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Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001.

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Immunobiology: The Immune System in Health and Disease. 5th edition.

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Other signaling pathways that contribute to lymphocyte behavior

Lymphocytes are normally studied in terms of their responsiveness to antigen. However, they bear numerous other receptors that make them aware of events occurring both in their immediate neighborhood and at distant sites. Among these are receptors that detect the presence of infection and receptors that bind cytokines produced by the cell itself or reaching it from elsewhere. In the absence of infection, lymphocyte populations are also kept remarkably constant in numbers. This homeostasis is achieved by a host of extracellular factors that interact with receptors on lymphocytes, the most important of which is the antigen receptor. Other receptors and ligands that come into play include Fas and its ligand, various cytokine receptors and their ligands, and intracellular proteins such as Bcl-2 that modulate survival.

6-15. Microbes and their products release NFκB from its site in the cytosol through an ancient pathway of host defense against infection

As we saw in Chapter 2, pathogens that infect the body are detected by the germline-encoded receptors of the innate immune system. One family of receptors that signals the presence of infection by microbes is known as the Toll family of receptors. The Toll receptor was originally identified in the fruit fly Drosophila melanogaster on account of its role in determining dorsoventral body pattern during fly embryogenesis. Later, it was found to participate in signaling in response to infection in adult flies. A close homologue of Toll has been identified in mammals, and similar proteins are also used by plants in their defense against viruses, indicating that the Toll pathway is an ancient signaling pathway that is used in innate defenses in most multicellular organisms.

In mammals, the Toll pathway leads to the activation of a transcription factor known as NFκB. Microbial substances such as lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, and many other microbes and their products can activate NFκB in lymphocytes and other cells through this pathway. LPS signals through a mammalian Toll-like receptor known as TLR-4, with which it interacts indirectly, through association with another cellular receptor (CD14) for LPS (see Section 2-17). Other microbial substances can interact directly with other Toll-like receptors; gram-positive bacteria, for example, interact with TLR-2. Toll-like receptors signal for NFκB activation through a pathway that seems to operate in most multicellular organisms. The cytoplasmic domain of the Toll receptor is known as a TIR domain because it is also found in the cytoplasmic tail of the receptor for the cytokine interleukin-1 (IL-1R). Ligand binding to the extracellular portion of the Toll receptor induces the TIR domain to bind and activate an adaptor protein known as MyD88. The IL-1 receptor also binds and activates MyD88 on binding IL-1. As the ligands recognized by the various Toll-like receptors and their downstream connections are now fully worked out, we illustrate this pathway in Fig. 6.20 and describe it below; the pathway from the IL-1R is very similar.

Figure 6.20. The transcription factor NFκB is activated by signals from receptors of the Toll-like receptor (TLR) family.

Figure 6.20

The transcription factor NFκB is activated by signals from receptors of the Toll-like receptor (TLR) family. The cytoplasmic domain of TLR-4 is homologous to that of the receptor for the cytokine IL-1 and is called a TIR (Toll/IL-1R) domain. This domain (more...)

At one end, the MyD88 protein also has a TIR domain through which it interacts with the TIR domain of Toll or IL-1R. At the other end of the MyD88 protein is another domain that mediates protein-protein interactions. This is known as a death domain because it was first found in proteins involved in programmed cell death. Through this death domain, the bound adaptor protein interacts with another death domain on a serine/threonine innate immunity kinase (SIIK) known as IRAK, or the IL-1R-associated kinase. This initiates a kinase activation cascade through which two kinases known as Iκkα and Iκkβ are activated to form a dimer (Iκk) that phosphorylates an inhibitory protein known as IκB. This protein is bound in a complex in the cytosol with the transcription factor NFκB and inhibits its action by retaining it there. When IκB is phosphorylated, it dissociates from the complex and is rapidly degraded by proteasomes. After removal of IκB, NFκB enters the nucleus and binds to various promoters, activating genes that contribute to adaptive immunity and the secretion of pro-inflammatory cytokines. Also activated is the gene for IκB itself, which is rapidly synthesized and inactivates the NFκB signal.

The responses activated by this pathway depend upon the cell type and organism concerned: in adult Drosophila melanogaster, Toll-family receptors induce the expression of antimicrobial peptides in response to bacterial or fungal infection. In mammals, as we saw in Chapter 2, the Toll signaling pathway contributes to the initiation of an adaptive immune response by inducing the expression of co-stimulatory molecules on tissue dendritic cells and the migration of these cells from a site of infection to a local lymph node. Here they can function as antigen-presenting cells and provide a naive lymphocyte that recognizes its antigen with a co-stimulatory signal that combines with the antigen-receptor signal to drive clonal expansion and differentiation.

6-16. Bacterial peptides, mediators of inflammatory responses, and chemokines signal through members of the seven-transmembrane-domain, trimeric G protein-coupled receptor family

Another way in which cells in the innate immune system are able to detect the presence of infection is by binding bacterial peptides containing N-formylmethionine, or fMet, a modified amino acid that initiates all proteins synthesized in prokaryotes. The receptor that recognizes these peptides is known as the fMet-Leu-Phe (fMLP) receptor, after a tripeptide for which it has a high affinity, though it is not restricted to binding just this tripeptide. The fMLP receptor belongs to an ancient and widely distributed family of receptors that have seven membrane-spanning segments; the best-characterized members of this family are the photoreceptors rhodopsin and bacteriorhodopsin. In the immune system, members of this family of receptors have a number of essential roles; the receptors for the anaphylotoxins (see Section 2-12) and for chemokines (see Section 2-20) belong to this family.

All receptors of this family use the same mechanism of signaling; ligand binding activates a member of a class of GTP-binding proteins called G proteins. These are sometimes called large G proteins, to distinguish them from the smaller Ras-like family of GTP-binding proteins, or heterotrimeric G proteins, as each is made up of three subunits—Gα, Gβ, and Gγ. Roughly 20 different large G proteins are known, each interacting with different cellsurface receptors and transmitting signals to different intracellular pathways. In the resting state, the trimeric G protein is inactive, not associated with the receptor and has a molecule of GDP bound to the α subunit. When the receptor binds its ligand, a conformational change in the receptor allows it now to bind the G protein, displacing the GDP molecule from the G protein and replacing it with a molecule of GTP. The G protein now dissociates into two components, the α subunit and the combined βγ subunits; each of these components is capable of interacting with other cellular components to transmit and amplify the signal. Binding of the α subunit to its ligand activates the GTPase activity of this subunit, cleaving the molecule of GTP to GDP, and thus allowing the α and βγ subunits to reassociate (Fig. 6.21).

Figure 6.21. Seven-transmembrane-domain receptors signal by coupling with trimeric GTP-binding proteins.

Figure 6.21

Seven-transmembrane-domain receptors signal by coupling with trimeric GTP-binding proteins. Seven-transmembrane-domain receptors such as the chemokine receptors signal through trimeric GTP-binding proteins known as large G proteins. In the inactive state, (more...)

Important targets for the activated G protein subunits are adenylate cyclase and phospholipase C, whose activation gives rise to the second messengers cyclic AMP, IP3 and Ca2+. These in turn activate a variety of intracellular pathways that affect cell metabolism, motility, gene expression, and cell division. Thus activation of G protein-coupled receptors can have a wide variety of effects depending on the exact nature of the receptor and the G proteins that it interacts with, as well as the different downstream pathways that are activated in different cell types.

6-17. Cytokines signal lymphocytes by binding to cytokine receptors and triggering Janus kinases to phosphorylate and activate STAT proteins

Cytokines, which we encountered in Chapter 2, are small proteins (of ~20 kDa) that each act on a specific receptor. They are secreted by a variety of cells, usually in response to an external stimulus, and they can then act on the cells that produce them (autocrine action), on other cells in the immediate vicinity (paracrine action), or on cells at a distance (endocrine action) after being carried in blood or tissue fluids. Cytokines affect cell behavior in a variety of ways and, as we will see in subsequent chapters, they play key roles in controlling the growth, development, and functional differentiation of lymphocytes, and as effector molecules of activated T cells. Many cytokines bind to receptors that use a particularly rapid and direct signaling pathway to effect changes in gene expression in the nucleus.

Cytokine binding to such receptors activates receptor-associated tyrosine kinases of the Janus kinase family (JAKs), so-called because they have two symmetrical kinase-like domains, and thus resemble the two-headed mythical Roman god Janus. These kinases then phosphorylate cytosolic proteins called signal transducers and activators of transcription (STATs). Phosphorylation of STAT proteins leads to their homo- and heterodimerization through interactions involving their SH2 domains; STAT dimers can then translocate to the nucleus, where they activate various genes (Fig. 6.22). The proteins encoded by these genes contribute to the growth and differentiation of particular subsets of lymphocytes.

Figure 6.22. Many cytokine receptors signal by a rapid pathway using receptor-associated kinases to activate specific transcription factors.

Figure 6.22

Many cytokine receptors signal by a rapid pathway using receptor-associated kinases to activate specific transcription factors. Many cytokines act via receptors that are associated with cytoplasmic Janus kinases (JAKs). The receptor consists of at least (more...)

In this pathway, gene transcription is activated very soon after the cytokine binds to its receptor, and specificity of signaling in response to different cytokines is achieved by using different combinations of JAKs and STATs. This signaling pathway is used by most of the cytokines that are released by T cells in response to antigen. Although cytokines are not in themselves antigen specific, their effects can be targeted in an antigen-specific manner by their directed release in antigen-specific cell-cell interactions and their selective action on the cell that triggers their production, as we will see in Chapter 8.

6-18. Programmed cell death of activated lymphocytes is triggered mainly through the receptor Fas

When antigen-specific lymphocytes are activated through their antigen receptors in an adaptive immune response, they first undergo blast transformation and begin to increase their numbers exponentially by cell division. This clonal expansion can continue for up to 7 or 8 days, so that lymphocytes specific for the infecting pathogen increase vastly in numbers and can come to predominate in the population. In the response to certain viruses, nearly 50% of the CD8 T cells at the peak of the response are specific for a single virus-derived peptide:MHC class I complex. After clonal expansion, the activated T cells undergo their final differentiation into effector cells; these remove the pathogen from the body, which terminates the antigenic stimulus.

When the infection has terminated, the activated effector T cells are no longer needed and cessation of the antigenic stimulus prompts them to undergo programmed cell death or apoptosis. Apoptosis can probably be induced by several mechanisms, but one that has been particularly well defined is the interaction of the receptor molecule Fas on T cells with its ligand, Fas ligand. Fas ligand is a member of the tumor necrosis factor (TNF) family of membrane-associated cytokines, whereas Fas is a member of the TNF receptor family. Both Fas and its ligand are normally induced during the course of an adaptive immune response. TNF and its receptor TNFR-I can act in a similar way to Fas ligand and Fas but their actions are far less significant.

All pathways inducing apoptosis lead to the activation of a series of cysteine proteases that cleave protein chains after aspartic acid residues and have therefore been called caspases. In the case of activated lymphocytes, apoptosis is initiated by stimulation of the receptors Fas or TNFR-I. The ligands for these receptors are in the form of trimers, and when they bind, they induce trimerization of the receptors themselves (Fig. 6.23). The cytoplasmic tails of these receptors share a motif known as a death domain which, as we saw in Section 6-15, is a protein-protein interaction domain. The adaptor proteins that interact with the death domains in the cytosolic tails of Fas and TNFR-I are called FADD and TRADD respectively. These in turn interact through a second death domain with the protein caspase 8 (also known as FLICE), whose carboxy-terminal domain is a procaspase (the inactive form of a caspase). Binding activates the enzymatic activity of caspase 8, leading to a protease cascade in which activated caspases cleave and activate a succession of downstream caspases. At the end of this pathway a caspase-activated DNase (CAD) enters the nucleus and cleaves DNA to produce the DNA fragments characteristic of an apoptotic cell. These fragments can be labeled by a procedure known as TUNEL (see Appendix I, Section A-32) that selectively stains and detects only those cells that have undergone apoptosis.

Figure 6.23. Binding of Fas ligand to Fas initiates the process of apoptosis.

Figure 6.23

Binding of Fas ligand to Fas initiates the process of apoptosis. The Fas ligand (FasL) recognized by Fas is a homotrimer, and when it binds it induces the trimerization of Fas. This brings the death domains in the Fas cytoplasmic tails together. A number (more...)

Mutations in the genes encoding Fas or Fas ligand have now been identified in both mice and humans. These mutations are associated with an excessive accumulation of abnormal T cells that lack both co-receptor proteins and express the CD45 isoform usually expressed by B cells. It is thought that these cells have been activated but subsequently failed to die. The mutations that cause this phenotype are mostly recessive; that is, both copies of the gene for either Fas or Fas ligand must be defective to produce an effect. However, in some cases in humans, the mutant phenotype is seen in heterozygous individuals. The production of an effect in heterozygotes is likely to reflect the need for trimerization of Fas for efficient operation of the Fas-Fas ligand interaction, and the fact that if one of the members of the trimer is mutant, the trimer cannot transduce a signal.(Image clinical_small.jpgAutoimmune Lymphoproliferative Syndrome, in Case Studies in Immunology, see Preface for details)

6-19. Lymphocyte survival is maintained by a balance between death-promoting and death-inhibiting members of the Bcl-2 family of proteins

Apoptosis plays a major role in the development and maintenance of all multicellular organisms. The apoptotic program is present in all cells and may be triggered by an absence of appropriate survival signals as well as by external stimuli as described for Fas-induced cell death above. Thus it is not surprising that all cells also possess a separate set of proteins that can inhibit programmed cell death.

The first member of this family of proteins was discovered as an oncogene in B cells. When tumors form in the B-cell lineage, they are frequently associated with chromosomal translocations in which the chromosomal DNA is broken and an active immunoglobulin locus is joined to a gene that affects cell growth, usually activating that gene in the process. By cloning the DNA breakpoints, one can isolate the gene that has been activated by the translocation. One such gene is bcl-2, which was isolated from the second B-cell lymphoma to have its breakpoint identified. bcl-2 is homologous with the Caenorhabditis elegans gene ced-9, which is a cell death-inhibitory gene. In cultured B cells and in transgenic mice, the expression of bcl-2 protects against cell death. bcl-2 is a member of a small family of closely related genes, some of which inhibit cell death whereas others promote it. These genes can be divided into death-inhibiting genes, such as bcl-2 and bcl-XL, and death-promoting genes, such as Bax and Bad. The proteins encoded by these genes act as dimers, and as Bcl-2 and Bax proteins can dimerize with each other to form heterodimers, the more abundant protein determines whether the cell lives or dies. One way in which Bcl-2 acts to prevent cell death is shown in Fig. 6.24.

Figure 6.24. Bcl-2 inhibits the processes that lead to programmed cell death.

Figure 6.24

Bcl-2 inhibits the processes that lead to programmed cell death. In normal cells, cytochrome c is confined to the mitochondria (first panel). However, during apoptosis the mitochondria swell, allowing the cytochrome c to leak out into the cytosol (second (more...)

The balance between death-promoting and death-inhibiting gene expression is critically important in lymphocytes, because lymphocyte populations are regulated so that a person will, in the absence of infection, maintain a constant level of T and B cells despite the production and death of many lymphocytes each day. The fate of individual lymphocytes is set by signals delivered mainly or entirely through the antigen-specific receptors, as we will learn in Chapter 7, which deals with the production of the mature repertoire of receptors on B and T lymphocytes. In the last section of this chapter we will look at the evidence for a continued role of antigen-receptor signaling in maintaining the survival of mature T and B cells.

6-20. Homeostasis of lymphocyte populations is maintained by signals that lymphocytes are continually receiving through their antigen receptors

Most of what we know about signaling through the B-cell receptor and the T-cell receptor derives from observations in cultured B-cell and T-cell lines. In these cells, as discussed in Section 6-9, the tyrosine kinases Syk and ZAP-70 are recruited as a consequence of receptor clustering and the subsequent phosphorylation of ITAMs in the receptor subunit tails. However, in T cells isolated directly from lymph nodes or the thymus, ZAP-70 is already bound to partly phosphorylated ζ chains but is not yet activated. The same state can be produced in both normal naive T cells and cultured T cells by exposure to altered peptide ligands (see Section 6-12). It therefore seems likely that the phosphorylated ITAMs and bound ZAP-70 seen in mature naive T cells in vivo reflect the receipt of signals from self MHC molecules bound to self peptides that behave like altered peptide ligands.

As we will see in Chapter 7, the ability to interact with self peptide:self MHC ligands is a criterion for survival during T-cell development in the thymus. Developing T cells are subject to stringent testing once the α:β T-cell receptor is expressed. This selection process retains those cells whose receptors interact effectively with various self peptide:self MHC ligands (positive selection), and removes those T cells that either cannot participate in such interactions (death by neglect) or recognize a self peptide:self MHC complex so well that they could damage host cells if allowed to mature; such cells are removed by clonal deletion (negative selection). Those T cells that mature and emerge into the periphery have therefore been selected for their ability to recognize self MHC:self peptide complexes without being fully activated by them.

A number of studies have now shown that naive T cells in the peripheral lymphoid tissues continue to receive signals through interactions with self MHC:self peptide complexes. These signals enable the cells to survive and are delivered most effectively by the cells that are most capable of T-cell activation, namely the dendritic cells. Thus, the T cells in mice transgenic for a T-cell receptor that is positively selected on a particular self MHC:self peptide complex can survive only if they receive similar signals from self MHC:self peptide complexes in the periphery. It seems as though the peripheral T-cell repertoire is maintained by a continuous dialogue between dendritic cells presenting self peptides on self MHC molecules and recirculating naive T cells that were positively selected by signals from the same self peptide:self MHC molecules during development. This dialogue is likely to account for the state of partial phosphorylation of the ζ chains discussed above. A requirement for repeated interaction with dendritic cells resident in peripheral lymphoid tissue could also account for the tight regulation of the numbers of naive CD4 and CD8 cells. For memory T cells, the factors that govern survival are less well defined. It is clear that self MHC:self peptide recognition is not required for the survival of memory T cells; in fact, the presence of MHC molecules does not seem to be required at all. Rather, it is likely that the levels of particular cytokines serve to maintain the memory T-cell pool.

In B cells, it is also clear that signaling through the antigen receptor determines cell survival from the time that it is first expressed on the cell surface. As we will see in Chapter 7, autoreactive B cells are induced to die on binding antigen. However, the expression of a functional B-cell receptor at the cell surface is also essential for cell maturation and survival. The role of the B-cell receptor in signaling for survival in the periphery was demonstrated quite dramatically using a conditional gene knockout strategy (see Appendix I, Section A-47). Mice were made transgenic for a rearranged immunoglobulin VH gene flanked by loxP sites. This construct was ‘knocked-in’ to the site at which the rearranged VH gene normally occurs in such a way that only the transgene could produce the heavy-chain V region. The animals were also made transgenic for the enzyme Cre recombinase, which can excise loxP-flanked genes; the transgene encoding the Cre recombinase was made inducible by interferon. After treatment with interferon, the induced Cre recombinase removed the transgenic rearranged VH gene, preventing the production of a heavy chain. Most of the B cells in the treated animals lost their receptors; these receptor-negative B cells rapidly disappeared. Thus, the B-cell receptor is clearly required to keep recirculating B cells alive, and must have a role in perceiving or transmitting survival signals to each B cell. Evidence also shows that receptor specificity can affect this process. However, the ligand or ligands responsible for signaling for B-cell survival are not yet known.

The identity of the ligands responsible for delivering survival signals to T and B cells through their antigen receptors remains an important question in immunology. There is also much to learn about the signaling process itself. Survival signals are likely to regulate the level of proteins in the Bcl-2 family because, as we discussed in Section 6-18, increased levels of Bcl-2 and Bcl-XL promote the survival of lymphocytes, whereas increased levels of Bax and Bad have an opposing effect. As yet, however, a direct link between antigen-receptor signaling for survival and the regulation of the Bcl-2 family has not been shown.

Summary

Many different signals govern lymphocyte behavior, only some of which are delivered via the antigen receptor. Lymphocyte development, activation, and longevity are clearly influenced by the antigen receptor, but these processes are also regulated by other extracellular signals. Other signals are delivered in a variety of ways. An ancient signaling pathway with a role in host defense leads rapidly from the IL-1 receptor or a similar receptor called Toll to initiate the detachment and degradation of the inhibitory protein IκB from the transcription factor NFκB, which can then enter the nucleus and activate the transcription of many genes. Many cytokines signal through an express pathway that links receptor-associated JAK kinases to preformed STAT proteins, which after phosphorylation dimerize through their SH2 domains and head for the nucleus. Activated lymphocytes are programmed to die when the Fas receptor that they express binds the Fas ligand. This transmits a death signal, which activates a protease cascade that triggers apoptosis. Lymphocyte apoptosis is inhibited by some members of the intracellular Bcl-2 family and promoted by others. Working out the complete picture of the signals processed by lymphocytes as they develop, circulate, respond to antigen, and die is an immense and exciting prospect.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2001, Garland Science.
Bookshelf ID: NBK27094