ALLERGY AND IMMUNOLOGY

Understanding immunology is fundamental to understanding the cause, diagnosis, and management of many diseases. A basic understanding of the immune system is essential for all physicians. Besides helping to understand disease processes, knowledge of the immune system is essential in the evaluation of recurrent bacterial infections. Most determinations in diagnostic immunology laboratories are based on well-established principles of antigen-antibody reactions. This chapter is an overview of immunology and the allergic response. The field of immunology continues to evolve rapidly, as indicated by the identification of more than 161 cluster of differentiation surface antigens and more than 18 interleukin molecules.
The immune system differentiates self and nonself. It identifies and destroys elements foreign to the body and recognizes and protects self components. Immune surveillance is the mechanism by which the immune system determines on a cellular and molecular level how to deal with foreign invaders or with deviations of self-constituents. If the immune system detects something foreign on a cell surface, a reaction aimed at eliminating that cell begins. The immune system is anatomically and functionally divided into three compartments—primary lymphoid organs that produce lymphocytes; lymph nodes and the spleen, which provide a microenvironment for efficient interactions between lymphocytes and antigens; and the extralymphoid or tertiary lymphatic tissues.
The cells primarily responsible for immune recognition are lymphocytes, which have surface-specific receptors for antigenic determinants, or epitopes, of foreign molecules. Because each lymphocyte bears several copies of the same receptor, the body needs millions of lymphocytes, each with different receptors, to recognize the myriad foreign substances that humans encounter during their lives.
The clonal selection theory of immune cell origin and development suggests that, first, specific antigens select only the appropriate lymphocyte clone and, second, the specificity of lymphocytes develops before the introduction of antigen. When an antigen contacts and binds to a receptor, the lymphocyte becomes activated and then proliferates. Proliferation or clonal expansion leads to production of a large number of lymphocytes with the same receptors as those of the parent cell. If the body contacts the same antigen in the future, the number of cells that can recognize it increases, and the reaction becomes faster and more effective; that is, there is a positive immunologic memory. Sometimes the first exposure to an antigen reduces the likelihood of a response to a second stimulus; that is, there is negative memory or immunologic tolerance. The other transformation that lymphocytes undergo is differentiation, by means of which they initiate protein synthesis of lymphokines and antibodies.
The immune system has nonspecific effector mechanisms that amplify the specific responses—the innate immune system (1). These nonspecific features include the response of mononuclear phagocytes, polymorphonuclear leukocytes, and the complement system as well as enzymes, such as lysozyme, physiologic mechanisms, such as ciliary motion, interferons, and proteins, such as acute-phase proteins.
DEVELOPMENT OF THE IMMUNE SYSTEM
The cells involved in immune reactions are derived from pluripotential hematopoietic stem cells (2). These pluripotential stem cells arise from the bone marrow and give rise to precursors in the erythroid, myeloid, and lymphoid lines. Molecules known as cluster of differentiation (CD) on the surface of immune cells serve to identify subpopulations, and they function in cell differentiation. For example, marrow hematopoietic stem cells display CD34, whereas T cells can display CD2, CD3, CD4, CD5, CD6, CD7, CD8, or CD28. The lymphoid precursors become either pre-B or pre-T cells.
Mature B cells have antibodies on their surface that act as antigen receptors. During early stages of development, B cells are inactivated by contact with self components (clonal abortion). Mature B cells, which escape clonal abortion, leave the bone marrow and migrate to germinal centers within the lymphoid follicles of lymph nodes and the spleen.
Pre-T cells initially travel from the bone marrow to the thymus to complete their maturation. Prethymocyte receptors are generated by random gene arrangements and must bind to class I or class II antigens of the major histocompatibility complex (MHC) to survive. Cells that do not recognize self (MHC) are destroyed, as are cells that bind too tightly to the MHC (these have potential for inducing autoimmune disease). Maturation involves interactions among T cells, thymocytes, and maturational hormones such as thymosin, thymopoietin, and thymulin, produced by the thymic stroma. There are positive and negative selection mechanisms for immature T cells, which maximize their functioning. Positive selection is mediated by termination of each cell by programmed cell death (apoptosis), which proceeds through intracellular messages. If there is little or no binding of the T-cell receptor (TCR) to the peptide-MHC complex, apoptosis ensues, and the cell is eliminated. The mature T cells leave the thymus and become localized in the deep cortex of lymph nodes and in the perivascular areas of the splenic medulla. This distribution optimizes interaction among T cells, B cells, and macrophages.
Lymphocytes (mainly T cells) recirculate between lymph nodes, blood, lymphatic channels, and some organs, providing immune coverage of the whole body. The process in which immune cells migrate into areas of inflammation is vital to host defense. Coordination of the sometimes rapidly fluctuating relocation of immune cells involves a number of molecules. For lymphocyte migration from the bloodstream, integrins (glycoproteins) on the cell surface mediate cellular attachment to endothelium. Inflammation incites endothelial cells to signal lymphocytes to activate their integrins by elaborating a family of molecules called chemokines. When an immune cell surface receptor contacts its complementary antigen, the activity of molecules involved in adherence to endothelium greatly increases. Once the lymphocyte adheres to endothelium, it “rolls” along the vessel, allowing sustained membrane contact. The rolling feature is mediated through an interaction of selectins, which are another family of surface glycoproteins on immune cells. After exhibiting the rolling feature, the cell penetrates between endothelial cells. Extracellular matrix proteins (fibronectin, laminin), intercellular adhesion molecules (ICAM-1), fibrinogen, and vascular adhesion molecules (VCAM-1) mediate cellular movement through tissue.
The process of cellular adhesion is similar to the complement cascade, in which one molecular interaction follows another, and the final outcome can be disrupted by the ineffectiveness of any step. Understanding adhesion of lymphocytes and of other circulating cells provides several approaches to inhibition of this cascade. These include receptor-ligand binding by monoclonal antibodies, binding of small molecules to ligands, and antisense oligonucleotides to target endothelial cell adhesion molecules and to inhibit nuclear factor-kb, which regulates gene expression of several adhesion molecules, such as ICAM-1, VCAM-1, and E-selectin. These approaches must balance the importance of adhesion in host defense against the tissue damage induced by an overzealous response. Persons deficient in adhesion molecules are at risk of severe bacterial infection.
CELL-MEDIATED IMMUNITY
Monocytes, macrophages, dendritic cells, Langerhans cells, and B cells can function as antigen-presenting cells (APC), in which engulfed antigens, such as proteins, viruses, and bacteria, are partially degraded in their phagolysosomes and presented on the cell surface (3). Fragments of these antigens reappear later on the phagocyte surface. With its receptor, the T cell recognizes both the presented antigen and the markers of self (MHC) attached to the phagocyte surface. These markers of self originate in the MHC, located on chromosome 6.
Two kinds of MHC antigens exist. Class I is composed of two polypeptide chains, one constant from person to person and the other highly variable. Class I antigens appear on the surface of all nucleated cells in the body and have CD8 as the TCR. Class II antigens, composed of two variable polypeptide chains, are present on the surface of APCs and B cells and have CD4 as the TCR. Major histocompatibility complex antigens can be induced (class I) or repressed (class II) by the same cytokine (small proteins). This shows that cells can have differential responses to the same immune mediator. Cytokines are released from the APCs and alter the immune function of the presenting cell and other cells in the immediate area. For example, interleukin-1 (IL-1), which is primarily monocyte-macrophage derived, stimulates proliferation of B cells and some T cells, hematopoiesis, and synthesis of tumor necrosis factor a (TNF-a). The redundancy of cytokine functions combined with their proinflammatory and anti-inflammatory activities makes it difficult to understand the role of these substances in disease.
Antigens on the surface of APCs contact helper T (TH) cells. The TH cell recognizes foreign antigens and class II MHC antigens. To become activated, a TH cell needs not only antigen and MHC binding but also IL-1, a growth factor produced by the APCs. Costimulatory molecules binding through CD40 also play a role. The TH cell secretes other growth factors, such as IL-2, which can stimulate the TH cells to exhibit IL-2 receptors on their surface. The up-regulation of IL-2 receptors produces an amplification mechanism. Most immune responses require soluble growth and differentiation factors such as IL-2. Persons deficient in these factors have severe impairment of the immune system.
Helper T cells have been classified into subsets, TH1 and TH2, on the basis of their distinct lymphokine secretion profile and function. The TH1 clones secrete IL-2, IL-3, IL-6, IL-10, TNF-a, TNF-b, interferon-d (IFN-d), and granulocyte-macrophage colony-stimulating factor (GM-CSF). They also proliferate in response to antigen presented by both B cells and macrophages without a requirement for IL-1. Helper T cells in subset 1 induce IgM, IgG, and IgA but not IgE responses, and they stimulate cell-mediated immune responses such as eradication of intracellular bacteria and viruses and delayed-type hypersensitivity. The TH2 clones proliferate suboptimally in response to antigen presented by B cells, unless IL-1 is added. The TH2 clones, which secrete IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, TNF-a, and GM-CSF are more effective than TH1 clones at assisting antibody secretion. In particular, IL-4 and IL-13 promote the switch of B cells to IgE production. The cytokine microenvironment and the type, amount, and site of antigen exposure affect the type of TH cell that develops. Helper T cells in subset 2 have been recovered from nasal and bronchial tissues of patients with allergies after antigenic challenge. The differentiation of TH0 cells to TH1 or TH2 cells may explain how different immunization conditions can preferentially or selectively induce either humoral or cell-mediated immune responses, and it may explain distinct patterns of disease, such as the polar forms of lepromatous and tuberculoid leprosy. The switch from a TH2 cell to a TH1 cell response has been postulated as the mechanism underlying effective allergen immunotherapy and the reason for the increasing prevalence of allergic rhinitis (4).
Delayed-hypersensitivity T cells (TDH cells) also recognize antigen. Along with class II MHC products, TDH cells become activated by IL-2 and secrete lymphokines. Some of these lymphokines activate and attract macrophages, which ingest and destroy the antigen. This is the basis of skin testing for evaluation of delayed hypersensitivity. Antigen, such as tuberculin, mumps vaccine, or candida, is injected intradermally. If the antigen is recognized, local inflammation occurs, and cytokines are released that increase expression of the adhesion molecules ICAM-1 and VCAM-1 on the vascular endothelium. Adhesion molecules and surface molecules on TDH cells, such as very late activating antigen 4 (VLA-4), interact during T-lymphocyte movement to the area. The site becomes indurated 24 to 48 hours later. A positive response means that the patient has adequate APCs in the skin, as well as adequate numbers of functioning TDH cells and macrophages. CD8 T lymphocytes, like CD4 T lymphocytes, have been shown to have subsets (Tc1 and Tc2). Tc1 cells secrete IFN-g, and Tc2 cells secrete IL-4. The role of these cells is just beginning to be appreciated.
Cytotoxic, or killer, T cells (TC cells) recognize antigen coupled to class I MHC products (4). They kill the body’s own cells that have undergone change, such as viral or malignant transformation. For activation, TC cells must recognize a class I MHC plus antigen specific for virus and then get help from TH cells. Once activated, the TC cells circulate in the body and kill all cells that bear the new antigen through the transfer of cytolytic compounds (perforin and granzymes). Perforins and granzymes primarily destroy some viruses and intracellular bacteria, and they mediate rejection of grafts and tumors.
T cells can cause cell death (apoptosis) by binding to a molecule, Fas (CD 95), attached to the surface of most cells. Binding of Fas to its ligand leads to apoptosis of the cell expressing Fas through activation of an intracellular cascade of proteases. Fas molecules are involved mostly in tolerance induction and regulation of T-cell maturation. Fas expression on T and B cells increases after an encounter with an antigen. Fas ligand begins to be expressed on mature CD4 and CD8 T cells after activation. Toward the end of an immune response, they can induce apoptosis in cells that have up-regulated Fas. Abnormalities in this process are responsible for a childhood disorder of lymphoproliferation and for autoimmune thyroiditis. Fas ligand also is permanently expressed in the eye and testis and is thought to be responsible for immune privilege at these sites. That is, anatomic sites where transplanted foreign tissues survive for an extended time in a person with normal immune function. How tumors provide their own environment of immune privilege is an active area of investigation (5).
Suppressor T cells (TS cells) also participate in the normal regulation of immunity. Their role in human immunology, however, has not been defined precisely. They probably down-regulate TH cells and secrete suppressing lymphokines in an antigen-specific or nonspecific manner. Evidence suggests that excess TS cell function occurs in some immunodeficiency states, whereas TS cell deficiency occurs in some autoimmune diseases (6).
gdT cells play a role in microbial immunity. Although the precise role is not defined, these cells appear to be involved in terminating the host immune response to infection and preventing chronic disease. After activation, gdT cells acquire cytotoxic activity and kill stimulatory macrophages (7).
Unlike TC cells, which appear in the tissues in response to antigen, another class of killer cells, called natural killer (NK) cells, does not depend on previous immunization. They kill some types of tumor cells, primarily of the hematopoietic system, by means of release of perforins and proteases and by means of induction of apoptosis. It is possible that they regulate lymphocyte development and that their ability to kill tumors represents a cross-reaction. In animals, tumors that are not susceptible to NK cells have greater malignant potential than do NK-susceptible tumors. The activity of NK cells increases greatly with exposure to interferon. These cells have an as yet undefined role in the control of viruses, bacteria, and parasites. Natural killer cells lyse specific cells when antibody is present, a method termed antibody-dependent cellular cytotoxicity. Natural killer cells look like large, granular lymphocytes, but differ from mature T cells or B cells. They display CD2, CD16, and CD56 markers, which are not classic B-cell and T-cell markers, and they do not express surface immunoglobulins. Most can be identified by the monoclonal antibody Leu-11. Interleukin-2 causes activation and proliferation of NK cells, now called lymphokine-activated killer cells (LAK cells), which exhibit antitumor activity (8).
T-cell receptors for antigen are unique markers on the surfaces of T cells and are heterodimers. Monoclonal antibodies to these surface markers can identify T cells and their subpopulations. For example, CD3 and CD2 are antibodies that recognize all T cells, whereas CD4 identifies helper and TDH cells and CD8 identifies cytotoxic and suppressor T cells. T cells represent about 65% of lymphocytes in the peripheral blood. Unlike B-cell receptors, which increase their affinity to antigens by means of mutation, TCRs do not. Genetic recombinations of TCRs can produce a nearly infinite number of antigen receptors. This is important in the distinction between self and nonself (a T-cell function) and in prevention of autoimmunity. Once processed, antigen and associated MHC bind TCR, and CD22 on B cells binds CD45 on T cells, which activates intracellular phosphatyrosine phosphatase. This activates tyrosine protein kinase, which phosphorylates components of the CD3 complex and leads to hydrolysis of membrane phosphoinositides. The diacylglycerol and inositol 1,4,5-triphosphate produced then act as second messengers to mobilize calcium. This activates protein kinase C, which phosphorylates other proteins, and this causes the genetic effects that produce cell activation and elaboration of proteins.
T-cell receptors do not bind soluble antigen as do their antibody counterparts on B cells. Instead polypeptide antigens are internalized and processed by special APCs, which give rise to short peptides. These bind to molecules of the MHC, and the combination is recognized by the TCR. Superantigens, powerful microbial toxins, produced by Staphylococcus aureus and Streptococcus pyogenes organisms cause fever and shock by binding to class II MHC and TCR molecules. Binding to HLA-C antigen inhibits the lytic capability of NK cells. T cells have a graded response to antigens from activation to complete unresponsiveness.
HUMORAL IMMUNITY
Unlike T cells, B lymphocytes (B cells) have numerous receptors that bear striking similarities to the immunoglobulin molecules they later secrete. These receptors initially belong to the IgM and IgD classes, but later shift to IgG, IgA, or IgE. Each receptor reacts to one antigen, so millions of B cells are needed to ensure proper immune function. Unlike T cells, B cells do not need the joint recognition of self-markers and antigens (9). Eighty percent to 90% of all immunoglobulin-producing cells are located in the mucosa and exocrine glands. The adhesion molecules a4b7 and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) appear important in localizing B cells to these areas (10).
Some antigens can directly activate B cells (T-independent antigens), whereas other antigens require T-cell cooperation (T-dependent antigens). In T-dependent activation, the B cells first contact antigen through IgM and IgD molecules on the cell surface, then process and present the antigen. The processed antigen later appears on the surface of the B cells in association with class II MHC. An antigen-specific T cell binds to this combination with the help of several membrane proteins (TCR, class II MHC, leukocyte function-associated antigen 1 [LFA-1], and ICAM-1). The activated T cell then expresses gp39, which binds to CD40, a membrane protein on B cells, and which is a potent mitogen receptor. With the assistance of lymphokines, the B cell begins to proliferate and differentiates into an antibody-secreting cell (plasma cell) that no longer carries immunoglobulins on its surface. However, a percentage of B cells that have been clonally selected remain as memory cells. These cells possess a high density of high-affinity surface immunoglobulins, usually of the IgG, IgA, or IgE class. T-dependent activation occurs with complex antigens, because the antigenic determinants are not accessible to the B cell because of stereometric hindrance, forcing the antigen to be processed before being presented.
T-independent antigens have large structures with repeating antigenic determinants (epitopes), such as carbohydrates, which can cap and bridge immunoglobulins on the B-cell membrane. This mode of B-cell activation is inefficient and provides primarily IgM antibodies, because the isotype switch from IgM to IgG production in the same B cell requires T-cell factors such as IL-4 and IFN-d. Isotype switching is mediated by specific genetic rearrangements and maintains antigenic specificity. Independent antigens include carbohydrates from the capsule and cell wall components of bacteria. They do not include most protein antigens. This may explain why patients with severe compromise of T-cell function maintain antibody levels to bacterial pathogens.
IMMUNOGLOBULINS
Immunoglobulins are glycoproteins composed of 82% to 96% polypeptide and 4% to 18% carbohydrate components (2) (Fig. 8.1). They account for approximately 20% of the total plasma proteins. All immunoglobulin molecules contain an equal number of heavy (H) and light (L) polypeptide chains. Each polypeptide chain consists of a number of domains of constant size (100 to 110 amino acid residues) linked by intrachain disulfide bonds. The N-terminal domain of each chain, designated as variable region (Fab), shows more variation in amino acid sequence than does the C-terminal end (constant region, Fc). The antigen-binding site of the antibody molecule represents only a small number of amino acids in the V regions of H and L chains. However, because a number of gene segments can combine to form new V, D, and J segments, a nearly unlimited number of antigenic specificities are possible. These amino acids are brought into close relation by the folding of the V regions. Covalent interchain disulfide bridges hold the chains together and form a bilateral symmetric structure. The polypeptide chains fold into globular regions called domains. The domains in H chains are designated VH (variable region of heavy chain) and CH1, CH2, CH3, and CH4 (constant region of heavy chain), and those in L chains are designated VL and CL. Both ends of antibodies function in that the Fab portion (antigen-binding fragment) binds with specific antigens, whereas the Fc portion initiates a variety of secondary phenomena, such as complement fixation.
All of the L chains have a molecular weight of approximately 23,000 and can be classified into two types, k and l, on the basis of structural differences in constant regions. In humans, k chains outnumber l chains two to one. Any immunoglobulin molecule always contains identical k or l chains.
Five classes of H chains exist in humans. They are based on structural differences in the constant region. The different H chains, designated g, a, µ, d, and e, vary in molecular weight from 50,000 to 70,000. The µ and e chains possess five domains (one variable and four constant) rather than the four domains of g and a chains. The H chain determines the class of the immunoglobulin. There are five classes of immunoglobulins: IgG, IgA, IgM, IgD, and IgE. Most of the H-chain classes have been further subdivided into subclasses on the basis of differences in the constant regions. H chains representing the various subclasses, however, are much more closely related to each other than to the other immunoglobulin classes. There are four subclasses of g chains in humans, g1, g2, g3, and g4, which yield IgG1, IgG2, IgG3, and IgG4 subclasses of IgG molecules. In the same way, µ and a chains have two subclasses each.
Immunoglobulins are present not only in serum, but also in body secretions such as saliva, mucus, sweat, breast milk, and colostrum. Immunoglobulin A, the predominant immunoglobulin class in external secretions, usually exists in human serum as a four-chain unit of approximately 160,000 molecular weight. The IgA in secretions consists of two four-chain units associated with a secretory component and a J chain. The J chain, in contrast to the secretory component, is associated with all polymeric forms of immunoglobulins that contain two or more basic units. The presence of a J chain facilitates the polymerization of basic units of IgA and IgM molecules. Quantitative measurements indicate that there is a single J chain in each IgM pentamer or polymeric IgA molecule.
The secretory component, or polymeric immunoglobulin receptor, which mediates the transport of polymeric IgA, is an integral membrane protein expressed in the basolateral membrane of epithelial cells. From there, this receptor undergoes continuous endocytosis, is transported across the epithelial cell, and then is secreted at the apical membrane into mucosal secretions. Transport of polymeric IgA occurs after synthesis and secretion by B cells in the lamina propria (9). Polymeric IgA binds with high affinity to the SC on the epithelial cell and the complex is transported to mucosal secretions. Mucosal secretions therefore contain a mixture of secretory IgA and free secretory component, except in patients with IgA deficiency, who have only secretory component. Immunoglobulin M also can be transported by this process. Persons deficient in IgA often have a compensatory increase in secretory IgM.
Immunoglobulin G
In normal adults, IgG, which has the most prominent role in memory immune responses, constitutes approximately 75% of total serum immunoglobulin. The relative concentrations of the four subclasses are as follows: IgG1, 60% to 70%; IgG2, 14% to 20%; IgG3, 4% to 8%; and IgG4, 2% to 6%. IgG can cross the placenta and provides protection of the newborn during the first months of life. No other immunoglobulin has this property. Immunoglobulin G can fix complement, with the subclasses functioning unequally: IgG3 greater than IgG1, which is greater than IgG2, which is greater than IgG4. Immunoglobulin G4, although completely unable to fix complement by the classic pathway, can use the alternative pathway. Other complement components adhere to the bacterial surface and promoting phagocytosis through C3b receptors and through those that bind IgG1, IgG3, and their Fc fragments. The coating of the bacteria with antibodies (opsonization) makes it easier for phagocytes to capture them and increases the efficiency of phagocytosis several hundredfold. Immunoglobulin G is involved in cytotoxicity with NK cells. Antibody response to proteins yields primarily IgG1 and IgG3, whereas polysaccharides elicit mainly IgG2.
Immunoglobulin A
Immunoglobulin A, most of which is produced locally, predominates in body secretions. There are two subclasses, IgA1 and IgA2. The TH cells in the lymphoid tissues of the gastrointestinal and respiratory tracts switch the B cells from IgM to IgA secretion. Secretory IgA, because of its abundance in saliva, tears, bronchial secretions, nasal mucosa, prostatic fluid, vaginal secretions, and mucous secretions of the small intestine, provides the primary defense mechanism against local mucosal infection. Its main function may be to prevent access of foreign substances to the general immunologic system. Besides its traditional role in extracellular antibody function, IgA can neutralize viruses intracellularly, can provide an internal mucosal barrier by intercepting antigens and ferrying them through the epithelium, and after binding to the surface of some leukocytes can activate the alternative pathway of complement activation. Immunoglobulin A, however, is primarily believed to be an anti-inflammatory antibody. Immunoglobulin A normally exists in the serum in both monomeric and polymeric forms, constituting approximately 15% of total serum immunoglobulin.
Immunoglobulin M
Immunoglobulin M constitutes 10% of serum immunoglobulin and normally exists as a pentamer with a molecular weight of 900,000. Immunoglobulin M antibody predominates in the early immune response. Immunoglobulin M, with IgD, is the main immunoglobulin expressed on the surface of B cells. It is the most efficient complement-fixing immunoglobulin, but its huge size makes it dangerous in high concentration. The IgM response declines and is replaced by IgG of the same antigen specificity. Fetuses make IgM before birth, but maternal IgM does not cross the placenta. Immunoglobulin M antibody to a specific organism in newborn serum indicates intrauterine infection.
Immunoglobulin D
Immunoglobulin D is a monomer normally present in serum in trace amounts (0.2% of total immunoglobulin). The main function of IgD is unknown. Immunoglobulin D, with IgM, predominates on the surface of human B lymphocytes. Its most important role may be as a receptor.
Immunoglobulin E
Immunoglobulin E constitutes only 0.004% of total serum immunoglobulin, but it binds with high affinity to mast cells and basophils through the Fc region. When combined with allergens, IgE antibodies trigger the release of inflammatory mediators such as histamine from mast cells and basophils. Immunoglobulin E also binds to macrophages, platelets, and eosinophils by means of low-affinity receptors. Like IgD and IgG, IgE normally exists in monomeric form.
COMPLEMENT
The complement system is the primary humoral mediator of antigen-antibody reactions (Fig. 8.2) (11). It consists of at least 20 chemically and immunologically distinct plasma proteins, which can interact with each other, with antibodies, and with cell membranes. The biologic activity of complement is manifested in three ways. First, complement proteins bind or opsonize to particles. Specific cellular receptors for these complement proteins then mediate the binding and uptake of the opsonized particles by polymorphonuclear leukocytes and monocytes. Second, the small fragments of proteolytic cleavage from complement proteins diffuse readily and can bind to neutrophils and macrophages, causing chemotaxis and cell activation. Similar receptors on lymphocytes and APCs bind complement-opsonized antigen in the form of immune complexes and enhance specific immune responses. At least 12 regulatory proteins and at least five complement receptors regulate the function of complement. Third, complement causes lysis by the insertion of a hydrophobic “plug” into lipid membrane bilayers and allows osmotic disruption of the target. Deficiencies in complement frequently cause severe infection or autoimmune disease. Most of the proteins in the complement system are clustered on chromosome 1q and within the MHC region on 6p (12).
The proteins of this system circulate as functionally inactive molecules and compose approximately 15% of the globulin fraction of plasma. Many of these proteins are zymogens, that is, proenzymes that need proteolytic cleavage to acquire enzymatic activity. Each protein of the classic pathway and membrane attack complex is assigned a number, and the proteins react in the following order: C1q, C1r, C1s, C4, C2, C3, C5, C6, C7, C8, and C9. The proteins of the alternative pathway are assigned letters preceded by the letter F (factor).
Activation
The most important step in differentiation of self from nonself by complement is the covalent binding of C3 to particles. Bound C3 functions as an opsonin and as an inciter of lytic membrane attack. Cell surfaces contain molecules that effectively limit C3 deposition, whereas nonself surfaces allow rapid deposition of many C3 molecules. The second mechanism whereby complement differentiates self and nonself is specific direction of C3 deposition to antigen-antibody complexes.
The Classic Pathway
The classic pathway is the main antibody-directed mechanism for the triggering of complement activation. C1q binds to the CH2 domains of IgG in immune complexes or to the CH3 domains of a single IgM molecule, which has been modified by antigen binding. The next steps both amplify the response and concentrate the site of activation to the particle that initiated activation. C1s cleaves C4 into C4a and C4b. Zymogen C2 binds to C4b and is cleaved to C2a and C2b. C4b2b, the classic pathway C3 convertase enzyme, cleaves C3 into C3a and C3b.
Alternative Pathway
An initial requirement for activation of the alternative pathway is the presence of C3b, which is generated continuously in small amounts. C3b reacts with factors B and D to generate an enzyme (C3bBb) that cleaves C3 into C3a and C3b. The newly generated C3b interacts with additional factors B and D to form more C3bBb. The C3bBb enzyme dissociates rapidly unless it binds to properdin (P). Forming the complex C3bBbP stabilizes it.
Membrane Attack Complex
Formation of the membrane attack complex begins with enzymatic cleavage of C5. C5 binds to C3b for cleavage by the C5 convertase enzyme (the trimolecular complex C4b2b3b for the classic pathway and C3bBbP for the alternative pathway). Subsequent formation of the membrane attack complex is nonenzymatic and follows successive binding of C6 and C7 and C5b to form the C5b67 complex. C8 and C9 then bind sequentially to this complex, resulting in formation of the lytic plug.
Breakdown products of this cascade (anaphylotoxins C3a and C5a) stimulate chemotaxis of neutrophils and degranulation of basophils and mast cells. The anaphylotoxins have a powerful effect on blood vessel walls, causing contraction of smooth muscle and an increase in vascular permeability, probably mediated indirectly by release of histamine from mast cells. Bound C3 and C4 fragments act as opsonins to enhance phagocytosis and stimulate exocytosis from neutrophils, monocytes, and macrophages of granules that contain powerful proteolytic enzymes and free radicals. A link between complement activation and adaptive immunity is becoming recognized.
PHAGOCYTIC CELLS
Both polymorphonuclear leukocytes and monocytes phagocytize microorganisms during inflammatory reactions. Although monocytes show greater diversity in function and response, both types of cells recognize and ingest particles and soluble ligands through receptors on their cell surfaces and digest them in their lysosomes. They also have a number of oxygen-independent mechanisms that include lactoferrin, lysozyme, major basic protein, and defensins. Defensins are antimicrobial cationic peptides that are divided into a and b subfamilies. The a defensins are produced by neutrophils; epithelial cells produce the b defensins. The a defensins play a role in inflammation, wound repair, and specific responses. They increase bacterial adherence and induce histamine release. In contrast, b defensins regulate complement and inhibit proteases (13).
Monocytes
Monocytes originate in the bone marrow from pluripotential stem cells and are released into the blood. Tissue macrophages arise by maturation of monocytes that have migrated from the blood. In proliferation of immature macrophages, mitogens such as colony-stimulating factor (CSF), which is produced by fibroblasts, lymphocytes, and monocytes, play an important role. During inflammation, both of these processes increase dramatically. Giant cells arise either by fusion of macrophages or by failure of cytokinesis during mitosis. The most important functional property of the macrophage is its ability to recognize and ingest foreign and damaged materials. The capability of macrophages to recognize opsonized particles resides in their receptors, which bind the Fc portion of immunoglobulins and the C3 components of complement. Macrophages possess surface MHC molecules and have receptors for activation by lymphokines and for CSF, which regulates their function and proliferation. Monocytes also produce complement components, prostaglandins, interferons, proteases, and cytokines. Langerhans cells, another type of APC, are interspersed in the epithelial layer of the nasal mucosa and skin and help to induce T-cell responses. They present antigen to T cells.
During phagocytosis, particles bound to specific or nonspecific membrane receptors are surrounded by the cell membrane to form phagocytic vesicles. Endocytic vacuoles become secondary lysosomes after fusion with primary lysosomes. Within the lysosomal compartment, the contents are digested at acid pH by more than 40 hydrolytic enzymes. After ingestion of particles, macrophages and neutrophils undergo a respiratory burst. The burst is observed as a dramatic increase in consumption of oxygen and activation of membrane-associated oxidase. This oxidase reduces molecular oxygen to superoxide anion, which undergoes dismutation to hydrogen peroxide. Superoxide and hydrogen peroxide interact to give rise to hydroxyl radicals and singlet oxygen. These reactive metabolites of oxygen exert antimicrobial and antitumor effects.
Another group of effector molecules synthesized by macrophages includes nitric oxide and reactive nitrogen intermediates. The macrophage itself is protected from these oxygen metabolites by glutathione peroxidase and catalase. Many soluble agents, including antigen-antibody complexes, C5a, ionophores, and tumor promoters, can trigger the respiratory burst without phagocytosis.
Substances chemotactic for macrophages include C5a anaphylatoxin, bacterial products such as N-formylmethionyl peptides, and products from stimulated B and T lymphocytes. Also important are substances that inhibit migration away from sites of inflammation: lymphokines (macrophage inhibitory factor and macrophage activation factor) and proteolytic enzymes produced during activation of complement (factor Bb).
Macrophages are important in the initiation and regulation of the immune response. Macrophages that produce IL-12 increase bronchial responsiveness associated with eosinophil migration. Macrophages that produce IL-1 stimulate T-cell function, and they present immune molecules to lymphocytes. This function requires display of the same MHC determinants by both T cells and macrophages. The presence of IL-1 increases production of prostaglandins and leukotrienes, which can alter vascular permeability and bronchial tone. Interleukin-1 also induces production of acute-phase proteins, including complement components, fibrinogen, and clotting factors, and increases the activity of adhesion proteins such as ICAM-1.
Granulocytes
Polymorphonuclear leukocytes (neutrophils) accumulate at sites of acute inflammation. This requires a series of coordinated steps that include adherence to endothelium, extravascular migration, chemotaxis, membrane recognition and attachment to particles, phagocytosis, fusion of lysosomes and degranulation, and a burst of oxidative metabolism. Some of the genes responsible for the oxidative burst have been identified and associated with chronic granulomatous disease. A genetic locus on chromosome 1q42-q44 has been identified in patients with Chédiak-Higashi syndrome, a disease characterized by giant granules in neutrophils.
Blood neutrophils are composed of two interchangeable subpools: the circulating pool and the marginal pool. One of the early events in acute inflammation is an increase in neutrophil margination and adherence to the vascular endothelium. C5a, a component of complement, mediates neutrophil chemotaxis, although other chemoattractants from bacteria, stimulated leukocytes, products of coagulation or fibrinolysis, and oxidized lipids exist. The chemokines plus selectins assist with neutrophil adhesion to vascular endothelium. Neutrophils recognize particles by opsonins attached to them. These opsonins include immunoglobulins to which the neutrophil exhibits Fc receptors and the C3b fragment of complement. After phagocytosis, the processes described for mononuclear phagocytex applies to the neutrophil.
Eosinophils are produced in the bone marrow, circulate in the blood, and reside predominantly in tissues. Eosinophils have receptors for several cytokines and for IgG and IgE. They possess several adhesion molecules (ligands), which assist in chemotaxis. Their function is attributed to elaboration of a variety of cytokines, proteins, peroxidases, and enzymes. One of these, major basic protein, is cytotoxic and helminthotoxic. Also elaborated are eosinophil peroxidase, eosinophil-derived neurotoxin, Charcot-Leyden crystal protein, and eosinophil cationic protein. Survival of eosinophils in tissues is based on their need for several growth factors, such as IL-5, IL-3, and GM-CSF. In the absence of growth factors, eosinophils undergo programmed cell death or apoptosis.
Basophils are granulocytes that possess high-affinity IgE receptors. They contain histamine and other mediators, including cytokines. Basophils are believed to contribute to anaphylaxis by releasing histamine and are known to ontribute to allergic reactions at tissue sites, such as the nose, lungs, and skin.
IMMUNE SENESCENCE
With life expectancy increasing, investigations into the effects of aging on the immune system have increased. Results of unavoidable exposure to a large number of potential antigens (viruses, bacteria, foods, and self-molecules) influence the immune response. Changes reported with aging include (a) dysregulation of peripheral B and T cells with production of large clones, (b) alterations in lymphocyte subset distribution, signaling, and cytokine production, (c) thymic involution with a decreased output of T cells, (d) a void of virgin T cells to respond to new infectious and noninfectious disease, (e) an increase in the percentage of NK cells with a mature phenotype and associated impairment of their cytotoxic capacity and their response to IL-2, (f) decreased phagocytic capability of neutrophils, and (g) an increase in cell adhesion molecules. Low numbers of NK cell among elderly persons is associated with mortality and the risk of severe infection. Innate immunity, mediated by genes that remain in the germline configuration and encode for proteins that recognize conserved structures on microorganisms, is a much more ancient system of host defense. Innate immunity (chemotaxis, phagocytosis, defensins, and complement) is conserved with aging (14).
IMMUNOPATHOLOGY
Immunopathology is the study of an adaptive immune response that occurs in an exaggerated or inappropriate form and causes tissue damage. The response also has been called hypersensitivity, and it manifests itself only after a second contact with a particular antigen. Autoimmunity involves formation of IgG and IgM antibodies to self and reflects a deviation from the principle of self-nonself discrimination. There are autoantibodies to essentially every organ in the body. Several mechanisms may explain the presence of autoantibodies. For example, a foreign antigen can cross-react with a normal body structure, as in cross-reaction of specific streptococci and heart antigens in rheumatic heart disease. In other cases, the release of a previously sequestered self-antigen appears, as in postmumps orchitis. Still other cases involve a deficiency of suppressor T cells. Autoimmune mechanisms occur in myasthenia gravis, autoimmune hemolytic anemia, pernicious anemia, and Goodpasture syndrome.
Antibody (IgG and IgM) and antigen form soluble complexes that precipitate in basement membranes of blood vessels with considerable outflow of plasma. These vessels include those lining serosal surfaces, such as the peritoneum and pleura, joints, kidneys, and skin. The complexes activate complement and set in motion an inflammatory response characterized primarily by the influx of neutrophils. The inflammation harms the blood vessel walls and adjacent tissues.
Delayed-type hypersensitivity is the pathologic variant of normal T-cell-mediated immune response. Often the T-cell response to an environmental antigen can be overly enthusiastic. Much of the damage to the lung in tuberculosis is caused not directly by Mycobacterium tuberculosis but by the macrophages attracted by T-cell-derived cytokines. The macrophages specialize in destroying ingested material but are not adept at differentiating foreign antigens from host antigens. Adjacent tissues can be damaged as innocent bystanders. Delayed-type hypersensitivity also occurs in graft rejection and allergic contact dermatitis.
ALLERGY
Pollens and other potential allergens are deposited on the mucosal surface (Fig. 8.3). Antigens extracted from the pollens penetrate through or between epithelial cells and interact with APCs spread throughout the mucosa. Initial stimulation of the IgE mucosal immune response usually occurs in the tonsils and adenoids. Chronic exposure to low doses of antigen favors IgE over IgG production.
Production of IgE by B cells involves APCs and TH cells. Locally produced IgE first attaches to local mast cells. Excess IgE enters the circulation and binds to receptors on both circulating basophils and tissue-fixed mast cells throughout the body. Although the serum half-life of IgE is only 2½ days, mast cells may remain sensitized for many weeks after passive sensitization with atopic serum containing IgE.
Persons with a family history of asthma, eczema, hay fever, and urticaria and a positive skin test result are referred to as atopic. About 20% of the U.S. population have positive immediate wheal and flare skin reactions to common inhalant allergens. Parents with allergies have a higher than usual proportion of children with allergies—50% of children with two parents with allergies have an allergy. When only one parent has an allergy, the chance is about 30%. A family history of allergy is an important risk factor for allergic disease.
The incidence of allergic rhinitis is increasing in many industrialized nations. There is no obvious cause of this increasing occurrence. Pollution with diesel exhaust fumes and the prevalence of new antigens do not account entirely for this increase. The possible reduction of infection could be shifting T-helper responses from TH1 to TH2 cells (Fig. 8.4). Immunostimulatory DNA sequences that favor a TH1 response have been proposed as a means of immunotherapy to shift the allergic TH2 response toward a TH1 response.
The total amount of IgE alone is not predictive of an allergic state because genetic and environmental factors, such as parasitic infestation, affect the levels. The mode of inheritance of high levels of total IgE is not yet known, but many of the cytokines that control its regulation map to chromosome 5. Specific antigen-specific IgE responses frequently are associated with particular human leukocyte antigen (HLA) markers.
Mast cells are important in the allergic reaction. Mast cells are subtyped according to content of proteases. Both subtypes contain histamine and exist in the nasal mucosa. The final phenotype depends on local microenvironmental factors. The number of mast cells increases in the nasal mucosa after seasonal exposure to allergens. Evidence suggests that some microorganisms interact directly with mast cells and activate them to elicit an inflammatory response that clears bacteria—a possible link between mast cells and innate immunity.
Immunoglobulin E binds through its Fc receptor on the cell surface of mast cells. Cross-linking of IgE on the surface triggers degranulation. Activation of mast cells causes an influx of calcium ions. This process causes, first, exocytosis of granule content with the release of preformed mediators, such as histamine, heparin, and proteolytic enzymes (tryptase and b-glucosaminidase). Second, mast cell activation induces the synthesis of newly formed mediators from membrane-bound phospholipids. This results in production of prostaglandins, leukotrienes, and platelet-activating factor. Cytokines such as IL-3, IL-4, TNF2, and GM-CSFE also are produced by mast cells.
Minutes after antigen exposure, increases are detected in the levels of mast cell–associated mediators. Concurrent with the release of inflammatory mediators in nasal secretions, sneezing, rhinorrhea, nasal itching, and congestion begin. Localized changes around mast cells are amplified by neuronal reflexes. For example, stimulation of one side of the nasal cavity with antigen causes local histamine release, which stimulates sensory nerves. The sensory information travels to the central nervous system and stimulates parasympathetic signals that cause bilateral nasal secretion. The nervous system also potentially influences the reaction through release of neuropeptides.
The early response of mast cell degranulation does not entirely explain the symptoms of patients with allergic rhinitis. The following observations support this notion: (a) The duration of the early reaction to antigen is measured in minutes, whereas clinical disease is more prolonged, patients reporting nasal symptoms hours after pollen exposure. (b) Systemic glucocorticoids, although useful in refractory cases of allergic rhinitis, do not inhibit the early reaction. (c) Biopsy of the nasal mucosa during the allergy season shows inflammatory cellular infiltration, whereas study of the early reaction shows only mast cell degranulation and tissue edema. (d) The dose of pollen necessary to induce symptoms during experimental provocation exceeds severalfold the amount needed to produce a response during the allergy season. (e) Changes in reactivity to nonspecific irritants occur during seasonal exposure but not during the early response. Allergic rhinitis therefore cannot be strictly considered an immediate hypersensitivity reaction. The concept of the pathophysiologic mechanism of allergic rhinitis must be expanded.
The late response is defined as recurrence of symptoms and the appearance of mediators in nasal secretions hours after antigen exposure. Depending on the variable analyzed, the incidence of late reactions varies from 16% to 53% among patients with allergies with an onset within 3 to 11 hours after antigen challenge. Among these patients, the symptoms recur spontaneously in concordance with an increase in the levels of some but not all of the same mediators of the early reaction.
Hours after the early response, a marked overall increase occurs in the number of cells recovered in lavage of collected nasal secretions and in biopsy specimens obtained from the nasal mucosa. The increase is specific for persons with allergies and is easily detected for about two thirds of persons with allergies. The influx of eosinophils, neutrophils, and lymphocytes is maximal 4 to 11 hours after exposure to antigen and is mediated by adhesion molecules on the endothelium (ICAM-1, VCAM-1, selectins, and integrins) and by their counterligands on the cells (VLA-1, LFA-1, macrophage-1 antigen [Mac-10], and platelet-endothelial cell adhesion molecule 1 [PECAM-1]) (15) (Fig. 8.5). There appears to be a greater eosinophil influx among persons with late reactions. The nasal mucosa and surface secretions show similar but not identical changes. For example, the mucosa contains greater numbers of TH2 lymphocytes.

In addition to their function as a barrier, cells in the epithelium are involved in the immune process. Epithelial cells secrete IL-6, IL-8, and GM-CSF. Langerhans cells are interdigitated with epithelial cells and present antigens to and induce activation and differentiation of T cells. Intraepithelial lymphocytes are predominantly T cells and play a role in the immune response. These cells secrete IL-2, IL-5, IFN-t, and transforming growth factor b and have cytotoxic functions (16).
Rechallenge with allergen 11 hours after the initial provocation increases the amount of inflammatory mediators in a pattern suggestive of both mast cell and basophil activation. More important, the dose of antigen necessary to induce a clinical reaction is markedly reduced. Oral glucocorticoids inhibit this increased reactivity as well as the late reaction and the cellular influx, supporting the importance of this reaction. Repeated exposure to antigen can maintain a constant inflammatory process in the nasal mucosa. Progressively smaller doses of antigen induce the same allergic response (priming), explaining the persistence of strong symptoms even beyond the peak of the pollen season. With perennial antigens, this phenomenon can be constant, and patients have symptoms all year.
There also is an increase in nonspecific nasal airway reactivity to histamine, methacholine, and cold, dry air after antigen exposure. Such reactivity correlates with an increase in the number of eosinophils and with an increase in vascular permeability in the nasal mucosa. This increase in nonspecific nasal reactivity probably is related to the inflammatory cellular infiltration that occurs after antigen stimulation. Topical steroids have been shown not only to inhibit the early and late responses to antigen but also to inhibit the nonspecific reactivity due to antigen and the accompanying eosinophil influx, even when given after challenge.