Neutrophils are the first circulating phagocytic cells recruited to the site of infection and inflammation to ingest, kill, and digest pathogens. These cells are produced from myeloid stem cells in the bone marrow (Fig. 1-2). Neutrophils constitute the large number of leukocytes in the blood. After stimulation, mature neutrophils display more motility, adherence, phagocytic activity, and intracellular killing than any other type of cell (Fig. 1-5). Neutrophils persist in the circulation for only several hours. Then, they are either removed by the RES or migrate into inflammatory sites.
Figure 1-5. Development and function of neutrophils.
Adherence: The transmigration of leukocytes through the intercellular junctions of endothelium and their adherence to endothelium are dependent in part upon membrane glycoproteins such as LFA-1 and Mac-1. These belong to the integrin family of proteins and consist of alpha/beta heterodimers, which are restricted to leukocytes. Their beta-chains are identical, whereas the alpha-chain of each class of protein is distinct. Other adherence molecules distinct from integrins are L-selectin and ELAM-1.
Chemoattraction: Chemoattractants play a very important role in the recruitment and activation of leukocytes. The movement of leukocytes within the interstitium is largely adherence- independent and due mainly to hydraulic forces. Once neutrophils enter the interstitium, they may be further activated by chemoattractant agents released by invading microorganisms or produced by the host in response to injury. These chemoattractants include N-formylmethionyl peptides from bacteria, a proteolytic fragment of the fifth component of complement (C5a) (see section on complement system), an inflammatory mediator leukotriene B4 produced from the metabolism of arachidonic acid, and interleukin-8.
Opsonization: Molecules that coat the surface of foreign particles and are ligands for receptors on the surface of phagocytes (opsonins) aid in the ingestion of those particles (opsonization). Four major types of opsonins are fibronectin (a cold-insoluble globulin), specific IgG antibodies, and active fragments of the third component of complement, C3b and C3bi. The antibodies and complement fragments facilitate the adherence of microorganisms to neutrophils by binding to specific receptors in the external membranes of the leukocytes. Mac-1 not only aids in adherence but also in phagocytosis by its role as the C3bi receptor.
As a result of the membrane perturbation caused by foreign particles adhering to the external membrane of the phagocyte, a chain of events is initiated that culminates in the engulfment of the particle (i.e., phagocytosis or the formation of a phagosome), the fusion of the phagosome with primary (lysosomal or azurophilic) and secondary (specific) cytoplasmic granules, and the assembly of the major intracellular microbicidal system. The sequence of events is as follows. As the plasma membrane of the phagocyte invaginates, microfilaments accumulate in the nearby cytoplasm. Consequently, the invagination closes to form a phagosome. Simultaneously, a signal is transduced from the receptor-ligand complex through a guanine nucleotide binding protein to activate phospholipase-C in the plasma membrane. As a result, two secondary messengers are produced. The first, inositol triphosphate, stimulates the flux of intracellular calcium. The second, diacylglycerol, participates in the activation of protein kinase C and phospholipase A2. Primary granules contain a high content of acid hydrolases and proteolytic enzymes that inactivate or digest microorganisms; secondary granules contain lactoferrin, gelatinase, complement receptors CR1 and CR3, and an essential part of the intracellular killing machinery, cytochrome b558.
Microbicidal Mechanisms: Neutrophils produce chemicals that are capable of inactivating ingested microorganisms. Once neutrophils are activated, intracellular mechanisms are turned on that lead to the conversion of oxygen to superoxide and then to hydrogen peroxide in the presence of superoxide dismutase. The process includes the assembly of NADPH oxidase, and up-regulation of cytochrome b558 from membranes of specific granules. Hydrogen peroxide then reacts with chloride ions in the presence of myeloperoxidase to form chlorinated derivatives. In addition to the formation of microbicidal agents, simultaneously, primary and secondary granules extrude from the cell where they attack extracellular pathogens, or if the process is excessive, host tissues.
Eosinophils play a major role in the killing of parasites, particularly hemoflagellates, echinococcus, and enteric nematodes. This killing is due to a basic protein and a cationic protein contained in large cytoplasmic granules that are unique to eosinophils. These cells also play a prominent role in the pathogenesis of the allergic inflammation. These cells are induced to grow and differentiate by interleukin-5 and are recruited to inflammatory sites by agents such as platelet-activating factor from the lipoxygenase segment of the arachidonic acid pathway.
Basophils and their tissue counterparts, the mast cells, play a major role in defense against parasites and in allergic inflammation. These cells are distinguished by many large cytoplasmic granules that contain heparin and histamine and by high affinity receptors for IgE antibodies. If these cell bound IgE antibodies are cross-linked by antigens, the cells degranulate and are activated to produce and secrete a group of low-molecular weight vasoactive mediators and certain proinflammatory cytokines, e.g. tumor necrosis factor alpha (TNF-alpha) and interleukin-5 (IL-5).
Some functions of macrophages such as phagocytosis and intracellular killing are similar to those of neutrophils, whereas others are distinct. The distinctive features are as follows: a) They are able to reside in the RES for long periods. b) They process protein antigens and present the resultant peptide fragments to T cells in the context of MHC class II molecules. They produce cytokines (Table 1-2). These cells are also highly adherent, motile and phagocytic. These properties are greatest in activated macrophages, somewhat less in unstimulated macrophages, and least in monocytes. The role of these cells in processing and presenting antigens is dealt with in the next section.
Monocytes and macrophages are activated by bacterial products such as endotoxin (lipopolysaccharides); autocrine agents, such as TNF-alpha, IL-1, and IL-8; cytokines such as interferon-gamma (IFN-gamma) and a special group of mediators called chemokines. Activated macrophages play a prominent effector role in cellular immunity by 1) ingesting and killing pathogens, 2) clearing immune complexes, and 3) aiding in the genesis of specific immune responses by antigen presentation.
These cells are responsible for the development and maintenance of specific immunity. Lymphocytes are comprised of three separate populations, T cells, B cells, and NK cells, each of which express different phenotypic and functional properties (Fig. 1-3). Two major types, T and B cells, produce and express specific receptors for antigens.
T lymphocytes are thymus-derived lymphocytes and play a central role in the generation and regulation of the immune response to protein antigens. T cells originate from bone marrow stem cells (Fig. 1-2) that develop into T precursor cells that migrate to the thymus where they multiply and differentiate (Fig. 1-6). The rate at which the thymus produces T cells is very high in childhood and declines thereafter. Because mature T cells are long lived and recirculate (Fig. 1-7), they comprise about 70-80% of lymphocytes in blood and lymph, and they are responsible for much of the immunologic memory.
Figure 1-6. Ontogeny of B and T lymphocytes.
Figure 1-7. Lymphocyte circulation pathways. T cells are principally recirculating; B cells are principally sequestered in peripheral lymphoid organs.
Maturation: The maturation of T cells takes place in the thymus and is characterized by a sequential appearance of certain cell surface molecules. Among the first surface molecules to appear are CD3, T cell receptors (TcR), which are alpha/beta positive (Fig. 1-8) ; CD4; and CD8. Thus immature thymocytes are CD3+CD4+CD8+. Cortical T cells lose either CD4 or CD8 molecules to become CD3+TcR+CD4+ or CD3+TcR+CD8+. Mature T cells migrate to the medulla of the thymus from where they exit into the systemic circulation.
Figure 1-8. The TcR-CD3 complex on helper (CD4+) or cytotoxic/suppressor (CD8+) T cells. The TcR receives peptide fragments from antigen presenting cells. CD3 is a signaling molecule.
The TcR recognizes protein antigen determinants that are presented by MHC molecules (see below). In addition, TcR are physically associated with CD3. This association with CD3 is required for transmembrane signaling that culminates in T cell activation.
Role of MHC in T Cell Development: One major function of MHC molecules is to present antigens to T cells. Lymphocytes in the thymus are exposed to various endogenous (self) proteins, particularly the products of MHC (see below). Some nascent T cells that have specificity towards self MHC molecules are eliminated (negative selection), while remaining T cells become "educated" to recognize foreign antigenic peptides that are associated with self MHC (positive selection). Thus, antigen recognition by T cells becomes "MHC restricted," that is, the mature T cell recognizes its specific antigen only if that antigen is presented by the correct MHC molecule.
Two kinds of MHC genes, class I and class II (see Fig. 1-9 for their protein products) (see section on MHC), are involved in the development of T cells. In the course of selective adaptation, T cells learn to recognize foreign antigens in association with protein products of either MHC class I or II genes. MHC class I-restricted T cells express CD8 molecules that bind to the invariant portion of MHC class I, whereas MHC class II-restricted T cells express the CD4 molecule that binds to MHC class II molecule. Thus, mature T lymphocytes leaving the thymus are either CD4+ or CD8+ (single positive) and express CD3 and TcR molecules.
Figure 1-9. Structures of MHC class I and II molecules. Binding sites in the molecules are shown for processed protein antigens for presentation to T cells. Leters N and C represent N and C termini of the polypeptide, respectively.
T-Cell Subpopulations: Both CD4 and CD8 molecules participate in T cell activation. CD4+ T cells are principally regulatory cells, which control the functions of other lymphocytes. Based on the lymphokines they produce, CD4+ Th cells are divided into two subsets, namely Th1 cells that promote cellular immunity (Fig. 1-10), and Th2 cells that help antibody production (Fig. 1-11). CD8+ T cells are cytotoxic/suppressor cells which participate in cell-mediated immunity against viruses, fungi, bacteria, and against certain tumors and play a role in immune regulation.
Figure 1-10. Genesis of cellular immunity and T-cytotoxic cells by activation of Th1 cells.
T Helper (Th) Cells: These cells are involved in the regulation of both T cell and B cell- mediated immune responses. IgG, IgA, and IgE antibody responses against T-dependent antigens require Th2 cells. Th2 cells aid antigen-activated B cells to proliferate and differentiate into antibody-producing plasma cells and to undergo class switching (Fig. 1-11). They recognize foreign antigens complexed with MHC class II molecules on antigen-presenting cells (B cells, macrophages, dendritic cells and Langerhans cells).
Figure 1-11. Immunoglobulin isotype switching. Reconfiguration of genes for IgM to IgA while retaining the same antigen binding specificity. According to which switch sites combine, the intervening DNA is looped out and eventually deleted. In this illustration, an IgA antibody gene containing VDJ genes and C-alpha gene is formed.
Antigens are presented to Th2 cells in two ways. In the first (Fig. 1-12), the antigen is taken up and processed by accessory cells, such as macrophages or B cells, that present the Ag/MHC complex to Th2 cells. Activated T cells then produce lymphokines that recruit and activate B cells to produce antibodies. Unlike phagocytic cells, B cells bind the antigen by specific antibodies, then they internalize and process the antigen (Ag), and express a fragment of it bound to MHC class II molecules on the cell surface in the context of MHC class II molecules. Antigen-specific Th2 cells that bind the Ag/MHC complex on the antigen-presenting cells become activated and produce helper factors for adjacent B cells. Furthermore, macrophages may process and present antigens without MHC products to B cells or, in the case of complex polysaccharides, the antigen may be presented directly to B cells (Fig. 1-12) without the aid of other cells. Which pathway is used depends on the nature of the antigen.
Figure 1-12. Antigen presentation mechanisms.
Helper T cells (Th1) also aid effector T lymphocytes (vide infra) in cell-mediated immunity. This process occurs according to the pathway depicted in Figure 1-12, except that the recipients of the helper factor are effector T cells.
B and T cells require different cytokines for growth and differentiation. The pattern of the production of those particular factors define whether the cells are Th1 or Th2. For example, Th1 cells produce IFN-gamma, a cytokine that activates macrophages. Those activated macrophages in turn participate in delayed hypersensitivity, a major aspect of cell-mediated immunity (Fig. 1-10). In contrast, Th2 cells produce cytokines such as IL-4 and IL-10, which activate certain phases of antibody production and inhibit the genesis of delayed hypersensitivity.
Delayed Hypersensitivity: Cell-mediated antibacterial resistance (delayed hypersensitivity) is mediated by CD4+ Th1 cells in concert with macrophages. T cells activate macrophages via the production of IFN-gamma and other lymphokines. Initially, antigen-specific Th cells migrate to the site of infection. After activation by antigen, the cells produce a myriad of cytokines that attract and activate monocytes, macrophages, and other lymphocytes. If the infection is not resolved promptly (as in Mycobacterium tuberculosis infection), it could lead to chronic inflammation or even to granuloma formation.
Suppressor T Cells: These cells are involved in antigen-specific suppression and thus play an important role in maintenance of self-tolerance. T suppressor cells are less well understood than Th cells. T-suppressor lymphocytes are usually CD8+.
Cytotoxic T Cells: These cells destroy virus-infected cells and certain types of tumor cells in an antigen-specific manner. Cytotoxic T cells (CTL) (Table 1-1) are usually CD8+ and MHC class I-restricted. Recognition of endogenous foreign peptide (i.e., viral antigenic peptide) in the context of MHC Class I molecule by TcR of CD8+ cells, stimulates the CD8+ cells to become CTLs. The CTL killing is antigen-specific and MHC class I restricted (i.e., target cells infected by a different virus or infected cells that do not express the correct MHC class I molecule are spared). Th cells could also influence the CTL function.
In special cases, alloreactive (reactivity against foreign histocompatibility antigen) CTL recognize and kill target cells expressing a foreign MHC molecule, as found in MHC-incompatible tissue transplants.
These cells provide innate protection by killing tumor cells and cells infected with intracellular pathogens. Natural killer cells are large granular lymphocytes that do not express CD3, TcR, or immunoglobulins (Table 1-1), but display a low-affinity surface receptor for the Fc fragment of IgG (CD16; e.g. CR3) and CD56 (Fig. 1-3). Natural killer cells account for 10-15% of blood lymphocytes and are found in low numbers in the peripheral lymphoid system.
Natural killer cells regulate certain aspects of T and B cell activation and hematopoiesis, and they defend against certain tumors and intracellular infections by killing the involved cells. In contrast to cytotoxic T cells, the NK cell-mediated cytotoxicity neither requires previous sensitization nor is MHC-restricted. The cytotoxicity of NK cells is increased after exposure to cytokines such as IL-2 or IFN-gamma. NK cells also mediate antibody-dependent, cell-mediated cytotoxicity via the CD16 Fc receptor. NK cells can be activated to produce cytokines (IL-2, IFN-gamma, IFN-alpha, TNF-alpha) that aid in immunomodulation.
These cells are primarily involved in antibody production and antigen presentation to T cells. B cells originate from lymphoid stem cells in the fetal liver and the bone marrow (Fig. 1-2). B lymphocytes are thymus-independent cells that express intrinsically produced immunoglobulins (vide infra) on their external membranes and upon stimulation by antigen differentiate into plasma cells that produce and secrete large numbers of antibody molecules (Fig. 1-6). Pre-B cells, the immediate precursors of B cells, are restricted to the bone marrow and are characterized by the presence of cytoplasmic µ chains (H chains for IgM) but no L chains. Mature but unstimulated B cells express monomeric IgM antibodies, MHC class II molecules, CD19, CD20, the Epstein-Barr virus/C3d (CR2) (CD21) receptor, and T cell interaction molecules, B7-1, B7-2, and the CD40 ligand, CD39 (Fig. 1-13). B cells account for 10-15% of blood lymphocytes. They, and their progeny, antibody-producing cells, primarily reside in peripheral lymphoid organs.
Figure 1-13. Surface markers on B cells.
Each B cell expresses and produces immunoglobulin molecules of one antigen-binding specificity. Clones expressing different specificities are involved in the production of antibodies to a complex immunogen because of the multiplicity of antigenic determinants (epitopes) on the molecules. Hence, many separate clones of B cells are required to produce the overall antibody response (a polyclonal response). If the immunogen has a very limited set of epitopes, the antibody response will be oligoclonal or monoclonal.
Development: The development of B cells from stem cells through mature B cells is antigen-independent. Antigen is, however, the initial trigger for B cells to transform into antibody-producing, secretory plasma cells. After antigens bind to immunoglobulins on the cell surface, the antigens are internalized and processed. This antigen/receptor interaction sends the first biochemical signal for the B cell activation. In the case of proteins, a fragment of the antigen is transported to the surface where it is expressed in a complex with MHC class II molecules. This allows B cells to interact with antigen-specific helper T cells. Consequently, cytokine receptors are expressed on the B cell surface and T cells are activated to produce cytokines, such as IL-2, IL-4, IL-6, and IL-10 (Table 1-2), that further stimulate proliferation and differentiation of B cells. In addition, certain bacterial products (generically called mitogens) such as lipopolysaccharides, activate B cells to proliferate regardless of their antigen specificity. That results in a non-specific polyclonal antibody response.
Isotype Switching: B lymphocytes switch their immunoglobulin production from IgM to IgG, IgA, or IgE, during the course of immune response against T cell-dependent antigens. Lymphokines from T helper cells are necessary for the class (isotype) switch that occurs in antigen-stimulated B cells. These events in B-cell differentiation are accompanied by immunoglobulin gene rearrangements, which will be described later in this chapter (Fig. 1-14). As a result of the recombination of the same VDJ genes with a different C region gene, a different isotype of immunoglobulin with the same antibody specificity is produced (Fig.1-11). Once the mature B cell encounters the appropriate antigen, the cell differentiates to form a plasma cell. Plasma cells are characterized by a lack of surface membrane immunoglobulin, but have an extensive production and secretion of antibodies of one isotype and specificity for a single epitope (idiotype).
Figure 1-14. Antibody diversity is principally generated by immunoglobulin gene rearrangement. H-chain gene rearrangement is depicted.