, and Th2 cells, which promote B cell division and differentiation.

Ficolins

A group of opsonins that recognise carbohydrate PAMPs.

Dendritic cells

A set of cells present in tissues, which capture antigens and migrate to the lymph nodes and spleen, where they are particularly active in presenting the processed antigen to T cells. Dendritic cells can be derived from either the lymphoid or mononuclear phagocyte lineages. In addition to macrophages and B cells, there is another group of cells called dendritic cells that are very effective in presenting antigen to T cells. In the context of immunology, dendritic cells are a specific group of leukocytes, derived from stem cells in the bone marrow. The term 'dendritic' describes the shape of a cell that has numerous filamentous processes. Since many cells in the nervous system are dendritic in shape, neuroscientists often use this term descriptively of nerve cells, but this is not the same as a leukocytic dendritic cell. Dendritic cells are very effective at presenting antigens to T cells, including naïve T cells, which have not previously been activated by antigen, something that macrophages and B cells do less well. Typically, dendritic cells are found in lymphoid tissues, where they are surrounded by T cells. Cells that belong to this lineage are also found in the blood, in very small numbers, and in the lymph. Dendritic cells can also occur in non-lymphoid tissue. For example, dendritic cells are found in the skin where they are called Langerhans cells. If an antigen contacts the skin, they can take it up, migrate out of the skin, and pass through the lymphatic channels to the local lymph nodes carrying the antigen. They then process the antigen and present it to T cells. Dendritic cells localise in the T cell areas of lymph nodes, in order to present their antigen to the T cells there.

Opsonin

A soluble molecule that binds to a pathogen or antigen and promotes its uptake by phagocytes.

Large granular lymphocytes

Although the majority of lymphocytes in the body are B cells or T cells, 15% of the lymphocytes express neither a B cell receptor nor a T cell receptor. Over the years these cells have had many different names, but they are most often described according to their appearance, as large granular lymphocytes (LGLs). It is only recently that the function of these cells has become clearer. It had been noted that these cells were sometimes able to kill tumour cells or cells which had become infected with virus, i.e. they acted as cytotoxic cells (Figure 1.9). As they performed this function without appearing to use an antigen receptor they had been described as natural killer (NK) cells but are now known to be an additional immune defence for recognising and dealing with infected cells. The lymphocyte recognises molecules on the surface of the target and can use a number of mechanisms to damage or kill it. A single lymphocyte can successively kill several target cells. However, they use a different recognition system from cytotoxic T cells. You can think of cytotoxic T cells and natural killer cells as the police and the secret police. They both have the same function of protecting the body from subversion by viruses, but they go about it in different ways.

Follicular dendritic cells (FDCs)

Antigen-presenting cells present in the B cell areas of lymphoid tissues, which retain stores of antigen.

Antibody forming cells

B cells are antibody-producing cells. We have already noted that they have antibody molecules on the cell surface which act as their antigen receptor. If a B cell binds to antigen and receives appropriate additional signals from Th2 cells it is driven to divide and differentiate, producing numerous plasma cells, a process which takes place primarily in the lymphoid tissues. Plasma cells produce a secreted form of antibody, which has an identical binding specificity to the surface antigen receptor on the original B cell from which the plasma cell was derived. It differs in that it lacks the portion of the molecule that keeps the B cell receptor bound to the cell membrane. Plasma cells are essentially antibody-producing factories. They live for a few weeks and then die. Their secreted antibodies are important components of the immune defence system. • Most B cells and plasma cells die after a few weeks. Most T cells also only survive for a few weeks. Most phagocytes die within days of production, and this is all perfectly normal. Why it is necessary to have such enormous levels of cell death in a normal immune system? • Recall that phagocytes are produced at the rate of 108 per minute, and that clones of lymphocytes can divide every 6 hours during an immune response. In order for the immune system to respond to infection (i.e. adapt by producing more cells) but still remain a constant size, cell production is balanced by cell death over longer periods of time.

B cell receptor

BCR The B cell surface immunoglobulin that, with its associated signalling molecules, CD79a and CD79b, forms the receptor complex.

Bacterial infection

Bacterial infections may be extracellular or intracellular depending on the species, and this determines, to a large extent, the type of immune response that is appropriate to deal with the infection. • Give some examples of extracellular bacterial infection and intracellular infection. What types of immune response would you expect to be effective against these two types of infection? • Staphylococci, streptococci, pneumococci, Haemophilus and clostridia are predominantly extracellular pathogens. Mycobacteria and many Salmonella species live inside cells. In considering effective immune responses, recall that soluble mediators (antibodies, complement, pentraxins, ficolins, etc.) are more effective against extracellular targets, whereas cytotoxic cells (Tc cells and NK cells) can recognise infected target cells.

reactive oxygen intermediates (ROIs)

Bactericidal metabolites produced by phagocytic cells, including hydrogen peroxide, hypohalites and singlet oxygen.

MALT

Because the mucous membranes are such important points of entry for pathogens, these sites have large concentrations of specialised lymphoid tissues to protect them. You will probably be familiar with tonsils, which are lymphoid organs found on the sides of the throat, and perhaps also know of the adenoids that lie in the nose. These are both examples of mucosa-associated lymphoid tissues (MALT). Generic term for lymphoid tissues associated with the gastrointestinal tract, bronchial tree and other mucosa.

Antibodies

B cells use a cell surface antibody molecule to recognise antigens, and if they become activated they will go on to produce a secreted form of that antibody. Secreted antibodies are essential components of immune defence, performing a variety of functions, but the primary function is always to recognise and bind to antigen. In fact, there are several different types of antibody, described as antibody classes. Each class of antibody has a distinctive structure and function. Antibodies are also called immunoglobulins (abbreviated to Ig) and the five antibody classes are called IgA, IgG, IgM, IgD and IgE. Briefly, the function of the different classes is as follows. • IgA is the main antibody in mucous secretions. It protects against infections of the gut, respiratory tree and the genitourinary tract. • IgG is the main antibody in blood and tissue fluids. It can directly neutralise many toxic molecules, prevent bacteria and viruses from attaching to cells, and it acts as an opsonin. IgG has many other functions in immunity, including protection of the fetus and newborn, since it is the only antibody class which crosses the placenta. • IgM is the first antibody to be produced in an immune response. • IgD is involved in the activation of B cells, but does not have a major direct role in defence against pathogens. • IgE is involved in the development and control of inflammation. We return to the functions of antibodies in detail later, but first we look at their structure and how it is related to function. All antibodies have the basic four-chain structure, which you encountered in Unit 1 (Figure 1.7). IgG can be taken as a prototype. It is the heavy chains of the molecule that determine the class of the antibody. So IgG molecules have one type of heavy chain (γ) while IgA molecules have another (α), and so on. The light chains of IgG are folded into two globular domains and the heavy chains into four domains. Each domain is stabilised by disulfide bonds. Looking at the amino acid sequence of antibodies that are derived from different clones of B cells shows that the domains lying at the distal tips of the molecules (i.e. the ends of the two arms) are highly variable and are therefore called V-domains. It is these V-domains, one from the heavy chain (VH) and one from the light chain (VL), which form the antigen-binding sites (Figure 2.2). Antibodies can be divided into two regions, the Fab region and the Fc region. The antigen-binding fragment of the antibody (the Fab region), consists of two domains from the heavy chain and two from the light chain; the Fc region can bind to receptors on different populations of leukocytes. Closer examination of the structure shows that the variability within the V-domains is mostly located in loops of polypeptides found clustered at the tip of the molecule. There are three of these loops in the VH domain and three in the VL domain, and these six loops (three from the heavy chain and three from the light chain) determine the antigen specificity of the antibody (Figure 2.3). So, the binding site of an antibody is formed by six highly variable loops, which are hence called hypervariable regions. Note, however, that since any single antibody has identical heavy and light chains, it will have two identical antigen-binding sites. It is the amino acid sequence in these hypervariable loops that determines the structure of the binding site and hence which epitope an antibody will recognise. The remaining domains of the antibody are much less variable, and so are called constant domains or C-domains. However, even the C-domains vary to a limited extent because, as noted above, the different antibody classes have different heavy chains. Also, you know that each of these antibodies can be produced as either a cell surface form that acts as a receptor on the B cell, or as a secreted form. These two types vary in the part of the molecule that attaches to the cell surface. Also of interest is the hinge region, which allows the molecule flexibility. This means that the two antigen-binding sites are not fixed in relation to each other, but can independently attach to two identical epitopes, such as might occur on the surface of a virus or bacterium. Now view the animation of an IgG molecule (Video 2.1). You should be able to see the overall symmetry of the molecule, the heavy and light chains, the domains, the hinge region and the two antigen-combining sites. The heavy chains are in dark blue and dark green. The light chains are light blue and light green. The two antigen-combining sites are at the ends of the arms of the Y-shaped molecule, and are each formed by V-domains of one heavy and one light chain. IgG has been used as the prototype here, but you should be aware that other classes of antibodies have different heavy chains. Sometimes the heavy chains can have five domains instead of four (IgM and IgE), but a more striking difference is that IgA and IgM are usually produced as polymeric forms of the basic four-chain structure (Figure 2.4). IgA is usually dimeric and thus has four antigen-combining sites, while IgM is pentameric and has ten. Although the diversity of antibodies may seem complex, the principle underlying antibody structure is very simple: one part of the molecule (V-domains) binds specifically to epitopes on an antigen; other parts of the molecule interact with cells or other immune defences. In other words, antibodies act as adapters. • What other molecules in the immune system can act as adapters? • Pentraxins, ficolins and collectins of the innate immune system (Figure 2.1) act as opsonins (adapters) allowing macrophages to recognise bacteria. When an antibody binds to an epitope the two molecules are seen to have complementary shapes. The shape of the epitope is determined by the molecular groups present on the surface of the antigen, while the shape of the antibody's binding site is determined by the amino acid residues in the hypervariable loops of the heavy and light chains. In addition to having complementary shapes, the antibody forms many weak non-covalent bonds (e.g. hydrogen bonds) with the epitope. The bonds that are formed depend on the molecular groups on the antigen and antibody. Although each individual non-covalent bond is weak, the combination of bonds produces a strong overall bond. Video 2.2 is an animation of an antigen interacting with IgG-Fab. The model shows just one part of the antibody - a light chain (light blue) and part of the heavy chain (dark blue), bound to an antigen (green). The antigen and antibody interact with each other over an extended area that involves 16 amino acid residues on the antigen and 17 on the antibody, some from the light chain and some from the heavy chain. Most of the contact residues in the binding site come from the hypervariable loops on the antibody. • If one of the amino acid residues forming an epitope on an antigen was mutated, what effect do you think this would have on the ability of the antibody to bind to it? • It would affect the complementarity of the antigen and antibody shapes. It might also result in the loss of one of the non-covalent bonds between the two molecules. This might completely destroy the ability of the antibody to bind to the antigen, or at least it would reduce the strength of the bond between the two molecules. If an antibody loses its ability to bind to an antigen, it is ineffective in immune defence. • If a virus mutated one of its surface antigens, what effect would this have on the ability of that virus to survive in the host? • If the mutation affects part of the virus which is recognised by an antibody (an epitope), the antibody will bind less well and the mutated virus will be at a selective advantage as it can evade the immune response (for a while at least). If, however, the mutation affects an area outside the epitope, there is probably no advantage to the virus. (If the mutation adversely affected some critical function of the virus, e.g. attachment to cells, this would put the mutated virus at a disadvantage against the unmutated virus.)

B cell antigen presentation

B cells, like macrophages, take up antigen, process it and present it on the cell surface in association with MHC class II molecules. They do this by endocytosing antigens that have bound to their cell surface immunoglobulins. Because immunoglobulins bind specifically to antigen, this means that a B cell selectively concentrates the antigen to which it responds and thus presents just this antigen to T cells (Figure 3.2). • How do B cells and macrophages differ in the antigens that they present? • B cells and macrophages both present the antigens which they have taken up. A macrophage has many receptors, which allow it to take up a wide variety of antigens, such as bacteria and fungi, microorganisms that have been opsonised by antibody and complement as well as immune complexes. Therefore, a macrophage can present peptides representing many different antigens. In contrast, the antibody on the surface of a B cell binds to just one antigen and so only peptides derived from this antigen are presented. B cells process antigen in a similar way to macrophages; that is, they break it down into fragments which are then combined with MHC molecules in the MIIC compartment. • What determines whether a fragment of an antigen can be presented to the T cell? • The MHC class II molecule: if a peptide can bind in the groove on the class II molecule, it can be presented. Antigenic peptides form non-covalent bonds with amino acid residues lining the groove and so the structure of the MHC class II molecule is critical. As MHC molecules are structurally very variable, they will differ in what antigen peptides they can present. This affects the ability to make an immune response and consequently an individual's susceptibility to particular diseases. One contrast in the way in which cells process and present antigen is that B cells recognise one part of the antigen (the epitope), while T cells (recognising the same antigen) often recognise an antigenic peptide, and this is usually a different region of the antigen. It all depends on which peptides are presented. Here we have two simple observations: 1. T cells and B cells recognise different parts of the same antigen. 2. What constitutes an antigenic peptide depends on an individual's MHC genes. Simple as they are, these facts have far-reaching consequences when it comes to designing vaccines, an idea we shall return to later on. • What advantage might there be in producing low affinity IgM antibodies? Could they serve any function in protection against infection? • Although they are of low affinity they are usually of high avidity, because they are pentamers with ten binding sites and so can bind to antigen via a number of epitopes. IgM antibodies are produced early in immune responses, and they activate complement very efficiently, so would be particularly useful in the early stages of an infection. B cells that respond to such polymeric antigens do not require help from T cells in order to differentiate and synthesise secreted antibodies. They are therefore called T-independent antigens, to distinguish them from T-dependent antigens, which require both T cells and B cells to recognise the antigen. There are several important differences between T-dependent and T-independent antigens, listed in Table 3.1. Note in particular that T-dependent antigens induce high affinity IgG, IgA and IgE antibody production. In other words, T cells are required for antibody class switching (see the later parts of Section 1 for discussions of switching, and refer to Unit 2 Section 3.6 for a description of the functions of different antibody classes).

Lectin receptors

Cell surface receptors that bind to carbohydrate units.

Mast cells

Cells found distributed near blood vessels in most tissues, which are full of granules containing inflammatory mediators.

Cytotoxic T cells

Cells that can kill virally infected targets expressing antigenic peptides presented by MHC class I molecules.

Peyer's patches

Collections of lymphoid cells in the wall of the gut which form a secondary lymphoid tissue.

Lymphocytes

Cells of the immune system are highly mobile within the body. They move between the lymphoid organs and other tissues of the body, using the blood and lymphatics as routes of migration as you can see in Figure 1.2: • lymphocytes leave the primary lymphoid (1) • they enter the blood and colonise the secondary lymphoid tissues by crossing specialised endothelial cells that line the blood vessels in these tissues (2) • from there they can return to the blood via the lymphatics - the lymphatic system is a network of blind-ended vessels that permeate all tissues except the brain (3) • lymphocytes which have been activated following an encounter with antigen migrate more frequently through other tissues of the body (4) • but can return from there to the secondary lymphoid tissues via lymphatics (5) • lymphocytes can also migrate into and out of the spleen (6) • mononuclear phagocytes migrate from the primary lymphoid tissues to blood and then to other tissues, but can also recirculate via the lymphatics (1, 4, 5, 3) • the other principal type of phagocyte, the neutrophil, makes a one-way journey from the bone marrow to the tissues (1, 4). This means that the population of leukocytes seen in the blood is just that group of cells which is in transit from one place to another, and they constitute a tiny proportion of the whole. Infection can affect any organ of the body and, as you have seen in Block 1, infectious agents can enter the body by a number of routes. For this reason, lymphoid organs are strategically sited throughout the body to intercept the spread of infection, and leukocytes migrate through all the other tissues of the body to control any infection which has become established. Lymphocytes are the key cells controlling immune responses. They are responsible for the initial recognition of infectious agents and they organise an appropriate response against them. In fact lymphocytes do not recognise the whole of an infectious agent, they recognise antigens, which are individual molecules or fragments of molecules. The lymphocytes have particular receptors - antigen receptors - on their surface which allow them to recognise the antigen, and these receptors are clonally distributed. This means that an individual lymphocyte has just one type of antigen receptor, which recognises just one antigen. However, although each lymphocyte can only recognise one antigen, there are roughly 1012 lymphocytes in the body, and the population as a whole can recognise millions of different antigens - enough to recognise any molecule that the microbial world can throw at the immune system. • If you consider a single antigen - say the haemagglutinin from influenza virus - how many lymphocytes are there likely to be in an individual that can recognise it? Make an estimate on the assumption that the immune system can recognise 107 different antigens and there are 1012 lymphocytes in the body. • The number is relatively small, between a few thousand and a few million in an individual who has never been exposed to the antigen previously. From the figures above this is, on average, 1012/107 = 105, although the range is very great. Antigenic molecules are generally large and complex, so that they have several potential antigenic regions, which are recognisable by different clones of lymphocytes. In addition, each pathogen will have several, or indeed many, antigenic molecules. Leukocytes that specifically recognise and may react against antigens. The three principal populations are T cells, B cells and large granular lymphocytes (LGLs).

Phagocytes

Cells that can internalise large particles or microbes. In the presence of serum yeast cells cause the production of peptides that attract macrophages (chemotaxis). The process by which cells internalise material by incorporating it into intracellular vacuoles is called endocytosis. If a cell takes up a large particle, such as a bacterium or yeast cell, it is called phagocytosis (Figure 1.10). The large particle, for example a yeast cell, is enclosed within a membrane-bound vesicle called a phagosome. Lysosomal granules fuse with the phagosome causing their contents to be poured into the phagosome, and producing a phagolysosome. Ultimately, opsonised bacteria, viruses, unicellular parasites and immune complexes are taken up for destruction by phagocytes. These may be the neutrophils that are mobilised to areas of acute infection and inflammation. It could be the fixed mononuclear phagocytes that reside in the tissue, or the mobile macrophages that are recruited during infection. Typically, macrophages are more prevalent at sites of chronic infection. These cells are very well equipped to deal with pathogens; they contain an arsenal of molecular killing systems that they can turn onto pathogens they have phagocytosed. Mononuclear phagocytes also secrete a large number of molecules, including cytokines, which bolster the local immune defences. These cells do not work in isolation: in particular, macrophages are very much dependent on Th1 cells for recognition of the pathogen and require cytokines released by Th1 cells in order to develop their full range of effector functions. This process is called macrophage activation. Both macrophages and neutrophils can phagocytose and destroy pathogens, but macrophages, as long-lived cells, carry out many functions that neutrophils do not, and neutrophils have one or two special antibacterial defences that macrophages do not possess. The first step in phagocytosis is binding to the pathogen that is to be internalised (Figure 4.3). You have already seen how important antibody is in this process, and macrophages have two types of receptor for IgG: FcγR1 and FcγRIII. They also have three different types of receptor for activated complement. The complement receptors (CRs) that are present on macrophages are designated CR1, CR3 and CR4, and these allow the cell to bind to anything with C3 fragments bound to the surface. (Two of these molecules, CR3 and CR4, are integrins, and CR3 doubles up as an adhesion molecule used for migration across the endothelium.) The receptors allow the cell to target anything that has been identified by antibody or complement, and when they bind to targets they promote phagocytosis and activation of the killing mechanisms described in the following section. • Macrophages also have receptors that recognise generic structures found on microbial pathogens or on dead cells. Name some of these receptors. • These are pattern recognition receptors (PRRs) and include the lectin-like receptors that bind to microbial components and the scavenger receptors which bind to cell debris and dying cells. Once the pathogen has bound to the receptors, pseudopods extend around it so that it becomes enclosed in a phagosome - a membrane-bound vesicle, which contains phagocytosed material. The macrophage will now direct its biochemical arsenal towards the phagosome, where the pathogen is contained (Figure 4.4). In the first instance, the cell assembles an enzyme in the phagosome membrane that pumps electrons into the phagosome, thereby generating highly reactive superoxide anions. These can go on to generate further reactive molecules including hydrogen peroxide, and hypochlorite (bleach).

Chemotaxis

Directional movement of cells in response to an inflammatory mediator. Cells are highly sensitive to, and migrate up, concentration gradients of molecules such as C5a.

Innate

Evolutionarily ancient immune defense mechanisms that do not improve with repeated encounter with the same pathogen.

Th1 versus Th2 responses

How does the body 'know' whether to make a Th1 response or a Th2 response? This partly depends on the genetic make-up of the individual and partly relates to the way in which the immune system first encounters the antigen. This in turn is related to how the antigen is first presented to the different populations of T cells. • If you are designing a vaccine for a pathogen, does it matter whether you produce a Th1 response or a Th2 response, or is any type of immune response good enough? Explain your answer. • An appropriate type of immune response is needed. For example, a Th1 response and macrophage activation

Auto-immune disease

Immune defences will be directed against the host's own tissue, producing a so-called autoimmune disease. This is rare, since there are numerous controls on immune responses to prevent it happening, but it can occur in diseases such as rheumatic fever, which are discussed later.

Costimulation

Let us consider the interactions that occur between Th2 cells and B cells. Like the interaction between Th1 cells and macrophages, this involves costimulatory molecules and cytokines. The initial interactions involve adhesion molecules on each cell type, and this allows the B cell to present antigenic peptides on its MHC molecules to the T cell. Costimulation mediated by B7 and CD28 is also important. However, in the T cell-B cell interaction, an additional pair of costimulatory molecules is crucial. After activation, T cells transiently express a molecule CD40L (CD40 ligand) which binds to a costimulatory molecule CD40 on the B cells. Ligation of CD40 sends a strong activation signal to the B cell and induces it to express B7, which acts to costimulate the T cell. Thus there is a mutual activation between the two cell types.

Sepsis (aka septicaemia)

Life-threatening infection characterised by circulation and multiplication of bacteria in the blood.

Mononuclear phagocytes

Mononuclear phagocytes are representatives of the myeloid lineage in every tissue of the body. Some of them are fixed cells and some are mobile within the tissues. For example, in the liver there is a group of fixed phagocytic cells (Kupffer cells) which take up bacteria and cell debris from the blood as it passes through the liver. In the brain, the phagocytic cells are the microglia and they constitute 10% of the total cells in the brain. Every tissue needs a permanent population of phagocytes to clear up debris, and these fixed phagocytes may become activated for defence if the organ becomes infected. In addition, mobile phagocytes are important in patrolling through tissues. The precursor of this lineage is the monocyte, a cell type that constitutes about 5% of the leukocytes in the blood. Monocytes can migrate out of the blood vessels into tissues where they differentiate into macrophages, for example the lung macrophages. Tissue macrophages can differentiate further into distinctive types, depending on the cytokine environment, and the way in which they develop is markedly affected by infectious agents and the immune responses induced against them. Like the neutrophils, macrophages have a powerful but distinct range of mechanisms for killing bacteria and parasites. But unlike the neutrophils they are long-lived and have several important additional functions, one of which is antigen presentation. Mononuclear phagocytes are able to take antigens that they have internalised, process them and present them to Th1 cells. • Recall how T cells recognise antigen, using their T cell receptor. What does the receptor recognise? From this, you should be able to predict two of the events that occur as a macrophage processes antigen for recognition by Th1 cells. • T cell receptors recognise antigen fragments bound to MHC molecules. So the antigen processing involves first the fragmentation of the antigen, and second the combination of antigen fragments with MHC molecules. A lineage of bone marrow derived long-lived phagocytic cells.

Acute phase proteins

Serum protein whose level increases during infection or inflammatory reactions.

Immunorepelents

Signaling molecules that inhibit chemotaxis.

Toll-like receptors

TLRs A group of receptors, mostly located on the plasma membrane, that recognize PAMPs and transduce signals for inflammation.

peri-arteriolar lymphatic sheath (PALS)

The accumulations of lymphoid tissue constituting the white pulp of the spleen.

membrane attack complex (MAC)

The assembled terminal complement components C5b-C9 of the lytic pathway that becomes inserted into cell membranes.

T cell receptor

TCR. The T cell antigen receptor consisting of either an αβ dimer (TCR-2) or a γδ dimer (TCR-1) associated with the CD3 molecular complex. The T cell receptor (TCR) was introduced in Figure 1.7, and it has many similarities to the Fab region of an antibody molecule (see Figure 2.2). It is formed from two polypeptide chains, each of which has two domains, with the outer domains being highly variable between different T cells. Each of the variable domains has three hypervariable loops, so that the binding site for the MHC/antigen complex is principally formed from six hypervariable loops - very similar to a single antigen-combining site on an antibody (Figure 2.17). The hypervariable loops are numbered and shown coming together at the distal tip of the molecule to form the binding site for the MHC/antigen peptide complex. The binding site is principally formed by the six hypervariable loops (1-3) clustered at the top of the molecule, although additional polypeptide loops (4) can also contribute. The letters a-g indicate the successive strands of β-pleated sheet making up each of the domains. The domain structure is similar to that present in antibodies. Also, although the mechanisms have not been covered in detail, the way in which T cells generate their repertoire of different TCRs is very similar to the way in which B cells generate their repertoire of different antibodies. Like the B cells, the T cell receptors are clonally distributed, meaning that each T cell only has one type of receptor, even though the population of T cells as a whole has an enormous range of receptors. The antigen/MHC receptor shown in Figure 2.17 is associated with a group of molecules (CD3) which form a non-variable component of the receptor, which signals to the cell. This molecular model of a T cell receptoris depicted with the polypeptide backbone of the TCR α and β chains as ribbon diagrams. The antigen binding site (hypervariable loops of the V-domains) is at the top of the model. You have seen that T cells recognise antigen fragments presented on MHC molecules using a T cell receptor (TCR) (Figure 1.8). In this section, you will find out more about this process and the functions of antigen-presenting cells. Understanding the cell biology of antigen presentation allows you to understand: • how T cells help B cells to make antibody and activate macrophages to destroy the pathogens they have phagocytosed • why some people are more susceptible to particular infections than others • why infection can lead to secondary autoimmune disease in some people • how some pathogens have evolved to evade immune responses. The events that follow T cell activation depend on cytokine signals, and on where the interactions take place. In lymph nodes or other secondary lymphoid tissues, T cells generally go on to divide. An important cytokine for T cell proliferation is IL-2. When a T cell is activated it synthesises IL-2 receptors and expresses them on its surface, and it also synthesises IL-2. Thus it can respond to the cytokine it produces, or which other T cells in the vicinity are producing. This is an example of autocrine stimulation. With this final signal, the T cell undergoes a number of cycles of cell division (Figure 2.23). Activation of the T cell therefore depends on the availability of IL-2 and whether the cell is expressing IL-2 receptors. The IL-2 receptor itself is not a simple molecule. It can exist in high and low affinity forms. At rest, a T cell expresses just the low affinity form in small numbers, so it takes a high concentration of IL-2 to stimulate the cell to divide. When the cell has been activated by antigen it synthesises an extra chain for the receptor, which in turn then converts the receptor into a high affinity form. Many more of these high affinity IL-2 receptors are produced, so that the cell will respond much more readily to low amounts of IL-2 (Figure 2.24). This means that the response of a T cell will depend, not just on the levels of IL-2 present, but also on the numbers and type of IL-2 receptors. The expression of the IL-2 receptors can be enhanced by other factors, such as the cytokine IL-1. If a T cell is activated in non-lymphoid tissues, it will still produce IL-2 and express IL-2 receptors, but the level of cell division is very limited. On the other hand, in non-lymphoid tissues the other cytokines produced by the T cells such as IFNγ are very important in orchestrating the immune response to infection. Clearly a T cell cannot go on dividing forever. T cells that have undergone several rounds of cell division fall back into resting phase, unless they are once more stimulated by antigen that is presented by an antigen-presenting cell. This is very logical. The immune response is driven by lymphocytes responding to antigen. When the source of the infection and the antigens are gone, the response is no longer needed, and cell division stops. Costimulation, or more precisely the lack of it, determines whether T cells will fall back into resting phase. As a T cell is activated, the CD28 molecule interacts with B7 on the antigen-presenting cell. After a number of rounds of cell division, if the cell has not been restimulated with antigen, the CD28 is replaced with an analogous molecule, CTLA-4, which also binds to B7, but with higher affinity than CD28. So CD28 cannot engage B7 and therefore the T cell does not receive its costimulatory signal. Hence, the CTLA-4 effectively transmits an inhibitory signal to the T cells (Figure 2.25).

Toxic molecule production

The collection of molecules generated is collectively termed reactive oxygen intermediates (ROIs), also known as reactive oxygen species (ROS). They are very toxic for pathogens. Indeed, you will probably have noticed that these are the same kind of molecules that we use out of a bottle when we want to kill bacteria. The macrophage just got there first. Macrophages that have been activated by the inflammatory cytokines IFNγ and TNFα can generate a separate group of toxic molecules called reactive nitrogen intermediates (RNIs). When the macrophage is activated it synthesises an enzyme called inducible nitric oxide synthase (iNOS) and this can catalyse the production (from arginine) of nitric oxide (NO), another highly reactive, toxic molecule. Combination of the ROIs and NO produces another batch of toxic compounds, the peroxynitrites, which can kill fungi, and many other eukaryotic parasites and bacteria. • Children are sometimes born with genetic defects that affect the ability of their macrophages to produce reactive oxygen intermediates. What effects do you think this would have on their ability to fight infection? • Children with these defects are susceptible to serious bacterial infections, particularly with streptococci, staphylococci, pneumococci and Haemophilus. This susceptibility results in pneumonia, infections of the lymph nodes and abscesses in the skin and internal organs. The range of infections is similar to that seen in patients who lack antibodies.

MHC molecules

The class I and II molecules are structurally similar integral membrane proteins (Figure 2.15). They have two domains lying next to the cell membrane, and the distal part of the molecule consists of a grid of β-pleated sheet, surmounted by two long loops of α-helix, forming a cleft - the antigen-binding cleft. The class I molecule has one chain encoded within the MHC that folds into three domains α1, α2 and α3, and a second smaller chain β2-microglobulin (β2m) that is encoded on another chromosome. The antigen-binding site is formed by two loops of α-helix supported on a grid of β-pleated sheet and it is occupied by a peptide of eight amino acid residues. The cleft is closed at each end and can accommodate peptides of eight amino acid residues in length. In class II molecules, however, there are two MHC-encoded peptides and the cleft is open at the ends and can accommodate polypeptides of 12-14 residues. The overall structure is clearly very similar. The molecular model is shown as a ribbon diagram of the polypeptide backbone. The MHC-encoded α-chain is in green and the single-domain polypeptide of β2-microglobulin is in grey. The antigenic peptide (eight amino acid residues) from influenza virus is shown as a space-filling model (blue), in the antigen-binding site at the top of the model. The molecular model is shown with the MHC-encoded α and β chains as ribbon diagrams of the polypeptide backbone. The antigen, shown as a space-filling model (blue) is a 12 amino-acid peptide derived from the nucleoprotein of vesicular stomatitis virus (VSV). There was evidence in the crystallographic data that the MHC class II molecule was a dimer, and the polypeptide backbone of the second MHC molecule is shown as a single strand alongside the space-filling model. When an MHC molecule presents an antigen to a T cell, it actually presents a peptide fragment of the antigen. The receptor on the T cell recognises not just the antigenic peptide but the combination of a specific peptide associated with a particular MHC molecule. In practice this means that the amino acid residues which form the T cell antigen receptor interact with both residues of the antigenic peptide and residues on the α-helices of the MHC molecule. The 'view' which the T cell receptor sees as it approaches the MHC/antigen complex is shown in Figure 2.16 where the antigenic peptide (purple) nestles in the groove at the top of the molecule. The peptide in the class I groove is shorter and is anchored by non-covalent hydrogen bonds at each end. The peptide in the class II groove is longer and is anchored to residues on the MHC molecule along its length. • What advantage is there (if any) in having a variety of different MHC molecules, each with a slightly different structure, but each capable of presenting antigenic peptides? • The peptides which can bind to an MHC molecule depend on the particular allelic variant. If a person has a variety of MHC molecules, their cells can present a wider variety of antigens. This means that their immune system can respond efficiently to a wider variety of pathogens. Antigen presentation mediated by MHC class I and class II molecules has been understood since the early 1980s, but as scientists started to unravel the genome, it became clear that there were many other molecules that resembled MHC class I molecules, for which the functions were often unclear. These are sometimes called non-classical MHC class I molecules, to distinguish them from those encoded by the HLA-A, -B and -C loci, which have been discussed so far. Some of these molecules are encoded in the major histocompatibility complex, but others are encoded in odd places throughout the genome. Interestingly, however, some of these molecules can also present antigen. In particular, one set of molecules called CD1 has a very deep, hydrophobic antigen-binding groove, and they are able to bind to lipid or glycolipid antigens. Long acyl groups can occupy the deep groove, leaving other groups such as carbohydrate exposed to be recognised by the TCR. In addition, one member of this family of molecules is able to bind lipo-arabinomannan. • Where might lipo-arabinomannan come from? • It is a component of the cell wall of mycobacteria. Thus, it seems that whereas the standard MHC class I and class II molecules present polypeptide antigens, some of the non-classical MHC molecules present other types of antigen. First, we must distinguish two different pathways of antigen presentation. The pathway by which all nucleated cells sample their internal molecules and present them on the cell surface is called the class I pathway or internal pathway, because the cell is taking its own intrinsic, internal molecules and placing them on its cell surface. To do this it uses 'class I molecules' encoded within the major histocompatibilty complex (MHC). The presented molecules are reviewed (i.e. potentially recognised) by cytotoxic T cells (Tc cells, see Figure 2.13). In the second pathway, the cell takes up molecules from the outside by phagocytosis and presents them on the cell surface associated with MHC 'class II molecules'. Because the presented molecules come from outside the cell, this is called the external pathway or class II pathway. The presented molecules are reviewed by helper T cells (Th cells, see Figure 2.13). This pathway is used by a more limited group of cells, including macrophages, B cells and dendritic cells. The MHC is a large group of linked genes, located on chromosome 6 in humans. The gene complex was originally identified because of its role in determining whether tissue would or would not be rejected when grafted between individuals, but clearly this is not a physiological role. There are at least 150 genes in this region with a variety of functions, but we shall concentrate on the so-called class I and class II genes that are involved in antigen presentation. Figure 2.14 shows the location of the class I and II genes within the MHC. In humans the gene locus is called HLA, for historic reasons. You will see that there are three class I loci (HLA-A, -B and -C) and three class II loci (HLA-DP, -DQ and -DR). The MHC genes are highly variable between the different loci and there are numerous allelic variants in the population. Since each person has a maternal and a paternal chromosome 6, this means that most people will be able to produce six different class I molecules and six different class II molecules because the genes are codominantly expressed. (The term MHC molecule is usually restricted to just the class I and II molecules encoded within the MHC.)

Classical pathway

The pathway by which antigen-antibody complexes can activate the complement system, involving components C1, C2 and C4, and generating a classical pathway C3 convertase. The classical pathway is activated by immune complexes containing antibodies of the IgM or IgG classes (but not IgG4). The classical pathway is so called because it was the first to be discovered. Activation occurs when antibody binds to the first component of the pathway (C1), which causes the cleavage of two components of C1 (C1r and C1s), which in turn activate C4 and C2 to form a C3 convertase (C4b2a) which cleaves C3.

Immune complexes

The product of an antigen-antibody reaction that may also contain components of the complement system.

Class switching

The process by which an individual B cell can link immunoglobulin heavy chain C genes to its recombined VDJ gene to produce a different class of antibody with the same specificity. This process is also reflected in the overall class switch seen during the maturation of an immune response. In order to understand the processes of immunoglobulin class switching, we need to look at the genes that encode the immunoglobulin heavy chain. Look first at the gene for IgM (Figure 3.5). Like most mammalian genes, it consists of a number of coding segments (exons, shown as labelled boxes) which are separated by non-coding segments (introns). The coding and non-coding segments are transcribed as one long piece of RNA that is then spliced, to bring the coding segments together, forming messenger RNA (mRNA). The diagram shows which part of the gene encodes which part of the protein. Generally, there is one exon for each domain. A small exon (SS) encodes a signal sequence that directs the translation of the IgM heavy chain into the endoplasmic reticulum. Next come exons encoding the variable and constant domains. The exon encoding the variable domain is formed during B cell development in the bone marrow, a process which involves reassortment and recombination of several gene segments, referred to as 'V', 'D' and 'J'. You do not need to know the details of this process, only that the end result is that the gene that encodes the variable domain of a heavy chain is often called a VDJ gene segment. The variable domain of the heavy chain is encoded by one exon (VDJ), and each of the constant domains is encoded by another exon (CM1 to CM4). Two other small exons (M1 and M2) encode the transmembrane segment and intracytoplasmic portions of the polypeptide (m and c). A switch sequence in the DNA is important in switching between production of different antibody classes. Further downstream (3′) of the IgM exons on the same chromosome are genes for the constant domains of the heavy chains of all the other immunoglobulin classes (Figure 3.6). Each of these sets of genes (except IgD) is preceded by a switching sequence. Note, however, that there is only one VDJ exon encoding the variable domain. In naïve B cells, the VDJ exon is transcribed together with the exons for the IgM constant domains, so that IgM is produced. As the B cell develops and with T cell help, a rearrangement of the genes takes place so that genes for one of the other immunoglobulin classes are moved up to replace the IgM exons. This means that the VDJ gene is now transcribed together with constant domain genes for one of the other immunoglobulin classes. In the example shown in Figure 3.6 the gene for IgM lies closest to the VDJ gene that encodes the V-domain. Genes for other heavy chains of immunoglobulin classes lie downstream. (Note that each of these loci will contain several exons.) When a B cell switches the class of antibody it produces, the switching sequence preceding the new gene recombines with the switching sequence preceding the IgM gene, bringing it next to the VDJ gene segment. The intervening genes, including the IgM genes, are lost during this process. This is the genetic event which underlies antibody class switching by the cell and which underlies class switching in an immune response. • What effect will class switching have on the antigen specificity of the antibody which the B cell produces? • There is no effect. The specificity of the antibody is determined by the amino acid sequence in the variable domains which is encoded by the VDJ gene segment. Since this segment stays the same during class switching, there will be no change in the antibody specificity. Now view Video 3.2, which summarises the molecular genetics of how B cells and T cells develop to produce a single type of antibody or T cell receptor, respectively. One event that occurs as an antibody response develops is the switch from production of IgM to other antibody classes IgG, IgE and IgA, and this is called class switching. The interaction between Th2 cells and B cells is essential both for the development of the antibody response and for the class switch, which occurs during a secondary immune response. This phenomenon can be demonstrated in rare individuals who genetically lack CD40L. They have a very high level of IgM in the blood, but low levels of IgG, IgA and IgE. Because the T cells cannot pass the costimulatory signal to the B cells, the B cells fail to switch from IgM production to other classes. This is a key point: • individual B cells can switch from producing IgM to IgG • and this cellular event underlies the switch in antibody classes seen in an immune response. One other thing is notable in individuals who lack CD40L - their lymph nodes lack germinal centres. This gives us an indication that class switching occurs in the germinal centres of lymphoid tissues, which is indeed true.

Immunology

The reason for the complexity of the immune system is, however, easy to understand: infectious agents vary enormously in size, lifestyle, location within the body and in the damage which they produce, so different types of immune responses are appropriate for each group of pathogs. The principal cells of the immune system are leukocytes or white blood cells, which are distributed throughout all the other organ systems of the body, and are also located in specialised lymphoid organs. You will find that immunologists refer to two types of lymphoid tissues. • The primary lymphoid tissues are the bone marrow and thymus, where the cells of the immune system are first produced and trained. • The secondary lymphoid tissues include the spleen, lymph nodes and mucosa-associated lymphoid tissues (MALT) including Peyer's patches (Figure 1.1 and Video 1.1). Note that bone marrow contains mature cells and can therefore also be considered a secondary lymphoid tissue. MALT (boxed descriptions in Figure 1.1) contain mature lymphocytes that can respond to infection. Since many pathogens enter the body through mucosal surfaces, the MALT is particularly important in protecting these potential sites of entry. Lymphoid tissues contain the leukocytes that initiate the immune response. There are numerous different types of leukocyte, but two major groups of cells are the lymphocytes, which are responsible for recognising foreign material, and phagocytes whose main function is to internalise it and destroy it. Taken together, these cells constitute a major organ system of the body. To give you some idea, the bone marrow in an adult produces about eight million new phagocytes every minute. These are released into the circulation in order to patrol other tissues. The production of these cells requires a substantial amount of the total energy used by the body each day. After education within the thymus, T cells circulate throughout the lymphatic system and accumulate inside the lymph nodes, spleen, and other secondary lymphoid tissues. Other types of cells are also present, including B cells and macrophages, both of which can also act as antigen presenting cells. Consider a macrophage. Should it encounter an antigen for which it has no specific receptors, it can still engulf it, using, for example, its lectin-like receptors. This is termed non-specific internalisation. The pathogen bearing the antigen is contained within a phagolysosome in the macrophage. Other vesicles fuse with the phagosome, bringing proteolytic enzymes in. Elsewhere in the macrophage, major histocompatibility complex molecules are being synthesised at the endoplasmic reticulum. MHC class II molecules are transported by vesicles which intersect the pathways of vesicles, released from the phagolysosome. Proteolytic fragments of the antigen bind to the MHC class II molecules in a special compartment, displacing the invariant chain of the class II molecule. Loading is facilitated by another MHC-encoded molecule called DM. The vesicle containing the MHC class II antigen fragment combination migrates to the surface of the macrophage where the contents remain attached, presented to any Th1 cells that may be encountered. A Th1 cell that recognises the particular antigen will bind to the surface group of the macrophage using its T cell receptor, plus a CD4 molecule which recognises the MHC class II part of the presented antigen fragment. This binding alone is insufficient to trigger T cell activation. Other costimulatory molecules already on the surface of the T cell come into play. ICAM 1, (which stands for intercellular adhesion molecule 1) on the macrophage binds to LFA 1 (lymphocyte functional antigen 1) on the T cell while CD2 also on the T cell binds to LFA3 on the macrophage. Meanwhile an interaction between CD80 on the macrophage and CD28 on the T cell delivers a powerful costimulatory signal to the T cell. The macrophage releases cytokines, including interleukin 1, and the T cell makes interferon gamma. In this way, each cell activates the other. The macrophage is put into a more hostile state to destroy any bacteria it has taken up. The T cell's released interferon gamma will activate other as yet uninvolved macrophages, and other cytokines, such as interleukin 2, stimulate T cell replication. The macrophage is not the only cell capable of processing and presenting antigen. B cells are also able to perform this function. A B cell with the appropriate surface immunoglobulin receptor takes up the antigen. There is a huge variety in the types of receptors, so there will be a B cell suited to virtually any antigen. Recognition of most antigens depends on both T cells and B cells, so the B cell must present the antigen to T cells, to determine whether an antibody response is appropriate. The antigen is internalised, and degraded by proteolytic enzymes. These cleave off peptide fragments from the antigen. These fragments then become associated with the MHC class II molecules in a compartment where peptides from the endosome and class II molecules from the Golgi body are brought together. When the two vesicles meet, some of the peptides are able to bind to MHC class II molecules. Which of the peptides bind depends on the genotype of class II molecules available in that individual. The vesicles carry the assembly to the surface of the B cell where it is presented. This antigen-MHC class II complex is recognised by T-helper cells. On encountering it, the Th2 cell binds, using its T cell receptor and CD4 molecule. This is essentially the same as occurs between macrophages and Th1 cells. Where it differs is in the co-stimulatory molecules and cytokines involved. ICAM1 still interacts with LFA1, but is now joined by CD40 which pairs with the CD40 ligand on the T cell, and CD80 which pairs with CD28. CD40 is a molecule which stimulates B cell activation. The Th2 cell now releases cytokines, including interleukin 4 and interleukin 6. The signals cause the B cell to divide and differentiate creating a large population of antibody-producing cells specific to the antigen. These two examples involve leukocytes as antigen presenters. They bind the peptide fragment from the antigen to an MHC class II molecule, and so this is termed the class II pathway. In the case of a tissue cell becoming infected, for example, in the lung or mesopharynx, the infected cell can be its own antigen presenting cell. Within every tissue cell, proteasomes produce peptide samples from all molecules present in the cell, including the cell's peptides, as well as any foreign ones. MHC class I is synthesised by ribosomes on the endoplasmic reticulum. The peptide fragments are transported to the endoplasmic reticulum and loaded onto MHC class I molecules. The MHC plus antigen combination is then presented on the cell surface. Cytotoxic T cells circulate through the entire body after their education in the thymus. Their T cell receptors are specific to any non-self peptide, combined with an MHC class I molecule. When a cytotoxic T cell encounters a tissue cell presenting such an MHC antigen complex, it binds through its T cell receptor and CD8 molecule. This triggers a cytotoxic reaction by the T cell against the infected tissue cell. These may be mediated by direct interactions of surface molecules, or signals may be sent by cytokines. Perforin or granzymes may also contribute to target cell destruction. Any of these alone, or in combination, may instruct the infected cell to die. This mechanism, using the MHC class I protein, is called the class one pathway.

Follicular dendritic cells

We must finally introduce one more type of antigen-presenting cell - follicular dendritic cells (FDCs). These cells are different from dendritic cells and are permanently located in the B cell areas of lymphoid tissues, where they present antigen to B cells. They are important in the process of antibody production, and will be returned to later in the module.

T and B cells

To understand the immune response you need to know more about the lymphocytes. They fall into two major groups - T cells and B cells. B cells develop in the bone marrow, while T cells develop in the thymus - the two primary lymphoid organs. As they develop, each clone of B cells or T cells generates an antigen receptor and expresses it on the cell surface. Each clone of lymphocytes is able to produce a different receptor by a complex process that involves rearrangement of the genes that encode the antigen receptor. Cells that fail to make an antigen receptor because they have been unsuccessful in rearranging their genes die. At this point the surviving cells undergo a process of 'education'. Any cells that recognise self-molecules will either be deleted (eliminated), or turned off, so they cannot make an immune response subsequently. This leaves lymphocytes that are capable of recognising non-self molecules. These educated lymphocytes will now move from the primary lymphoid organs to the secondary lymphoid organs where they are ready to deal with infection. At this stage they have receptors which are capable of recognising an antigen, although they have not yet encountered any infectious agent. These cells are called naïve or virgin lymphocytes. Notice these two points about lymphocyte development: 1. Lymphocytes generate receptors before they encounter an antigen. 2. Cells that recognise self antigens are actually or functionally deleted. This process is not perfect, and some self-reactive lymphocytes do survive. However, reactions against self molecules are also restrained by a number of regulatory mechanisms, involving both lymphocytes and cells from the tissues. If these mechanisms also fail then autoimmune diseases can develop, in which the immune system attacks tissues of the body. This is an important area of clinical immunology, but it is mostly beyond the remit of this module. In a few instances specific infections can trigger autoimmune disease, but in the great majority of infections and in most individuals, this does not occur. The antigen receptors on B cells and T cells are quite distinct. You can see the structures of these receptors in Figure 1.7. The B cell receptor (BCR) for antigen is a cell surface form of antibody or immunoglobulin which consists of two identical heavy chains and two identical light chains, so it is bilaterally symmetrical. The T cell antigen receptor is unsurprisingly called the T cell receptor (TCR) and it consists of two chains of about equal size called α and β on most T cells. Both of these receptors are associated with a set of molecules that signal to the inside of the cell if the receptor binds to its antigen. So, for example, the CD3 molecule that you encountered as a marker of T cells, and the CD79 molecule on B cells, are actually the parts of the receptor complex which transduce the activation signal to the cell. It is absolutely essential to understand that B cells and T cells recognise antigens in different ways. B cells use surface-bound antibodies (immunoglobulins) as their receptor for antigen. Antibodies have two antigen-binding sites that recognise molecular shapes called epitopes that are present on intact molecules (antigens). In contrast, the T cell receptor recognises fragments of antigens that are presented on the surface of other cells of the body. The fragments are presented by a group of molecules that are encoded within a region of the genome called the major histocompatibility complex, and so are called MHC molecules. The function of these molecules is described as antigen presentation. Thus T cells recognise a combination of the antigenic peptide and the MHC molecule on the surface of other cells. Every cell in the body samples its own internal proteins and places fragments of them on the cell. If a cell has become infected, molecules of the pathogen will be placed on the cell surface and can be recognised by T cells. And this is the important point: T cells are responsible for recognising whether a cell has become infected.

Cytokines

We must now briefly introduce the topic of cytokines. This is the generic term for secreted molecules produced by a wide variety of cells (not just leukocytes) and which act as signalling molecules. In this sense they are similar to some hormones. Literally tens of different cytokines have been described, which fall into different categories (see Box 1.2). Fortunately, you do not need to know all these cytokines, but the ones that are particularly important in protective immune responses are highlighted here. Each cell type secretes a particular set of cytokines that depends on the cell type and whether it has been activated. Only cells with specific receptors can respond to any particular cytokine and whether a cell has particular cytokine receptors depends on its type and on its state of activation. To summarise what you have learned about T cell populations: Th1 cells produce a set of cytokines which allow them to interact with phagocytes, while Th2 cells produce cytokines which signal to B cells. Cytokines are small proteins that act as signalling molecules. Because cytokines were originally identified by their activity, they were given a variety of appropriate names. For example, one cytokine that caused T cells to divide was called T cell growth factor. In time, it was found that several different researchers could be working with the same molecule, but had given it different names because they had identified it as having different biological activities. It was therefore necessary to adopt a unified nomenclature. Immunologists now distinguish several families of cytokines and these are listed in Table 1.1, along with their abbreviations. Table 1.1 Cytokine families and their functions Cytokine family Function Interleukins IL-1 to IL-35 involved in signalling between leukocytes and other cells of the body Interferons IFNα, IFNβ and IFNγ involved in protection against intracellular pathogens and inflammation Colony stimulating factors M-CSF, G-CSF, GM-CSF promote the production of different populations of leukocyte Chemokines more than 30 molecules involved in controlling cell migration and activation Tumour necrosis factors TNFα, TNFβ a group of molecules involved in inflammation Transforming growth factors mediators of cell growth Most of the cytokines have specific receptors so, for example, IL-3 binds only to the IL-3 receptor. But some cytokines bind to several receptors and some receptors bind several cytokines. This promiscuity of receptor binding is particularly prevalent with the chemokines. The expression of appropriate receptors determines whether a cell can respond to particular cytokines or not. Receptors are not always present on a cell but can be induced when a cell is activated, or at particular stages of its development. Following T cell-B cell interaction, cytokines come into play. Particularly important is IL-4, formerly called B cell growth factor, which is released by Th2 cells. Activated B cells express an IL-4 receptor and respond to IL-4 by dividing. Other cytokines released by Th2 cells, including IL-5, IL-6 and IL-13, promote differentiation of the B cells into antibody-forming cells and, ultimately, into plasma cells and memory B cells. • The B cell's antibody is a cell surface molecule with a transmembrane region that acts as a receptor for antigen. The plasma cell produces a secreted antibody that lacks the transmembrane region, but is in other respects similar to the cell surface form. The cytokines not only promote the differentiation of B cells, but they can also affect the type of class switch that the B cell makes. For example IL-5 tends to promote switching from IgM to IgE production, in addition to promoting the release of eosinophils into the blood. Because cytokines like IL-4, IL-5, IL-6 and IL-13 are produced by Th2 cells, and because they promote the production of antibodies (and sometimes also allergic reactions), this type of response is often called a Th2-type of response. Macrophages produce and respond to a number of cytokines (Figure 4.5). Most importantly, they are activated by IFNγ, released by antigen-stimulated Th1 cells. This cytokine, once described as macrophage-arming factor, enhances all of the macrophage's killing mechanisms. If the Th1 cell recognises something that the macrophage has internalised and is presenting on its surface, then it will release IFNγ, which in turn enhances the ability of the macrophage to destroy it. Cooperative recognition and activation is crucial in defence against pathogens because there are many pathogens that can survive in macrophages that have not been activated, but which are killed by activated macrophages. IFNγ not only enhances the ability of macrophages to kill pathogens, it also increases their ability to present antigen to the T cells. It does this by inducing the synthesis of MHC molecules and costimulatory molecules. This is another example of a cooperative interaction between the Th1 cells and the macrophages. Macrophages themselves produce cytokines when they have been activated, including tumour necrosis factor-α (TNFα) and interleukin-12 (IL-12), which promotes the development of further Th1 cells. You have already encountered TNFα as a cytokine that induces adhesion molecules on endothelium and thereby promotes cell migration into sites of infection. Related to this, mononuclear phagocytes are important sources of the chemokines that are needed to trigger migration of additional leukocytes to the inflamed tissue. Many cells of the body can respond to TNFα, including the macrophages themselves This kind of response, where a cell produces a cytokine that acts on itself and similar cells in the area, is called an autocrine action. It means that an activated macrophage can signal to neighbouring cells to enhance their killing mechanisms too. Macrophages secrete many other molecules, in addition to the cytokines. For example, macrophages can produce complement system molecules, including C3. Most complement components are synthesised by the liver, but the ability of macrophages to produce them locally is extremely useful since complement molecules will be used up at sites of infection, and complement is required at these sites for opsonisation. • Name a complement component that macrophages can recognise and state how they recognise it. • They have receptors CR1, CR3 and CR4, which allow them to recognise bound C3 fragments (Figure 4.3). Another important molecule secreted by macrophages is the enzyme lysozyme, which is able to cleave peptidoglycans in the cell walls of bacteria. Macrophages constitutively produce lysozyme, and it is found in many secretions. If the cell wall of a bacterium is damaged the bacterium may burst by osmotic shock, and this allows other components of the immune system access to the inner membrane. • Describe whether there is any way in which lysozyme could damage Gram-negative bacteria, which have an outer membrane shielding their cell wall. • Lysozyme cannot normally access the cell wall of Gram-negative bacteria, but if the complement lytic pathway damages the outer membrane, then it allows lysozyme to access the cell wall (see Section 2.3). So complement and lysozyme work in concert.