IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 10. Cell-Mediated Immune Responses

■ Cytokines play a central role in the positive and negative Regulation of Immune responses, as well as in integrating Immunity with the physiological Functions of other body systems, namely the endocrine and hematopoietic systems.

■ Recognition of microbial structures occurs at the very onset of the body's reaction to infection, prior to The Development of a specific Immune Response. The Nature of the subsequent response depends largely on the cytokines released.

■ Immune regulation is orchestrated by helper T Cells (Th cells). In response to an antigen, they secrete various sets of cytokines and thereby initiate distinct effector functions. For instance, Th1 cells activate macrophages, whereas Th2 cells promote antibody production. If an inappropriate effector function is activated, pathogen elimination fails, leading to chronic immunopathology.

■ A Th1-type immune response suppresses a Th2-type response, and vice versa.

■ Most cytotoxic T cells recognize Antigens presented in association with Class I MHC molecules, whereas NK cells respond to targets that do not express these molecules.

■ The cytotoxic activity of killer cells results from a combination of direct Cell-to-cell contact, cytokine release, and the exocytosis of granule Proteins, notably perforin and granzymes.

■ Activated macrophages destroy phagocytosed microorganisms using highly reactive oxygen and nitrogen metabolites.

■ When cell-mediated immune responses fail to eliminate an infection or persistent antigen and thus cannot reach completion, a chronic destructive inflammatory process develops in the Tissues or granulomas are formed. In such cases, direct destruction of vital cells or secondary microvascular disturbances caused by excessive cytokine release can trigger immunopathology.

Originally, the term cellular immunity (cell-mediated immunity) was used to denote local reactions (typically against intracellular pathogens) carried out by lymphocytes and phagocytes without the involvement of Antibodies, which are the effectors of humoral immunity. Today, this term is often used in a broader sense to describe anti-infective or antitumor immune responses in which antibodies play an auxiliary rather than a leading role.

However, it is impossible to completely separate cell-mediated and humoral immunity: cells participate in initiating antibody production, and antibodies perform crucial bridging functions in certain cell-mediated reactions. Furthermore, cell-mediated immunity apparently does not exist without The production of antibodies, which are capable of modifying The Cell-mediated immune response in various ways. For instance, antigen-antibody complexes trigger the release of chemotactic Complement fragments that strongly attract leukocytes to the site of inflammation. In addition, through Fc receptors, antibodies can participate in binding antigens to cells and thereby influence cell-mediated reactions—specifically, by facilitating the attachment of phagocytes and cytotoxic T cells to target cells. In general, a coordinated immune response involves multidirectional signal exchange between various types of participating leukocytes and tissue cells.

Intercellular signaling in The Immune System is carried out through direct contact-dependent interactions involving surface molecules, or via cytokines, often referred to as "communicating proteins." These proteins act as soluble mediators of Intercellular Communication. Together with Hormones and Neurotransmitters, they form the foundation of the chemical signaling language that regulates morphogenesis, tissue regeneration, and immune responses in Multicellular Organisms. Alongside signals generated by cell-antigen or cell-cell interactions, a cytokine signaling network regulates innate and adaptive immune reactions, including inflammation, antiviral defense, clonal proliferation of antigen-specific T AND B cells, and their functions.

CYTOKINES AND THEIR CELLULAR RECEPTORS

Cytokines are small proteins (molecular weight ranging from 8 to 80 kDa) that act in an autocrine (i.e., on the cell that produces them) or paracrine (on nearby cells) manner. The production and release of these highly active molecules are usually transient and tightly regulated. To date, over a hundred different human cytokines have been identified, and reports of new ones are constantly emerging. Cytokines affect cells by binding to specific receptors on the cytoplasmic membrane, thereby triggering a cascade reaction that leads to the induction, enhancement, or suppression of a set of genes under their control.

Many cytokines have multiple names. This is because they were independently discovered in various fields of research—immunology, virology, hematology, cell biology, and oncology. Cytokines include interleukins (IL), currently designated from IL-1 to IL-18, interferons (IFN), colony-stimulating factors (CSF), tumor necrosis factors (TNF), growth factors, and chemokines (chemotactic cytokines) (Fig. 10.1). A major source of confusion in cytokine nomenclature is that they exhibit diverse activities, at least in vitro; a prime example is IL-6, which has very pleiotropic effects (Fig. 10.2). Moreover, in some cases, the same cytokine was independently isolated in several laboratories using completely different experimental systems. This nomenclature confusion is further compounded by overlapping activities among certain cytokines, creating an impression of functional redundancy. Additional difficulties in studying cytokines arise from the fact that these mediators are rarely produced individually and rarely act in isolation. In short, cytokines are characterized by a complex network mode of functioning, where the production of one influences the synthesis or activity of several others. In vivo, an individual cell in the body is rarely targeted by just a single cytokine. Much more frequently, individual cytokines act like letters of an alphabet that spell out a cytokine "word," and cellular responses are triggered by The impact of this exact "word" on the cell surface.

The most important functions of cytokines and their receptors in the immune response will be discussed below; first, it is necessary to examine the fundamental aspects of the molecular biology of these proteins.

Fig. 10.1. Cytokine nomenclature partly reflects the functional activity by which each was first discovered, as well as the chronological order of their discovery.

Fig. 10.2. The cytokine IL-6 exerts a typical pleiotropic effect on many Organ Systems. Specifically, it stimulates osteoclast formation and activity, particularly following a drop in estrogen levels.

Cytokines and their receptors are classified into several families

There is little similarity at the DNA and Amino Acid Sequence levels among individual cytokines or their groups, yet they can be classified into several major families based on Homology. Of these, the three most significant are: the first consisting of at least 15 α-interferons (IFNα), the second comprising over 50 chemokines (according to genomic analysis data), and the third including cytokines that bind to TNF receptors. However, it is much easier to group cytokines not by function, but by their three-dimensional Structure; this Classification clearly reflects intragroup similarities (in conformation and amino acid sequence) among cellular cytokine receptors. The largest family—a superfamily—of cytokine receptors is characterized by the presence of extracellular domains with a homologous sequence of approximately 200 amino acid residues. This superfamily includes receptors for IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). It also includes receptors for humoral factors that act predominantly outside the immune system, such as Growth Hormone and prolactin. The second largest family encompasses receptors for all types of interferons, as well as receptors for IL-1α, IL-1β, and macrophage colony-stimulating factor (M-CSF). This family is integrated as a subset within the superfamily of immunoglobulin-like molecules.

The third family of cytokine receptors binds TNFα and TNFβ, lymphotoxin, and A number of related cytokines, including nerve growth factor (NGF). The Fas molecule (CD95), whose binding to the FasL Ligand signals cell death, also belongs to this receptor family.

Most cytokine receptors are type 1 membrane Glycoproteins consisting of a single transmembrane domain. However, truly functional receptors generally consist of two or more subunits, which may even share identical structures across different receptor complexes. Typically, a receptor contains a "private" high-Specificity subunit capable of binding a specific cytokine, and a "common" subunit shared with receptors for other cytokines. For instance, the IL-2 receptor complex consists of three subunits (α, β, and γ). The IL-2Rβ subunit is also found in the IL-15 receptor, while IL-2Rγ is present in the receptors for IL-4, IL-7, and IL-9 (Fig. 10.3). Similarly, IL-6Rβ (known as gp130) serves as a subunit in receptors for cytokines such as LIF (leukemia inhibitory factor), oncostatin M, and IL-11. The similar functional activity of certain cytokines can partly be explained by the presence of identical subunits in their cellular receptors. This likely explains why IL-6, IL-11, and oncostatin M have identical effects on hepatocytes, megakaryocytes, and osteoclasts, whereas the duplicate role of IL-2 and IL-4 as T-cell growth factors is presumably due to the presence of an identical IL-2Rγ chain in both of their receptors. At the same time, thanks to the differential expression of private receptor subunits, each cytokine also exerts unique activities on specific cell types. For example, LIF can inhibit the differentiation of Embryonic Stem Cells, whereas IL-6 lacks this activity because these cells do not express the corresponding receptor.

Fig. 10.3. The high-affinity IL-2 receptor is composed of three polypeptide chains: the α and β chains are responsible for cytokine binding, while the γ chain transduces the resulting signal intracellularly. The IL-4 receptor features a unique α chain that specifically recognizes this cytokine, and a signaling γ chain identical to that of the IL-2 receptor.

All chemokines bind to a distinct class of receptors united by a unique structure known as the seven-transmembrane glycoproteins. Some of these receptors are so specific that they bind only a single chemokine, whereas others exhibit affinity for multiple chemokines. There is also a receptor (known as the Duffy Blood group antigen) that promiscuously binds many chemokines and likely participates in clearing excess mediators from sites of inflammation. Chemokines also bind to β-adrenergic receptors, providing further evidence of overlap between the cytokine network and other soluble mediator signaling systems.

Cytokine receptor binding activates intracellular signal Transduction pathways

MODERN CONCEPTS OF the Biological Role of cytokines are based on structural analyses of their molecules and studies of the Intracellular Signaling pathways they trigger. Such research now allows us to trace this chain of protein-protein recognition events in considerable detail—from the moment a cytokine binds to the cell surface to the mobilization of various METABOLISM/31.html">Transcription factors in the Cell Nucleus. As is well known, The First stage of cytokine signaling is the aggregation of receptor subunits induced by cytokine binding. The cytoplasmic "tails" of these subunits interact with one another to trigger a downstream signaling cascade. In the simplest case, identical receptor subunits bind a cytokine to form a homodimer; in other cases, a "unique" subunit upon cytokine binding induces either hetero- or homodimerization of "shared" subunits that transmit the signal intracellularly (Fig. 10.4).

Fig. 10.4. Simplified diagram of cellular activation by a cytokine (illustrating the interaction of IL-6 with its receptor). Upon binding to a cell-surface receptor, the cytokine induces dimerization or polymerization of its polypeptide chains, thereby activating intracellular signaling mechanisms (such as kinase cascades). This leads to the generation of active transcription factors that migrate to The Nucleus and bind to enhancers—nucleotide sequences that enhance the transcription of genes activated by the given cytokine.

All cytokine receptors belonging to the first family (superfamily), like representatives of several other receptor families, are associated with molecules called Janus Kinases (Jaks). Activation of cytokine receptors triggers Jaks, specifically Tyrosine kinases (Tyks). Most, if not all, functions of cytokine receptors are carried out with the obligatory activation of Jaks. In performing their main function—aggregating receptor subunits—cytokines simultaneously cause the aggregation of Jaks. Subsequently, these Janus kinases mediate the cytokine-induced phosphorylation of tyrosine residues within various signaling proteins, including signal transducers and activators of transcription (Stats). Dimers of Stats proteins translocate to the cell nucleus and bind directly to DNA. This type of signaling is illustrated in Fig. 10.5 using the binding of IFNα to its cellular receptor as an example.

Fig. 10.5. Diagram of intracellular signal transduction mechanisms activated by IFNα. Binding of IFNα induces the aggregation of two cellular receptor subunits, resulting in the activation and phosphorylation of the Jak kinases Jak1 and Tyk2, which then phosphorylate Stat1 and Stat2 molecules. These transcription factors form a complex with the p48 protein that binds to DNA. The resulting complex reaches the cell nucleus and induces the transcription of genes bearing the interferon response element (ISRE).

Each cytokine induces distinct intracellular signaling pathways depending on whether it exerts a shared or individual activity. For example, in T cells, each of the three interleukins—IL-2, IL-4, and IL-9—interacts with the IL-2Rγ subunit to activate Jak1 and Jak3; meanwhile, IL-10 activates Jak1 and Tyk2, and IL-12 similarly acts on Jak2 and Tyk2. Additional Variability in cellular responses to cytokines arises at the level of Stat phosphorylation, since each cytokine activates its own specific set of signal transducers and activators of transcription. This likely represents The primary function of Stats in cytokine responses. Other signaling pathways, primarily involving Ras/MAP kinase activation, drive cell proliferation in response to respective cytokines, although certain cytokines can trigger apoptotic pathways leading to cell death. The functional flexibility of signaling systems is further enhanced by the fact that Stats can be activated not only by Jaks, but also by kinases of alternative origin. For instance, in the case of the cytokines TNFα and IL-1β, intracellular signaling proceeds not via Jaks and Stats, but through MAP kinases, ultimately leading to the binding of transcription factors such as AP-1, NFkB, and NFIL-6 to DNA.

Both the specific and pleiotropic effects of chemokines ultimately influence cell motility, but this is a complex process: following chemokine binding to receptors, signals are transmitted to G proteins, leading to the mobilization of intracellular secondary messengers, Cytoskeleton reorganization, formation of focal adhesion contacts, cell surface attachment and detachment, and the extension and retraction of pseudopodia—all of which are essential for directed migration. Chemokines such as RANTES and MIP-1α can also activate Stats, thus forming a shunt between G protein- and Stat-mediated signaling pathways. Thus, chemokine research is gradually transforming from a tangled web of multiple activities into an increasingly coherent system of regulatory proteins with dedicated receptors and clear intracellular signaling networks.

Differentiation of T helper cells into subsets constitutes a crucial step in determining the effector mechanisms of the immune response

It is now established that There are two subsets of CD4⁺ Th cells differing in their profile of synthesized cytokines, and this profile dictates which of the two primary TYPES OF IMMUNE response will be executed (Fig. 10.6). In humans, Th1 cells typically produce IFNγ, TNFβ, and IL-2 and participate in cell-mediated inflammatory reactions. Some of the cytokines secreted by Th1 cells possess proinflammatory activity and also stimulate cytotoxic cells and delayed-type hypersensitivity T effectors. In contrast, Th2 cells synthesize IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, thereby enhancing antibody production, particularly of the IgE class, and promoting allergic reactions. Furthermore, cytokines secreted by Th1 cells suppress Th2 cell activity, and vice versa. Consequently, any immune response develops along either a Th1 or Th2 pathway (Fig. 10.7).

Fig. 10.6. Diagram of murine T helper Cell Differentiation into subsets with distinct secreted cytokine profiles. The cytokines IL-12, IFNγ, and TNFβ promote The Emergence of Th1 cells, whereas IL-4 stimulates Th2 maturation. The type and Functions of the activated effector cells depend entirely on the cytokine profile produced by the T helpers.

Fig. 10.7. By secreting different sets of cytokines, Th1 and Th2 cells not only stimulate distinct effector mechanisms of the immune response but also mutually suppress each other's immunoregulatory activity.

Differences in surface markers between Th1 and Th2 cells have recently been identified. The Th1 cell membrane is characterized by the presence of LAG-3, an antigen belonging to the immunoglobulin superfamily. Th2 cells express significantly higher levels of the CD30 marker, a member of the TNF receptor family, compared to Th1 cells.

The determination (Selection) of the response type—Th1 or Th2—is crucial for the development of effective immunity and appears to depend on numerous interacting factors. Among these, the following can influence CD4⁺ Th cell differentiation pathways and, consequently, the cytokine profile that dictates the response type:

✵ the profile and quantitative ratio of cytokines secreted in response to an antigen. For instance, IL-12 serves as a potent stimulus for interferon-gamma production by T AND NK cells and, consequently, for Th1 cell differentiation. Meanwhile, alpha-interferon synthesized during the early phase of viral infection can not only induce elevated IL-12 expression but also switch Th-cell cytokine synthesis from the Th2 profile to Th1. Conversely, early IL-4 production favors the emergence of Th2 cells;

✵ antigen dosage;

✵ antigen-presenting cells and the cytokines they secrete;

✵ the genotype of the host Organism; and

✵ The activity of Costimulatory molecules and hormones at the site of antigen interaction with Th cells.

Among the listed T-helper differentiation factors, the last one is of particular interest. For example, glucocorticoid hormones, the levels of which rise during physical or psychological stress (such as cortisol), can drive the immune response toward a Th2 pathway. This effect is counteracted by dehydroepiandrosterone (DHEA) derivatives, which promote a Th1-type immune response. The concentrations of both Adrenal hormones are regulated systemically and also depend on local metabolism in Organs where antigens encounter Th cells. Therefore, The ratio of cortisol to DHEA concentrations within lymphoid tissue or at a pathological site can act as a determinant of the immune response type.

The immune response is not always strictly polarized toward the Th1 or Th2 pathway, as other T-helper subpopulations likely exist. Nevertheless, the roles of these two response types in establishing protective immunity as well as in immunopathology are fundamentally different. It is now well established that many diseases are characterized by the activity of a specific T-helper subpopulation. For instance, organ-specific immunopathology, acute allograft rejection, recurrent spontaneous abortions, and multiple sclerosis are associated with a Th1-type immune response. In these conditions, Th1 cells are readily isolated and cloned from patients' blood. Conversely, all T-cell clones derived from patients with atopic asthma sensitized to plant pollen, much like clones from systemic lupus erythematosus patients, secrete Th2-profile cytokines.

CD8⁺ T cells are likewise differentiated into subpopulations with distinct cytokine secretion profiles

Many CD8⁺ cytotoxic T cells secrete the same set of cytokines as Th1 cells. There are also CD8⁺ T cells that secrete Th2-type cytokines, a subpopulation that exerts regulatory and suppressor functions. The differentiation of CD8⁺ T cells can be influenced by the cytokine profile of CD4⁺ T cells. For example, IFNγ and IL-12 promote the differentiation of CD8⁺ T cells into the Tc1 subpopulation, whereas IL-4 drives it toward the Tc2 subpopulation. However, cells from both of these subpopulations exhibit cytotoxic activity and eliminate their targets primarily by releasing granule contents (see below).