Inflammatory cytokines
Interleukin-12 and Interleukin-18
IL-12 is a critical cytokine that stimulates the differentiation of naive helper T cells into Th1 cells, and it stimulates NK cells and Th1 cells to produce IFN-y. IL-12 also enhances the cytolytic function of cytolytic T cells and NK cells. IL-12 is produced by activated macrophages and dendritic cells, in response to a variety of microorganisms. It has been used experimentally as an adjuvant in vaccines aimed at stimulating Th1-induced cellular immunity. As with most cytokines, binding of IL-12 with its receptor generates signaling through the Jak and STAT pathways.
IL-18, which is produced by macrophages, also stimulates the production of IFN-y but appears to be less necessary than IL-12; whereas IL-12-deficient mice are susceptible to Leishma-nia major infection, IL-18-deficient mice combat such infection normally, although their initial immune response is slow.28 Similarly, IL-12 is critical for immunity to cytomegalovirus, whereas IL-18 is not.
Tumor Necrosis Factor-a
TNF-a, a major inflammatory cytokine, is one of the most abundant substances produced by macrophages after stimulation with IFN-y, migration inhibitory factor (MIF), or bacterial lipopolysaccharide (LPS). TNF-a is also produced by activated T cells, NK cells, and mast cells.
At low concentrations, TNF-a enhances the protective inflammatory response, activating and enhancing the function of various leukocytes, including neutrophils, macrophages, and eosinophils. This can further stimulate macrophages to produce cytokines, including TNF-a itself, IL-1, IL-6, MIF, and a variety of chemotactic cytokines [see Chemokines, below]. TNF-a enhances expression of MHC class I molecules, potentiates cytotoxic T cell-induced cell lysis, functions as an endogenous pyrogen (i.e., induces fever, by direct and indirect actions on the brain), activates the clotting system and the production of acute-phase proteins by the liver, and can cause immunodeficiency through suppression of the bone marrow. When present for prolonged periods, TNF-a causes cachexia.
TNF-a plays a primary role in the host response to gram-negative bacteria. The LPS of these bacteria causes the release of MIF, which in turn enhances TNF-a production by macrophages. At low concentrations of LPS, TNF-a mediates a protective response. At high concentrations of LPS, however, TNF-a mediates disseminated intravascular coagulation—part of what is known as the Shwartzman reaction—and can cause death from shock.
Protective immunity to certain intracellular organisms, such as Leishmania, is enhanced by TNF-a, and TNF-a also has potent antiviral activity. However, many of the symptoms of malaria, especially of cerebral malaria, and some symptoms of HIV infection may be mediated by TNF-a. Antibodies to TNF-a have been approved for the treatment of Crohn disease and rheumatoid arthritis [see 4:IV Inflammatory Bowel Diseases and 15:II Rheumatoid Arthritis].30
Lymphotoxin
Produced exclusively by Th1 cells, lymphotoxin has many of the same biologic properties as TNF-a and utilizes the same cell receptor as TNF-a; it is also referred to as TNF-p. Like TNF-a, lymphotoxin lyses tumor cells but not normal cells, activates neutrophils, and increases vascular adhesion and extravasation of leukocytes. In addition, lymphotoxin plays a role in the development of lymphoid tissue.
Interleukin-1 and Interleukin-6
IL-1 is produced mainly by monocytes and macrophages but also by other cells, such as epithelial and endothelial cells. It is an endogenous pyrogen, and many of its functions are similar to those of TNF-a. It induces the production of additional IL-1 and of IL-6 from macrophages and induces glucocorticoid synthesis and the release of prostaglandin, collagenase, and acute-phase proteins. IL-1 increases the expression of surface molecules on en-dothelial cells, leading to adhesion of leukocytes and coagulation, and stimulates the production of macrophage chemokines that in turn activate neutrophils. IL-1 differs from TNF-a in that it does not produce necrosis of tumors or tissue injury, increase expression of MHC, or, by itself, mediate the Shwartzman reaction.
Macrophages produce an IL-1 receptor antagonist (IL-1ra) that, along with the IL-1 receptors shed from activated cells, inhibits IL-1 and thus acts as a regulator. Such natural inhibitors to IL-1 are now in clinical use to counteract certain inflammatory processes, especially in rheumatoid arthritis.
IL-6 is induced by IL-1 and by TNF-a from macrophages and, in turn, inhibits macrophage production of IL-1 and TNF-a. Like IL-1 and TNF-a, IL-6 is an endogenous pyrogen. IL-6 acts on hepatic cells to produce acute-phase proteins, such as fibrinogen, a2-macroglobulin, and serum amyloid A protein. This cytokine can also inhibit macrophage activation.
Interferon Gamma and Other Macrophage-Activating Factors
IFN-y, produced by T cells and NK cells, is the primary macrophage-activating factor (MAF). MAFs play an important role in cell-mediated immunity because activated macrophages produce many cytokines and chemokines intimately involved in inflammation, including TNF-a, IL-1, IL-6, and MIF. Other MAFs include granulocyte-macrophage colony-stimulating factor (GM-CSF) and MIF. IL-1 and TNF-a have weak MAF activity. IL-12 stimulates NK cells to produce greater amounts of IFN-y, enhancing IFN-Y-dependent reactions. Both on its own and by enhancing the effects of TNF-a, IFN-y causes the expression of adhesion molecules on the surface of vascular en-dothelial cells, leading to T cell adhesion and extravasation.
The inflammatory effects of IFN-y are countered by TGF-p and IL-10, which inhibit macrophage activation. IFN-y has been used successfully to treat chronic granulomatous disease and drug-resistant visceral leishmaniasis.31
Migration Inhibitory Factor
MIF, the first T cell cytokine to be discovered, derives its name from the fact that it inhibits the random migration of macro-phages in vitro. MIF acts as an endogenous hormone that coun-terregulates glucocorticoid action.32 Macrophages and T cells release MIF in response to glucocorticoids and other inflammatory stimuli. MIF then overrides the immunosuppressive effects of steroids on macrophage and T cell cytokine production.32,33
The gene for MIF is expressed in many different tissues.34 Large quantities of the gene are found in macrophages and pituitary cells. Indeed, LPS stimulates the release of MIF by the pituitary. When given to mice, MIF greatly enhances the lethality of LPS; conversely, anti-MIF antibodies completely reverse the lethality of LPS.35 MIF upregulates the receptor for LPS on macrophages (Toll-like receptor 4). Recombinant MIF activates macrophages to kill Leishmania and stimulates macrophages to produce TNF-a and nitric oxide. Mice lacking the gene for MIF show enhanced resistance to the lethal effects of high doses of LPS and Staphylococcus aureus enterotoxin B, as well as to Pseudomonas aeruginosa and Escherichia coli, but they are susceptible to Leishmania, Salmonella, and Cysticercosis .3639 MIF has been shown to play a pathogenic role in several experimental models of inflammation and autoimmunity, including glomer-ulonephritis, arthritis, inflammatory bowel disease, atherogen-esis, and acute lung injury; it also inhibits p53 and enhances carcinogenesis. Mechanisms of MIF action include the activation of the extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2), leading to activation of phospholipase A2, cyclooxy-genase-2, and prostaglandin E2; enhancement of Toll-like re-ceptor-4; and inhibition of p53 and apoptosis.
Anti-MIF therapies are under development. The goal is to increase the immunosuppressive and anti-inflammatory properties of endogenously released glucocorticoids, thereby reducing the need for steroid therapy in a variety of autoimmune and inflammatory conditions.32 Anti-MIF therapy should also have a role in treating some gram-negative infections and preventing septic shock.
Interleukin-5
IL-5 mainly affects eosinophil recruitment and activation. IL-5 is produced by Th2 cells and activated mast cells and stimulates the growth and differentiation of eosinophils. In addition to IL-5, other substances involved in the activation of eosinophils are TNF-a and an eosinophil cytotoxicity-enhancing factor derived from monocytes. Activated eosinophils produce tissue damage in allergic states and kill helminthic parasites.
Other Interleukins
DNA sequence information from the Human Genome Project has led to the identification of a number of new cytokines: IL-19 through IL-24. Their role in the immune response needs further exploration. Several of these cytokines appear to have some properties similar to those of IL-10, IL-12, and IL-15. Hematopoietic cytokines and growth factors are discussed elsewhere [see V:I Approach to Hematologic Disorders].
Anti-inflammatory cytokines
Interleukin-4 and Interleukin-13
IL-4 stimulates the expression of an adhesion molecule on endothelial cells, leading to the binding of eosinophils, lymphocytes, neutrophils, and monocytes and their subsequent extravasation. However, IL-4 also acts as an anti-inflammatory cytokine, inhibiting activated macrophages and diminishing the production of TNF-a and nitric oxide. IL-13 has the ability to take over some of the functions of IL-4. Both IL-4 and IL-13 induce IgE synthesis in B cells and differentiation of T cells to Th2 cells and can suppress inflammatory processes induced by Th1 cells. These cytokines also act on macrophages to suppress the inflammatory response. Activated mast cells and basophils produce additional IL-4. Of interest is that mutated IL-4 can inhibit IgE synthesis by IL-4 and IL-13 and may prove useful in treating some allergic states.
Transforming Growth Factor
TGF-| is produced by a variety of cells, including platelets, lymphocytes, activated macrophages, and placenta cells. It is an important anti-inflammatory cytokine because it inhibits the activation of macrophages and the maturation of cytotoxic T cells and thus controls the effects of many cytokines.
Interleukin-10
IL-10 is an important regulatory cytokine. It is produced by CD4+ and CD8+ T cells, B cells, macrophages, activated mast cells, and keratinocytes. Although usually associated with activity of Th2 cells, IL-10 can also be produced by Th1 cells. IL-10 suppresses lymphocyte responses by downregulating macrophage cytokines—including IL-1, TNF-a, IL-6, IL-8, GM-CSF, and granulocyte colony-stimulating factor (G-CSF)—and inhibiting nitric oxide production.
Chemokines
Chemokines are a superfamily of low-molecular-weight chemotactic cytokines that mediate the directional migration of leukocytes during normal and inflammatory processes.40 They play an important role in attracting granulocytes into sites of inflammation. There are four distinct families of chemokines, distinguished on the basis of the position of their first two conserved cysteine residues: CXC (the first two cysteines are separated by one amino acid), CC, C, and CX3C. The receptors for chemokines are all integral membrane G-protein-coupled receptors, which constitute one of the largest classes of signaling molecules.
CXC chemokines predominantly activate neutrophils.41 This family includes IL-8; | -thromboglobulin (| -TG); the growth-related gene products gro-a, gro-|, and gro-y; and platelet factor 4. They are usually produced by monocytes, but some are produced by other cells, including T cells, endothelial cells, and platelets. IL-8 induces expression of neutrophil-binding inte-grins on endothelial cells, resulting in the rapid accumulation of neutrophils in tissues. The chemokine gro also stimulates neutrophil accumulation, as well as the release of lysosomal enzymes that contribute to the local inflammatory response. Platelet factor 4 and | -TG are released from aggregated platelets and stimulate fibroblasts, which are required for repair at sites of hemorrhage or thrombosis.
The CC chemokines activate T cells, monocytes, and eosinophils. This family includes RANTES (regulated on activation, normal T cell expressed and secreted), macrophage chemotactic and activating factor (MCAF), macrophage inflammatory protein-1a (MIP-1a), and MIP-1|. CC chemokines are produced by activated T cells and monocytes. RANTES is a potent attractant for memory T cells (but not for naive T cells) and also attracts monocytes. MCAF acts exclusively on mono-cytes, attracting them, activating them, and regulating the expression of integrins on their surface. MIP-1a and MIP-1| attract only monocytes. The CC chemokines eotaxin, eotaxin-2, and monocyte chemoattractant protein-4 (MCP-4) predominantly activate eosinophils.
In addition to their role in inflammation, chemokines are important in the hemostasis of lymphocytes moving through the lymphatic system; in the location of T cells, B cells, and dendritic cells in the lymph node; in Th1 and Th2 cell development; and in angiogenesis, angiostasis, and metastasis of tumor. In fetal mice lacking the CXC chemokine receptor-4, the heart and cerebellum do not develop properly, indicating that chemokines also play a part in nonlymphoid organ development.
Chemokine receptors play an important role as coreceptors for HIV. The virus first interacts with CD4 on T cells but requires a coreceptor to penetrate the cell membrane. The CC chemokine receptor-5 (CCR5), which mediates activation of T cells and macrophages, is the major coreceptor for some HIV-1 strains. The natural ligands for CCR5 include RANTES, MIP-1a, and MIP-1|.44 The CXC chemokine receptor-4 appears to be important in late-stage HIV infection.45
Effector Mechanisms in Cell-Mediated Immunity
Cell-mediated immunity encompasses the killing of invading microorganisms, such as bacteria, viruses, fungi, and parasites; the destruction of tumor cells; the rejection of tissue grafts; and injury to tissues in various disease states, including au-toimmunity. Cell-mediated immune reactions can also be induced by contact with antigens, such as those found in poison ivy and numerous drugs. Drugs are more likely to provoke cell-mediated reactions when applied topically than when given systemically.
Most cell-mediated immune reactions involve initial interaction between sensitized T cells and antigens on presenting cells. This reaction can trigger several effector pathways, including activation of cytotoxic T cells, stimulation of T cell production of cytokines that activate macrophages and promote the proliferation of NK cells, and production of antibodies involved in antibody-dependent cell-mediated cytotoxicity by NK cells and other cell types. Although cell-mediated immune reactions other than antibody-dependent cell-mediated cytotoxicity do not require the presence of antibody or complement, they can be modified by these humoral factors. Subsequent events require cooperation between different subsets of T cells; the reactions involved are controlled by various cytokines.
Figure 5 Cytotoxic cells recognize surface markers on cells that are to be destroyed. (a) Apoptosis is triggered by the cytotoxic T cell through nonsecretory Fas-Fas ligand interaction. (b) Apoptosis is triggered by the cytotoxic T cell by means of secretory mechanisms initiated by perforin and granzymes.
The mechanisms of cell-mediated immunity involving T cell-macrophage interactions can be both protective (leading to the killing of invading microorganisms) and harmful (leading to inflammation and tissue destruction). Sometimes, the two go hand in hand; in tuberculosis, for example, both the killing of tubercle bacilli and the development of cavities in the lungs are consequences of T cell-macrophage interactions. In addition to the acquired cell-mediated immunity discussed above, innate immunity also involves the mounting of an immune response by cells directly stimulated by microorganisms. Macrophages, other granulocytic cells, and NK cells are involved in innate cell-mediated immune responses [see 6:II Innate Immunity].
Cytotoxic t cells
Cytotoxic T cells are antigen-specific effector cells that are important in resisting infectious agents, especially viruses that are present in cells other than macrophages; in killing tumors; and in allograft rejection. Most cytotoxic T cells are CD8+ T cells that recognize antigen presented by MHC class I molecules, although a considerable number of CD4+ T cells have the capability to kill target cells. Killing by a cytotoxic T cell begins with adhesion to the target cell (which requires magnesium ions), followed by the delivery of cytotoxic chemicals to the target cell (which requires calcium ions). The cytotoxic T cell then dissociates from the target cell; death proceeds in the absence of the cy-totoxic T cell, which recycles to attack another target cell. If the cytotoxic T cell adheres to a cell that does not carry the targeted antigenic peptide-MHC molecule combination, no cytotoxic chemicals are released and the cells dissociate more rapidly.
Cytotoxic T cells develop granules that contain cytotoxic molecules, including perforins (proteins that produce holes or pores in a cell’s surface membrane), serine proteases (granzyme A and granzyme B), and serine esterases.46 Of these, perforins are the most important, as has been shown in mice in which the gene encoding perforin has been deleted. A second killing mechanism involves the Fas ligand on the cytotoxic T cell and Fas on the target cell. Binding of these leads to apoptosis of the target cell. This is the only mechanism of killing available to mice that lack perforin, and it is used preferentially—but not exclusively—in CD4+ cytotoxic T cells [see Figure 5].
Viral infection results in the production of a large number of virus-specific cytotoxic T cells. This is most dramatically shown during the initial responses to B cells infected with Epstein-Barr virus. Cytotoxic T cell clones specific for some antigen-MHC complexes are extremely abundant, constituting approximately 50% of all cytotoxic T cells.9 When the cytotoxic T cell response diminishes, these abundant T cell clones are probably removed through apoptotic mechanisms.
Activated macrophages
Macrophages are usually activated by Th1 cells that have been stimulated by antigens or microorganisms. Those Th1 cells then express CD40 ligand (CD40L) and produce macro-phage-activating cytokines, especially IFN-y and MIF. These cytokines, in combination with CD40L interacting with the CD40 on the macrophage surfaces, induces intracellular signaling transcription pathways in the macrophage. These pathways result in activation of transcription factors leading to production of various proteins and surface markers that characterize the activated macrophage. Activated macrophages produce reactive oxygen intermediates, including nitric oxide, that are involved in the destruction of microorganisms or foreign cells.
Bacterial killing also involves phagolysosomal fusion, which mobilizes enzymes such as cathepsins.
Activated macrophages produce many of the inflammatory cytokines (e.g., IL-12, TNF-a, IL-1, and MIF) and chemokines (e.g., MIP-1) that are involved in immunity to microorganisms and foreign antigens and in enhancing the process of activation itself. Activated macrophages also express MHC class II and costimulatory molecules, which further amplify the process. In addition, activated macrophages produce the cytokines IL-10 and TGF-p; these counteract the activation, damping down the inflammatory process and acting as feedback regulators [see 6:I Organs and Cells of the Immune System].
