Basic Principles of Host Defense

Saturday, August 16, 2008

INTRODUCTION

Higher animals have evolved many host defense mechanisms that ensure the relatively long-term survival of individuals despite their coexistence with countless microorganisms. Healthy people are at equilibrium with the microbial world; their internal soma, rich in nutrients, remains free of replicating microorganisms. Disease can be viewed as progression away from this equilibrium, in which microorganisms invade the body from the environment and replicate, causing inflammation and destruction of the tissues, as well as depletion of the host's nutrients. Unchecked, the rapid expansion of the microbial population leads to the death of the host.

A similar but distinct scenario is that the internal environment contains a variety of viruses, bacteria, fungi, protozoa, and microscopic metazoa, which are acquired throughout life. Again, these organisms achieve equilibrium with the mammalian host. Under these conditions they do not replicate, nor do they produce toxins. Instead, they exist in a dormant or latent state, which is infection without disease. Disease is a progression away from the equilibrium. When the balance is disturbed, microbial replication begins; host nutrients are then consumed by the proliferating population of microorganisms. This process leads to disease and ultimately death unless microbial replication is blocked.

To maintain equilibrium with the microbial world—that is, to maintain a state of health—host-protective mechanisms must fulfill distinct functions. In a broad sense, the functions come in two forms.

The first form includes the antimicrobial defense mechanisms that have evolved throughout the vertebrate species. Specifically, this first form is composed of a variety of mechanisms to prevent invasion of sterile tissues from contiguous body sites that are teeming with microbial flora. These mechanisms involve the actions of specialized cells—phagocytes and lymphocytes. For example, the upper respiratory tract is colonized with large numbers of bacteria, yet further down into the respiratory tract, below the vocal cords and into the lungs, few or no bacteria are found. Those bacteria that do gain access to the lung are rapidly destroyed. In another example, microbes may gain entrance to sterile body sites when an open injury introduces bacteria into the skin, subcutaneous tissue, muscle, or bone. This is countered by mechanisms in which the injured tissue is walled off to prevent spread and is invaded by phagocytic cells that envelop and kill bacteria. This is bactericidal activity. Yet another example involves specialized microorganisms that have evolved into successful parasites and that gain ready access to sterile sites in the host. They may be highly resistant to host-killing mechanisms. Under these conditions, equilibrium between parasite and host is reached by establishing microbiostasis in the tissues. In this way, living microbes may remain dormant or latent within host tissues for years, or even decades. This scenario implies microbiostatic host-defense mechanisms. For example, the human tubercle bacillus (Mycobacterium tuberculosis) is inhaled into the lung. In the immunocompetent host, immunity is acquired, microbial replication ceases, and the mycobacteria become dormant, especially (but not exclusively) in the lung apices.

A second way to thwart infectious disease is through a set of uniquely human prevention schemes that have resulted from brain development and intelligence. Our understanding of infectious disease transmission has led to effective prevention methods, such as the addition of chlorine to the water supply and modern sewage treatment. Knowledge of immunology has led to vaccine strategies that have achieved results such as the elimination of smallpox from the human population. Understanding microbial physiology and metabolism led to the discovery of antibiotics for treating heretofore lethal infections.

This chapter deals exclusively with the first category of anti-infection mechanisms. These mechanisms can be subdivided into two broad categories: (1) innate or natural host defenses and (2) adaptive immune responses. There are distinct mechanisms characteristic of each of these two, but there is overlap as well. Some innate responses use mechanisms that, when analyzed at the cellular and molecular levels, were previously thought to reside exclusively as part of the adaptive immune response. Conversely, acquired immunity may lead to microbicidal mechanisms, which are not immunologically (antigen) specific and probably exist as an evolutionary blend of the innate and adaptive host defenses.

INNATE HOST DEFENSES

DEFENSE MECHANISMS AT THE PHYSIOLOGIC LEVEL

Introduction

Numerous physiologic factors influence susceptibility to infection. Prime examples of these factors are discussed in the sections that follow.

Normal Microbial Flora

Body surfaces in continuity with the environment typically support a complex but characteristic set of numerous microbial species (Table 2-1). This group of microorganisms is called the normal flora and includes microorganisms that inhabit the integument, upper respiratory tract, gastrointestinal tract, and urogenital tract. More than 200 different bacterial species occupy the human colon as normal flora. The normal flora fill these body sites to the exclusion of other species. Thus, the presence of the normal flora provides a protective function. A relatively virulent species such as the fungus Candida albicans cannot occupy the vagina unless the normal flora are eliminated, for example, by use of broad-spectrum antibacterial agents used to treat urinary tract infections. Sometimes virulent microorganisms colonize a body site and coexist along with the normal flora. For example, Streptococcus pneumoniae is often found in the oropharynx of healthy individuals. Colonization is a critical requirement for the pathogenesis of bacterial pneumonia caused by this pathogen. At some point microaspiration events introduce S pneumoniae into the lower respiratory tract. Then virulence mechanisms prevent the killing of these microorganisms by innate host defense mechanisms. This leads to microbial replication in a nutrient-rich environment that is devoid of competing microbial species, and bacterial pneumonia results.

What determines which species predominate as the normal flora at a particular site? Although this is a complex process, the phenomenon of attachment to host cells is an important requisite. Particular microbial species express structures on their surface that bind to receptors on host epithelial cells. For example, strains of viridans streptococci adhere (by special attachments) to pharyngeal epithelial cells and are able to flourish and exist as the predominant species in the oropharynx.

Anatomic & Physiologic Factors

The normal human anatomy provides numerous examples of anatomic barriers that prevent microbial replication and invasion. This is best illustrated when a breach in these barriers occurs and infection follows. The skin is a highly efficient barrier that prevents bacteria and fungi from entering into the subcutaneous tissue. In burn victims this barrier is lost. Without aggressive countermeasures, individuals with extensive burns succumb to overwhelming bacteremia because enormous numbers of bacteria invade the damaged tissue and thereby gain access to the circulatory system.

Physiologic mechanisms provide similar functions that prevent microbial invasion. Secretions contain antimicrobial molecules. For example, lysozyme in tears dissolves the cell walls of particular gram-positive bacteria. Salivary secretions contain bactericidal proteins. Smooth muscle peristaltic movements prevent the overgrowth of bacteria in the small intestine by creating a continuous flow, which has a cleansing effect; hydrochloric acid in the stomach kills numerous bacterial cells that enter upon swallowing. The respiratory tract contains a mucociliary carpet that is under constant motion outward, which helps remove inhaled bacteria from the respiratory epithelium.

Similar to a breach in an anatomical barrier, when physiologic processes malfunction, disease is usually the result. For example, autoimmune diseases that destroy the salivary glands lead to severe problems with dental caries and gingivitis, which are caused by overgrowth of oral bacteria. Periodontal disease and loss of permanent teeth follow. This is in part because the saliva normally contains antibacterial and antifungal compounds.

Host Nutritional Status

The nutritional status of the host ranks as a critical factor for both innate and acquired resistance to infection. Protein-calorie malnutrition in children is associated with severe measles (rubeola virus). Historical observations point to reactivation of dormant tubercle bacilli during acute food shortage in populations with a high prevalence of this pathogen. Pyogenic bacterial infections and periodontal disease are additional examples. The original description of Pneumocystis carinii as a human pathogen was based on observations of pulmonary disease in starving European infants during and after World War II. Experimental research shows that starvation impairs phagocytes and cell-mediated immunity (CMI) before its effects on antibody production are observed.

Hormonal Influences

Glucocorticoids exert profound effects on host resistance to infection. In severe bacteremia, physiologic cortisol secretion improves chances of survival. Yet it is well known that pharmacologic dosages of glucocorticoids depress the inflammatory response and increase susceptibility to primary infection as well as reactivation of latent infection. Pregnancy, as it progresses into the third trimester, results in immunosuppression, primarily of CMI. This leads to increased susceptibility to certain infections such as the food-borne infection listeriosis.

Aging

Aging affects many aspects of innate defenses. Consequently, pneumonia, urinary tract infections, cholecystitis, diverticulitis, and bacteremia caused by pyogenic bacteria are increased. The aged are more likely to reactivate latent organisms such as Herpes zoster virus and Mycobacterium tuberculosis, which suggests decreased CMI. Aged individuals have reduced ability to mount immunoglobulin (Ig) synthesis against polysaccharide antigens.

The Acute-Phase Response

The acute-phase response is a phylogenetically primitive, pleiotropic collection of responses designed by evolution to inhibit microbial replication and enhance acquired immune responses. The acute-phase response is triggered by invasion of microorganisms. Its complex, multiorgan involvement is fundamental for the inflammatory response characterized by swelling, pain, redness, heat, and loss of function. With the evolution of specific adaptive immune responses in vertebrates, the acute-phase response was preserved and is used as an activator of T and B lymphocytes.

Signal molecules that emanate from bacteria and other microorganisms (eg, endotoxin, muramyl dipeptide from the peptidoglycan of cell walls, glucans, mannans, and microbial toxins) bind to receptors on mononuclear phagocytes (MNPs) and endothelial cells [the reticuloendothelial system (RES)]. Secretion of a major proinflammatory cytokine, interleukin-1 (IL-1), and possibly others induces a pleiotropic response that involves a variety of different organs and functions (Table 2-2). The acute-phase response also leads to induction of specific immune responses, namely antibody synthesis by B cells and CMI, which are controlled by T cells. In a practical sense, measurements of the acute-phase response in blood samples are used in diagnosis of infections and other inflammatory diseases. Phenomena affected by IL-1, namely fever, increased erythrocyte sedimentation rate, high white blood cell count, and muscle wasting are important clues to the presence of infection, autoimmune inflammatory diseases, and neoplastic diseases that involve lymphoreticular cells.

INNATE DEFENSE MECHANISMS AT THE CELLULAR LEVEL

Introduction

Ever-present cells and molecules (constitutive mechanisms) form an on-the-spot defensive line against microbial invasion. Compromise of this innate multifaceted system results in predictable serious infections, usually with invading species of the microflora. Thus, the importance of constitutive nonspecific defense mechanisms is revealed. Phagocytes and natural killer (NK) cells are discussed as examples of innate cellular defenses.

Phagocytes

Two types of cells can be considered "professional phagocytes" or cells that consume microbes. These are the polymorphonuclear leukocytes (called neutrophils, or granulocytes) and the MNPs (monocytes and macrophages). These cells emanate from bone marrow precursors and enter the circulation to perform their antimicrobial functions. Lack of either of these cell lineages is incompatible with life.

Neutrophils live for 1-4 days after entering the circulation. Continued neutrophil production is essential for survival. Neutrophils are an important regulator of homeostasis between the body, which harbors sterile enclaves, and the microbial world. Neutrophils deal with pyogenic bacteria and fungi. Neutrophils possess tightly regulated microbicidal systems that are activated within seconds after encountering microorganisms. In healthy individuals, neutrophils are constantly at work destroying small numbers of microbes that enter the body by various routes. Severe neutropenia (< 500 cells/uL of blood) leads to overwhelming bacteremia from enteric bacilli. Neutrophils are constitutively bactericidal to a high degree for gram-positive and gram-negative cocci and enteric bacilli. To maintain this constitutive defense, neutrophils have special functional properties, to be discussed below. A defect in any one of these important functions invariably leads to a life-threatening infection. However, neutrophils have less or little activity against facultative or obligate intracellular pathogens such as mycobacteria, dimorphic fungi, and protozoans. The human immune system has evolved other mechanisms for dealing with these pathogens.

MNPs leave the bone marrow as blood monocytes. They mature into macrophages in the periphery. Stationed throughout all organs of the body, they compose the RES. The RES of the sinusoids of the liver and spleen provides an important clearance mechanism to remove a variety of microbes from the circulation. Thus, the spleen serves as a highly efficient, specialized RES site for removal of virulent bacteria. These bacteria avoid clearance in other RES sites, such as the lung or liver, because they possess virulence factors that have extraordinary antiphagocytic properties, such as particular serotypes of exopolysaccharide capsules. Individuals with no spleen may succumb to high-grade infection caused by bacteria that possess antiphagocytic capsules. The alveolar spaces of the lung are inhabited by macrophages, which form a first-line defense of the lung. These cells phagocytize and kill inhaled viruses, bacteria, and fungi. Macrophages and macrophage-like cells (eg, dendritic cells) contribute to the adaptive immune response. Macrophages, which function in concert with T lymphocytes, are responsible for CMI defense against intracellular pathogens. Macrophages process and present antigen to lymphocytes and receive "activating" signals during the immune response. These signals enable the macrophages to control the replication of intracellular microorganisms; they are also capable of killing these microorganisms (see below). Some of the functions listed in Table 2-3 for neutrophils are also relevant for MNPs.

To kill bacteria and fungi, phagocytes must make physical contact with them. This involves a coordinated sequence of regulated events: (1) production, (2) release into the circulation, (3) attraction to a site of infection, (4) adherence onto and translocation through (diapedesis) adjacent capillary walls, (5) chemotaxis, (6) attachment to and phagocytosis of the microbe, (7) activation of killing mechanisms, and (8) digestion of dead microorganisms (see Table 2-3). These events are depicted in Figure 2-1. Genetic or acquired conditions that interfere with one or more of these steps are associated with increased propensity to develop pyogenic infections caused by microorganisms which are usually constituents of the normal flora.

A. Phagocyte Production (Stage 1). Phagocytes are produced in the bone marrow (see Figure 2-1A). Neutrophils are produced in bone marrow from precursor stem cells under the mitotic and differentiation influence of several low-molecular-weight proteins called colony-stimulating factors (CSF). CSF for granulocytes (neutrophils) (granulocyte CSF), monocytes/macrophages (macrophage CSF), phagocytes (granulocyte-macrophage CSF), and multiple cell types (IL-3) are secreted by endothelial cells, fibroblasts, and cells of the immune system. A large number of granulocytes are produced each day (~ 1011 cells). Granulocytes are short-lived cells; once they enter the tissues their half-life is only 6-8 h. There may be as many as 1010-1011 granulocytes/ml of abscess fluid. Thus, the turnover of these cells is high, and, when production is inhibited, by anticancer drugs, for example, neutropenia may become severe and lead to invasive bacterial and fungal infections.

B. Phagocyte Release (Stage 2). Phagocytes are released into the circulation (see Figure 2-1B). IL-1, a cytokine product from macrophages, signals release of neutrophils from the marrow. The circulating neutrophil population increases the peripheral white blood cell count, a hallmark of acute infection. Neutrophils that normally adhere to the vascular endothelium form the marginated pool. Certain chronic stimuli (eg, drugs, hypoxia, stress, and exercise) cause demargination with increased white blood cell counts.

C. Chemokinesis (Stage 3). Phagocytes are guided to sites of infection by chemical signals called chemokines. Sentinel tissue macrophages and migrating immune cells, in response to invading microbes, secrete proteins called chemokines. Numerous distinct molecules have been identified—some are the ligands for specific receptors on different leukocytes, and some control the types of leukocytes that infiltrate inflammatory foci. The chemokines exert their effect by facilitating adherence of particular cell types to endothelium adjacent to an inflammatory site.

D. Margination, Spreading, and Diapedesis (Stage 4). For phagocytes to enter foci of infection, they must adhere to adjacent capillaries and migrate out of the bloodstream into the tissues. This is a multistep, tightly regulated process. The initial adherence step is mediated by surface molecules called selectins that bind neutrophil (L-selectin) to the endothelial cell (E-selectin) as well as to platelets (P-selectin). At this stage leukocytes roll along the endothelium in the direction of blood flow (Figure 2-1C). Tight adherence with leukocyte flattening is mediated by receptors (ß-integrins) that bind to intercellular adhesion molecules called ICAMs that are expressed on endothelial cells. This interaction is controlled by regulation of these surface molecules through inflammatory mediators that diffuse from an infected site. Cytokines and arachidonic acid metabolites up-regulate ß-integrin (on neutrophils) and ICAM-1 (on endothelial cells) expression. Specialized adhesion molecules localized at endothelial cell junctions (platelet/ endothelial cell adhesion molecules) guide neutrophil motility between endothelial cells into infected tissue by a process called diapedesis (Figure 2-1D).

E. Chemotaxis (Stage 5). Phagocytes seek pathogens by attraction to gradients of microbial products and signals generated by the host inflammatory response. Motility of neutrophils increases in response to environmental signals called chemotaxins, which include cytokines [IL-6, IL-8, granulocyte macrophage CSF, tumor necrosis factor a (TNFa), interferon-? (IFN-?)], leukotrienes, and chemical products from microorganisms themselves (eg, microbial oligopeptides such as formylmethionine-leucine-phenylalanine). When no concentration gradient is present, increased motility is random [chemokinesis (Figure 2-1E)]; however, in the presence of even weak gradients, neutrophil motility becomes directed toward an increased concentration of stimulus [chemotaxis (Figure 2-1F)]. The interaction between chemotaxins, their high-affinity receptors on the neutrophil surface, and cytoplasmic cytoskeletal events, which propel the cell forward, are not entirely worked out. However, it is important that phagocyte motility depends on a substrate upon which the phagocytes crawl. These cells cannot swim. Thus it becomes clear that whenever bacteria gain access to nutrient-rich fluid-filled spaces (eg, cerebrospinal, pleural, peritoneal, pericardial, or synovial fluid), phagocyte defenses are put at considerable disadvantage. Neutrophils must make physical contact with the bacteria they kill. In a fluid-filled space (eg, massive ascites), this contact depends on random collisions between bacterial and phagocytic cells. Indeed, in the preantibiotic era, pyogenic infections of fluid-filled spaces were frequently fatal. Because bacteremia is often a precursor to meningitis, pericarditis, peritonitis, or septic arthritis, one major function of the RES is to clear bacteria from the bloodstream, which thereby prevents seeding of fluid-filled spaces. Bacteria that most often cause these infections are notoriously difficult to clear from the bloodstream because they possess antiphagocytic factors, most often capsules (S pneumoniae, Streptococcus pyogenes, Neisseria meningitidis, and Haemophilus influenzae).

F. Phagocytosis (Stage 6). Phagocytosis is required to kill invading microorganisms. Phagocytosis is a two-step process: (1) attachment of the particle to the phagocyte occurs and (2) the phagocyte extends lamellipodia circumferentially around the particle to enclose it within a phagocytic vacuole called the phagosome (Figure 2-1G). As noted above, opsonic proteins (Igs and complement proteins) coat microorganisms and, by engaging their receptors on the surface of phagocytes, they mediate attachment. These receptor-ligand interactions also induce intracellular events, which activate the engulfment process. Engulfment itself is a highly energy-dependent process and is powered by an ATP-dependent cytoskeletal polymerization-depolymerization of actin microfilaments. Nonopsonic phagocytosis may occur for some microorganisms. Molecules on the surfaces of both target and phagocyte interact with oligosaccharide moieties, which mediate attachment and activate the engulfment "motor" of the phagocyte.

Another aspect of phagocytosis involves interaction between the phagocyte and the surrounding tissue substrate (ie, the surface on which the phagocyte is crawling). This involves specialized serum molecules (extracellular matrix proteins) such as fibronectin, laminin, and vitronectin. These proteins do not interact with microorganisms directly. However, facilitation of interaction between phagocyte and substrate by these molecules leads to substantial enhancement of phagocytosis.

G. Microbicidal Activity (Stage 7). There are numerous phagocyte microbicidal mechanisms (Figure 2-1H). As microorganisms are internalized within phagosomes, microbicidal functions are initiated. Two general categories of killing mechanisms are recognized. These are the oxygen-dependent and -independent mechanisms. Oxygen-dependent killing is by the oxidative burst, sometimes called the respiratory burst. There are multiple oxygen-independent mechanisms that are mediated by microbicidal proteins that are delivered into the phagosome by degranulation. Degranulation occurs when specific cytoplasmic granules in the neutrophil fuse with the phagocytic vacuole and discharge their contents on particles sequestered therein. A phagosome that has undergone this process is called a phagolysosome. The phagolysosomal membrane contains a proton pump that acidifies the compartment. The low pH (~ pH 4.5) is an ideal environment for many enzymes, acid proteases, defensins, cationic proteins, and other proteins (bactericidal permeability-increasing protein, azuricidin, indolicin, and bactenectins) that kill and digest (stage 8; see Figure 2-1H) bacteria.

The oxidative burst is based on a unique enzyme system, which in the resting cell is inactive. This enzyme is the NADPH/oxygen oxidoreductase of neutrophils and MNPs. The NADPH oxidase is activated upon stimulation induced by phagocytosis of a microorganism. Oxygen is reduced to superoxide with NADPH serving as the electron donor. There is abrupt, large-scale oxygen consumption by the enzymatic reaction—the respiratory or oxidative burst. Of the numerous biochemical events that ensue, the most important is the generation of several microbicidal products. All are formed ultimately from superoxide itself, the proximal enzymatic product. Hydrogen peroxide, singlet oxygen, and hydroxyl radical—all highly efficient microbicidal agents—are generated from superoxide. Hydrogen peroxide and chloride form hypochlorite and, ultimately, chloramines, through catalysis by myeloperoxidase. Thus the neutrophil relies on an arsenal of chemical weaponry to kill microorganisms. In the test tube, several million neutrophils undergoing the respiratory burst kill 10 million Escherichia coli within minutes; the same process occurs in the body, but only if neutrophils can phagocytose their targets and if substrates for the respiratory burst are available. It is evident that these highly reactive and toxic oxygen reduction products may destroy host cells along with invading microbes. Consequently, systems have evolved in mammals for protection of the surrounding host tissues at sites of inflammation. Catalase, superoxide dismutase, and glutathione peroxidase/glutathione reductase are detoxifying enzymes whose mechanisms scavenge oxygen intermediates. Other small organic molecules function as nonenzymatic scavengers. Neutrophil oxidative metabolism is extremely important for the maintenance of homeostasis in humans. Rare mutations that ablate NADPH oxidase activity lead to serious, frequent and invariably fatal pyogenic infections in children. Recent advancements have led to strategies that can augment other defenses and thereby prevent serious infections in these children.

Oxygen-independent microbicidal mechanisms compose a heterogeneous group. The most important are the microbicidal peptides and proteins that are released after bacteria are ingested during phagolysosomal fusion. An extensively studied example is the defensin family of molecules. By assembling into hydrophobic structures, which form pores through which bacterial components are lost, defensins lead to perturbation of bacterial membranes. This culminates in death of the bacterial cell.

Natural Killer Cells

NK cells are non-B, non-T lymphocytes that circulate in blood and function in constitutive host defense by identifying and destroying cells infected with viruses. Equipped with specialized proteins and enzymes packaged within intracellular granules, NK cells lyse virus-infected host cells and thereby interfere with viral replication. NK cells can bind yeast cells and destroy them. NK cells are an important source of cytokines, chemical messenger molecules that regulate the immune response. In rapidly developing infections, NK cell-derived IFN-? (one of the most important cytokines) activates macrophages to a microbicidal state.

DEFENSE MECHANISMS AT THE LEVEL OF PROTEIN MOLECULES

The complement proteins, designated collectively as C´, constitute a complex system of plasma zymogens that operate as part of innate host-defenses (the alternate pathway) and with antibodies, in adaptive immunity (the classical pathway). Although composed of different proteins, the complement system bears a striking analogy to the blood clotting system.

Complement proteins are produced primarily in the liver by hepatocytes. MNPs also synthesize and secrete all of the complement proteins, but the bulk of production depends on the liver.

The initial step of the alternative pathway is always active, but it occurs at a very low level. When bound to a microbial surface product, such as the lipopolysaccharide polymer from the cell wall of gram-negative bacteria, limited proteolysis proceeds stepwise until C3 is cleaved to C3b and C3a. C3b is a short-lived protein. It forms a covalent thioester bond near its formation site, namely on the surface of a bacterial cell. MNPs and polymorphonuclear phagocytes express a family of distinct C3 receptors. Ligand-receptor binding anchors the bacterial cell to the phagocyte. This attachment event initiates phagocytosis. In animal models, depletion of C´ proteins with cobra venom factor renders the host highly susceptible to infection with encapsulated bacteria such as S pneumoniae. Thus, the main function of the complement system is to generate opsonins, primarily C3b, molecules that increase the efficiency of phagocytosis.

Further in the complement cascade, C5 is cleaved into large C5b and small C5a proteolytic fragments. C5a diffuses from the site of inflammation, which sets up a concentration gradient that directs phagocyte emigration by chemotaxis.

Still later in the cascade a complex forms composed of C7, C8, and C9—the membrane attack complex (MAC). The MAC inserts itself into the outer lipid bilayers of gram-negative bacteria-forming pores visible by electron microscopy; this drastic perturbation leads to bacteriolysis. Bacteriolysis effected by the MAC is thus a mechanism for killing bacteria by fluid phase components—a phagocyte is not directly involved. However, bacteriolysis has limitations. First, gram-negative bacteria spawn mutants that are resistant to the lysing capability of the MAC. Second, gram-positive bacteria, mycobacteria, and fungi are naturally resistant to the MAC. Although protozoa may be killed by the MAC, CMI is the main defense mechanism against these pathogens.

The complement system performs three important host-defense functions: (1) it generates C3b, a major opsonic component that promotes phagocytosis; (2) it generates C5a, a chemotaxin that directs phagocyte motility toward an inflammatory stimulus; and (3) it kills some gram-negative bacteria through the MAC.

Humans with complement deficiencies, although uncommon, have been identified. These individuals exhibit characteristic susceptibilities to infection, such as disseminated infection with Neisseria meningitidis and N gonorrhoeae.

ADAPTIVE IMMUNE RESPONSES

INTRODUCTION

There are two general categories of adaptive immune responses. The first comprises the synthesis of specific Ig proteins (antibodies) by specialized lymphocytes called plasma cells. This is called humoral immunity. The second type of response, CMI, involves direct or indirect attack on microorganisms mediated by lymphocytes and other accessory cells (eg, macrophages). Both humoral immunity and CMI are characterized by two important features. First, the responses are directed toward specific biochemical moieties on microorganisms, called antigens. Second, the responses impart a "memory" in the host. This means that subsequent exposures to specific antigens are met with a much enhanced immune response.

HUMORAL IMMUNITY

Production of Antibodies

Antibodies are complex glycoproteins called Igs, which bind specifically to moieties called antigens. After an antigen is bound by an antibody, a variety of cellular and molecular mechanisms come into play. These mechanisms, which function to protect the host from microbial invaders, are discussed in the sections that follow. In conjunction with the complement system, antibodies are the main effector molecules of humoral immunity. Antibodies are used to inhibit or destroy foreign cells. In the circulation and on mucosal surfaces, antibodies help define acquired resistance to particular pathogens. The mechanistic bases for antibody-mediated resistance are varied and complex. It is necessary for the antibody alone to bind the antigen. Even this is not sufficient in all cases because additional mechanisms may be required to kill microorganisms (eg, participation by a phagocyte). Examples of antibody effector mechanisms include the following: (1) complement activation that leads to bacteriolysis; (2) opsonophagocytic function, in which antibodies attach microorganisms to phagocytes, which then kill the microorganisms; (3) antibody-dependent cellular cytotoxicity (ADCC), an antibody bridge between an effector cell (eg, lymphocyte or monocyte) and target cell (eg, virus-infected somatic cell) that activates a killing mechanism, whereby the target cell is destroyed; and (4) neutralization, whereby the antibody binds to a microbial toxin or virus, which renders it unable to bind to its receptor through stearic hindrance.

Two critical functions are encoded in the structure of the antibody molecule. The N-terminal end (variable or V region) of the molecule defines the antigen recognition site. There are many possible different amino acid sequences that lead to different antigen-binding specificities. The carboxy-terminal portion determines one of nine classes and subclasses of Ig isotypes. Each isotype carries a distinct functional attribute suited for various biologic tasks, such as complement fixation, opsonization, or distribution into certain body sites and surfaces. B lymphocytes produce antibodies that express surface-bound Igs of a single specificity. When these antigen receptors are engaged by specific ligands, B cells proliferate and differentiate into plasma cells. These cells then begin to secrete Ig molecules that are specific for the target antigen.

Immunoglobulin Structure

All Igs share the same basic molecular structure. This structure consists of two identical peptide heterodimers linked by disulfide bridges. Each heterodimer consists of a heavy chain (CH) and a light chain (CL) (Figure 2-2).

The antigen-binding site is in the Fab portion. Three hypervariable regions containing 10-12 amino acids with markedly viable sequences occupy the VK and VH domains. The quaternary structure of the CL and CH in this region determines antigen specificity. Because each Ig molecule has two heavy and two light chains, antibodies are bivalent for antigen binding. The portion of the antigen recognized is called the epitope. The complementary Fab-binding site is called the paratope.

A constant region of the CH is the product of distinct genes. This region defines the "class" of Igs. A hinge region determines flexibility of the molecule related to biological function. The Fc portion is critical for biological function, such as complement activation, the ability to bind to phagocyte receptors, and the ability to cross anatomic barriers such as placental membranes.

Immunoglobulins in Humans

Five Ig classes defined by their CH type are present in humans (Table 2-4). There are four IgG and two IgA subclasses. There are two classes of CLs (? and ?). Each B lymphocyte produces only one class of CL.

A. Immunoglobulin M. During the primary immune response, IgM antibodies are the first to be produced. The pentameric structure endows IgM with 10 antigen-binding sites. This assists in complement activation and aggregation of antigen and microbes. Antigen affinity for IgM is usually less than for IgG. Measurement of IgM antibody specificity and concentration is useful in diagnosis because its presence correlates with primary, active infection.

B. Immunoglobulin D. IgD is expressed on the surface of B cells and functions as antigen receptor. Membrane anchoring is a function of the extended hinge region of IgD molecules.

C. Immunoglobulin G. IgG is the most abundant Ig, making up ~ 75% of the total. It crosses the placenta and hence provides protection to the newborn during the first 6 months of life. It is present in the lower respiratory tract and in exudative secretions. There are four subclasses (IgG1, IgG2, IgG3, and IgG4), which are determined by the constant regions of their heavy chains. Each bears distinct biologic activities. IgG1 and IgG3 fix complement well. IgG3, but not IgG2 and IgG4, binds and activates leukocyte Fc receptors. IgG2 is produced against the polysaccharide determinants of bacterial capsules. IgG4 may compete with IgE for antigen and thereby alter immediate-type hypersensitivity reactions.

D. Immunoglobulin A. There are two subclasses of IgA—IgA1 and IgA2. IgA is produced in gut lymphoid tissue and is dimeric. It is transported from submucosa to the gut lumen. This is mediated by a special secretory component, expressed as an integral membrane protein on the basolateral surface of gastrointestinal epithelial cells. This secretory component is a receptor for dimeric IgA and initiates endocytosis, transcytoplasmic transport, and release of IgA dimers into the intestinal lumen. This process occurs at other mucosal sites in the respiratory and urogenital tracts, as well as in nasal secretions, tears, saliva, and colostrum. IgA interferes with attachment of microorganisms to mucosal surfaces. It neutralizes ingested toxins, and it may promote phagocytosis by leukocytes emigrating into mucosal epithelium.

E. Immunoglobulin E. Although only trace amounts are present in the circulation, IgE predominates on the surface of mast cells and basophils through noncovalent high-affinity receptor binding of its Fc portion. Immediate-type hypersensitivity or reaginic responses occur when antigen binds to cell surface IgE. Degranulation with release of histamine, eicosanoids, and peptide mediators of inflammation from mast cells may lead to urticaria, laryngeal and intestinal wall edema, and anaphylactic shock. The evolution of reaginic responses was driven by the necessity for a host-defense mechanism against invasion of tissues, especially epithelial surfaces, by helminths and ectoparasites. Monocytes, eosinophils, and platelets may cooperate with IgE in ADCC reactions against certain trematodes.

B Cells

Generation of an effective humoral immune response is vested in progenitor cells located in the bone marrow, which undergo differentiation to become mature B cells in the spleen and lymph nodes. Activation of mature B cells into antibody-producing plasma cells is a complex, highly regulated process. Regulation occurs through the expression of B-cell surface receptor molecules, which alter cell function upon binding ligand. Important B-cell receptors and their functions are shown in Table 2-5.

Antigen binding to its specific receptor on B cells leads to the formation of the B-cell receptor complex. When formed, the complex lowers the threshold for B-cell activation. Intracellular signal transduction through protein phosphorylation by Src kinases activates membrane phospholipase C?2. Inositol triphosphate and diacylglycerol are formed. Intracellular calcium is mobilized and protein kinase C is activated. Transcription of cellular genes is induced by regulating cell division, differentiation, and Ig synthesis. However, these events are usually not sufficient for high-level antibody production. T cells, which recognize processed antigen-derived peptide in association with complementary major histocompatibility complex (MHC) class-II determinants, physically aggregate with B cells and promote B-cell activation. This is called T-cell helper function. This is the main pathway for antibody production against protein antigens.

Certain polymeric antigens, especially bacterial polysaccharides bearing repetitive monosaccharide units, can activate B cells without T cells. This type of T-cell-independent antibody response may relate to cross-linking B-cell receptor complexes (and hence more efficient signal transduction) by long strands to linear polysaccharide antigen molecules. T-cell-independent antibody responses tend to produce low-affinity IgM and IgG2 isotypes in relatively low levels. Furthermore, memory B-cell formation is inefficient. Thus, bacterial polysaccharide vaccines may not be particularly immunogenic. By coupling a bacterial polysaccharide to a protein component (eg, tetanus toxoid) a T-cell-dependent B-cell response is induced, and memory cells become more abundant. Recently, lymphocytes not ordinarily classified as T or B cells, which bear the CD1 surface marker, were shown to help process polysaccharide antigens covalently attached to lipid moieties. This pathway of antigen presentation, which is not restricted by MHC, could prove useful for future development of polysaccharide vaccines.

Antibody Diversity

Antibody diversity is explained by rearrangements of DNA sequences within unique regions of Ig genes. Antibodies produced during a primary antibody response after an encounter with a new antigen are IgM in isotype. They appear 5-10 days after exposure to antigen, and their antigen affinity is relatively low. On reexposure to antigen, secondary antibody responses result in rapid production (1-3 days) of higher-affinity, higher-titer IgG, IgA, or IgE isotypes. Memory B cells formed during a primary response mediate the more efficient secondary response with the help of T cells. Switching of isotypes from IgM to IgG, for example, is controlled by T cells and the cytokines they secrete to communicate with B cells. Isotype switching and affinity maturation and epitope diversity of Igs are the result of four distinct molecular processes:

1. Multiple segments within the hypervariable region of the DNA encoding light and heavy chains (VL and VH; Figure 2-2) undergo recombination.

2. The segments are rejoined inexactly.

3. The quaternary association between VH and VL protein products leads to unique antigen-binding sites. These events lead to an enormous repertoire of B cells bearing different antigen receptor specificities. These specificities are directed against those portions of antigen molecules called epitopes.

4. A nonrecombinatorial mechanism involving somatic point mutations in the VH and VL regions leads to idiotype diversity. This mechanism is believed to account for maturation, when repeated administration of antigen results in antibody production with incrementally increased affinity for the eliciting epitope.

Immunoglobulin Fc Receptors

Antibodies function in host defense in conjunction with other proteins (eg, complement components) and cells (eg, phagocytes). Antigen bound to the Fab portions is connected to functional components by the Ig Fc portion. Thus, the specificity of Fc ligands interacting with Fc receptors on other immune cells determines the character of immune responses mediated by antibodies. Families of Fc receptors and their functional activities are shown in Table 2-6.

Function of Antibody-Mediated Host Defense

Through specific binding of antigen and Fc portion receptor-ligand interaction, antibodies facilitate destruction of microbes and neutralize toxins. They may act at a distance from their cell of origin, and their soluble properties allow for dispersion throughout the body in the circulation and lymph. The utility of Igs in host defense is multifactorial. However, the overriding feature is their functional capability for attaching microorganisms to phagocytes and lymphocytes bearing microbicidal mechanisms. The following sections summarize antibody-directed immune mechanisms.

A. Activation of Complement Proteins. The CH2 (IgG) and CH3 (IgM) domains (see Figure 2-2) of antibody Fc regions activate complement by binding to microbes and then interacting with multiple sites on C1q, the first component of the complement cascade (classical complement pathway). Activation of complement on microbial surfaces leads to bacteriolysis in some instances or, more commonly, to phagocytosis. Gram-negative bacteria are susceptible to bacteriolysis as are some protozoans. But gram-positive bacteria, fungi, and viruses cannot be lysed. Furthermore, gram-negative bacilli resistant to complement-mediated lysis (serum resistance) can be selected for during natural infection. Opsonophagocytic antibodies important for the resolution of infection have been identified during infection with S pyogenes, S pneumoniae, H influenzae, N meningitidis, and Staphylococcus aureus.

B. Promotion of Phagocytosis. The three classes of IgG Fc receptors mediate phagocytosis of IgG-coated particles. IgG2 and IgG3 subclasses are generally required. Mucosal phagocytes, namely macrophages and neutrophils, bearing IgA Fc receptors facilitate engulfment of microorganisms. Fc receptors alone are usually sufficient for phagocytosis of most bacteria and fungi; however, removal of encapsulated bacteria may require Fc and C3 receptors functioning in concert. In experimental S pneumoniae bacteremia caused by highly virulent strains, clearance of bacteria from the circulation failed when animals were depleted of complement proteins, even when anticapsular IgG antibody was present.

C. Antibody-Dependent Cellular Cytotoxicity. ADCC is a mechanism by which leukocytes destroy somatic cells infected with microorganisms. Antibody-coated microorganisms or parasitized host cells can be killed when Fc receptors on leukocytes are engaged. This reaction occurs without phagocytosis, but depends on contact between effector and target cells. Nonphagocytic immune cells (eg, NK cells) lyse antibody-coated virus-infected host cells through IgG RIII receptors. In this reaction, NK cells exocytose cytotoxin protein molecules called perforins onto target cells. Perforins assemble in the target cell membrane-forming pores similar to the MAC of complement. Rapid lysis ensues. Macrophages and neutrophils participate in ADCC reactions. IgE-coated metazoans such as parasitic helminths are destroyed by eosinophils bearing Fc receptor II molecules. Eosinophils exocytose a cytotoxic molecule, called major basic protein, onto the parasite surface on engagement and cross-linking of these Fc receptors.

D. Neutralization of Microbial Toxins. Microbial toxin neutralization is a striking example of protection mediated by antibodies. Lethal toxins produced by Clostridium spp. (eg, botulinum toxin and tetanus toxin) and by Corynebacterium diphtheriae (diphtheria toxin) are efficiently detoxified by specific IgG. Antibodies bind to toxin molecules and, through stearic hindrance, block their receptor-mediated uptake by susceptible cells. The antitoxin effect of antibodies is the basis for some of the most successful immunization practices in medicine.

Endotoxins are the lipopolysaccharide polymers that form part of the structure of the outer membrane of gram-negative bacteria. Their biologic effects are myriad, but they include the induction of lethal collapse of the circulatory system. Much effort has been made to produce protective antibodies against endotoxins for administration to patients in shock. Despite broad reactivity and monoclonal antibody technology, this strategy has not yet led to significant advances for the treatment of endotoxemia.

E. Neutralization of Viruses. IgM, IgG, and IgA antibodies bind to specific epitopes on virions and block virus access to host cells. The effective prevention of polio, smallpox, hepatitis A and B, and rabies by immunization is based on this principle. Other viruses, most notably herpes viruses and human immunodeficiency virus (HIV)-1, spread from cell to cell and hence evade neutralizing antibody. One exception may be the intracellular neutralization of viruses within mucosal epithelial cells as polymeric IgA is transported through these cells.

F. Blockage of Microbial Adhesion. Pathogenic microorganisms entering the body via mucosal surfaces first attach to epithelial cells by receptor-ligand binding. Specific IgA directed at these determinants may block adherence and hence abort microbial invasion.

G. Agglutination of Microorganisms into Large Aggregates. Polyvalent IgM, IgG, and IgA can cross-link microbes forming large aggregates. In the respiratory tract, the mucociliary clearance mechanism may then facilitate removal of these aggregates before invasion can occur.

CELL-MEDIATED IMMUNITY

Introduction

CMI refers to an immune response against organisms (usually facultative or obligate intracellular microorganisms) in which antibody has a subordinate or no role. CMI was first demonstrated experimentally as an immune response that occurred in passively immunized animals transfused with cells (lymphocytes) but not with specific Igs. Such strict division between cell-mediated and humoral immune responses is oversimplified because specific Igs and antigen-antibody complexes interact to both enhance and inhibit CMI reactions.

CMI involves signal transmission between cells participating in the response. Intercellular signaling is accomplished in two ways: (1) by cell-cell interaction involving surface molecules on the interacting cells and (2) via chemical messenger molecules called cytokines. These mechanisms operate both in innate immune responses and in antigen-specific CMI. Intercellular signaling mechanisms are then used to activate cytotoxic effector cells for microbiostatic and microbicidal functions. Cell signaling and activation are discussed individually in the next section.

Cell-Cell Interactions

A. Major Histocompatibility Complex Molecules. MHC molecules compose a group of polymorphic integral membrane proteins encoded by segments of clustered genetic loci. These molecules participate in binding antigenic peptides for T-cell recognition. Two structurally distinct types of MHC molecules exist. MHC class I molecules are involved in classic immune responses to virus-infected cells, foreign tissue grafts, and tumors. There are ~ 70 different alleles composing three separate subclasses of MHC class I genes. MHC class I molecules express peptides synthesized within the host cell (eg, a protein encoded by a viral gene) and transported from endoplasmic reticulum via the Golgi apparatus to the cell surface. ß2-Microglobulin and calnexin are required to stabilize MHC class I complexes. MHC class I antigens are presented as nonapeptides, and class I can be presented by all nucleated cells.

MHC class II molecules express antigens only on "professional" antigen-presenting cells (APCs), such as MNPs (macrophages) and related cells, dendritic cells throughout the body and Langerhans cells in the skin, and some B lymphocytes. MHC class II antigens are presented as 12- to 24-amino-acid peptides that are endocytosed from the environment and are expressed on the cell surface, having made their way through the endosomal/liposomal pathway.

B.T Lymphocytes. T cells interact directly with APCs. This cell-cell interaction drives specific immune responses by inducing clonal expansion of antigen-specific T cells. This expansion in turn results in secretion of signaling molecules, called cytokines. Cytokines direct the activities of additional immune response cells. Thus, the response to a specific antigen is vastly amplified. T cells bear receptors that bind to peptide antigen-MHC molecule complexes on antigen-presenting cells. Antigen-presenting cells bearing MHC class I antigens bind to T cells expressing the CD8+ T-cell receptor surface marker. MHC class II antigens bind to CD4+ T cells. This means that class I and II antigens stimulate separate populations of T cells. This separation directs the immune response toward different mechanisms adapted to deal with different antigenic challenges. CD4+ T cells function as cytokine secretors, and they help B cells produce Igs. CD8+ T cells become efficient cytotoxic cells, capable of lysing cells infected with viruses. Both CD4+ and CD8+ T cells bear the CD3 cell surface marker. The CD3 molecule transduces an up-regulating intracellular signal on binding to MHC class I or II antigens with CD8 or CD4 receptors, respectively. Mice with null mutations of the CD4 gene lack T-cell helper function; mice with null mutations of the CD8 gene lack cytotoxic T-cell function.

T-cell activation is maximized by coaggregation of the T-cell receptor with CD4 or CD8 bound to an MHC molecule-antigen complex on the APC. Other costimulatory molecules participate in T-cell activation, such as the CD28 T-cell marker bound to the B7/BB1 family of molecules on APCs. Upon aggregation and binding of these molecules between T cells and APCs, a complex cascade of intracellular events ensues. These events lead to T-cell differentiation and T-cell replication with clonal expansion. The latter is mediated by the T-cell growth factor, IL-2, which is secreted upon T-cell activation. The immunosuppressant antibiotic cyclosporin A inhibits IL-2 secretion by T cells by blocking the intracellular calcium-dependent functions in the complex cascade during T-cell activation.

Microbial superantigens activate T cells. Particular microbial toxins (ie, staphylococcal enterotoxin, toxic shock syndrome toxin, and streptococcal pyrogenic exotoxin A) are presented as MHC class II antigens, but they bind nonspecifically to the T-cell receptor. Up to 10% of T cells may be activated. This leads to widespread cytokine secretion and the systemic toxicity of a generalized cell-mediated immune response. Particular MHC class II genotypes and the T-cell receptor ß-chain genotypes are more prone to these reactions.

Memory T cells help mediate secondary cell-mediated immune responses. These T cells bear the CD29 surface marker. Memory T cells show enhanced secretion of particular cytokines, and they mediate rapidly developing cytotoxicity responses.

Lymphocyte adhesion molecules mediate trafficking and homing of T cells to particular body sites. Some T cells home to mucosal sites where they are strategically stationed to participate with APCs upon entry of foreign antigens into the tissues.

Cytokines

Cytokines function as chemical messengers, sending signals from one immune cell to another. Cytokines are glycoprotein molecules secreted by lymphocytes. These molecules were originally called lymphokines. As their identities and cells of origin were defined, the term lymphokine was replaced by cytokine to include signal molecules elaborated during CMI whose cells of origin included other cell types (eg, macrophages, endothelial cells, and fibroblasts). As cytokines became characterized molecularly through cloning, sequencing, and expression techniques, they were assigned numbers and designated as interleukins (Table 2-7). However, some cytokines retained their original names, based primarily on their immunologic activities (eg, IFNs, TNFs, and CSFs). During CMI, cytokines act locally, infiltrating tissue sites of inflammatory cells. However, they may exert systemic effects when released into the circulation. This occurs during widespread infection and certain toxemias (eg, in response to superantigens as noted above).

Cytokines act to amplify or attenuate the immune response coordinately. Their action is not antigen specific, but their secretion is often driven by antigen-specific reactions. Certain microbial products (eg, lipopolysaccharide of gram-negative bacilli) directly stimulate cytokine secretion (eg, TNF and IL-1). Cytokines regulate clonal expansion for both T and B lymphocytes. They mediate recruitment of immune cells to sites of inflammation. They activate effector cells (eg, macrophages) to microbicidal states. Some cytokines deactivate cells to prevent local tissue damage after the destruction of invading microbes. Cytokines interact with specific receptors on the cells they signal. The receptors act as transducers and relay signals into the cells, leading to additional secretions, replication, cell cycle arrest, and activation for microbicidal activities.

Cytokines secreted by MNPs are called monokines. Monokines help initiate CMI as macrophages phagocytose microbes and present their antigens to T cells. Two important cytokines come almost exclusively from T cells (hence they are lymphokines)—IL-2 and IFN-?. IL-2 leads to many effects including replication of T cells activated by specific antigen. IFN-? is the main lymphokine for activation of effector cells (macrophages) to destroy or inhibit pathogens. T cells respond with lymphokine secretion only if the T-cell receptor is engaged with specific antigen presented in context with MHC class I or class II molecules. An exception is the stimulation by superantigens. This is in contrast to monokine secretion, which occurs in response to a wide variety of stimuli. NK cells are also a source of IFN-? during primary infection, before T-cell activation.

CD4+ T cells change the profile of cytokines they secrete, and this directs the cell-mediated immune response toward activities suited best for defense against particular pathogenic agents. In the mouse during infection with intracellular pathogens, CD4+ T cells secrete IL-2 and IFN-?. This leads to macrophage activation and arrest of microbial intracellular replication by unknown mechanisms (TH1 response). The monokine, IL-12, directs CD4+ T cells into a TH1 phenotype. During systemic helminth infections CD4+ T cells secrete IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. This profile leads to expansion of B-cell clones for production of Igs, including IgE (so-called TH2 response). IgE participates in release of products from basophils, mast cells, and eosinophils that mediate cytotoxicity for multicellular animals that invade tissue usually via the intestinal tract. IL-10 directs CD4+ T cells into a TH2 phenotype.

Transforming growth factor-ß, IL-4, and IL-10 down-regulate CMI to intracellular pathogens. Mice lacking a functional transforming growth factor-ß gene die after birth with massive infiltration of lymphocytes and macrophages in their vital organs.

Other roles for cytokines include (1) control of hematopoiesis, (2) T-cell maturation and proliferation, (3) B-cell differentiation, and (4) T-cell suppressor function. Cytokines also regulate innate immune responses such as fever (IL-1) and the acute-phase response (IL-6, IL-1, and TNF). Cytokines released systemically cause shock (TNF) during septicemia.

Activation of Effector Cells

Effector cells of the cell-mediated immune response are microbiostatic and microbicidal for intracellular pathogens. These cells are cytotoxic T-cells, NK cells, and macrophages. Effector cells mediate and cytokines modulate antimicrobial activity during cell-mediated immune responses. Effector cells participating in CMI along with the functions they perform are listed in Table 2-8.

A. Cytotoxic T Cells. Cytotoxic T lymphocytes (CTLs) kill somatic cells that are infected with microbial pathogens. These T cells express CD8 and mediate antigen-specific, class I MHC-restricted cytolytic activity. Recall that class I recognition occurs for antigens synthesized within the host cell. This occurs during the replication cycle of viruses. CTLs are ideally suited for destroying virus-infected cells. Release of immature, unassembled virus components upon host-cell lysis blocks efficient viral replication. CTLs may lyse cells infected with bacteria. This thwarts the strategy of some facultative intracellular pathogens, whose survival in the host may depend on evasion of the immune system by sequestration within an intracellular niche. Differentiation of CTLs to the cytotoxic state is regulated by cytokines. Of those involved, IL-2 is most important. The biochemical mechanisms of cytolysis involve a Ca2+-dependent assembly of a protein present in CTL granules, called perforin. The protein is assembled into a cylinderlike structure that is inserted into the plasma membrane of the antigen-bearing target cell. Serine proteases within CTL granules, called granzymes, enter the target cell through perforin pores. The enzymes then become catalytically active and participate in target cell destruction.

B. Natural Killer Cells. NK cells participate in CMI by acting as effector cells and cytokine-secreting cells. NK cells are CD158+ lymphocytes that lack the T-cell receptor. They lyse certain neoplastic cells and virus-infected cells by a recognition mechanism that does not involve antigen-MHC restriction. These cells are active against virus-infected host cells, in particular the human herpes viruses. NK cells also function as an important source of IFN-? early during bacterial infections before CD4 cell activation. Their large granules contain granzymes and perforin monomeres, such as CTLs.

C. Mononuclear Phagocytes. MNPs include circulating monocytes and tissue macrophages. They play two key roles in the cell-mediated immune response, as APCs and as cytotoxic effector cells. Their latter role is discussed here. Activated MNPs are recruited from a pool of precursor cells in the bone marrow. These phagocytes are released into the circulation and adhere to capillaries adjacent to infected tissue by a process involving intracellular adhesion molecules. Chemokines specific for MNPs participate to attract these cells. MNPs migrate between capillary endothelial cells and crawl toward microorganisms by chemotaxis. Chemotactic molecules from the host and infecting microbes act as stimuli, setting up a concentration gradient sensed by MNPs.

In the inflammatory milieu, MNPs are exposed to activating cytokines, primarily IFN-? and TNF. Macrophages phagocytize microorganisms via a variety of receptor interactions involving IgG, IgA, and complement components, as well as via lipopolysaccharide and carbohydrate ligands present on bacteria, protozoa, fungi, and virus outer envelopes. Lysosomal granules within MNPs fuse with phagocytic vacuoles forming phagolysosomes. Granule components, including proteases, lysozyme, and microbicidal proteins, are released as the phagolysosome is acidified.

Activated MNPs inhibit and kill phagocytized microorganisms by a variety of mechanisms involving reactive-oxygen and reactive-nitrogen (from nitric oxide) intermediates, microbicidal proteins, liposomal proteases, and as-yet-unknown biochemical mechanisms. Activated MNPs play an important host defense role in combating a wide variety of bacteria, protozoans, fungi, and metazoans that have adapted virulence mechanisms for intracellular survival. Activated MNPs are critical for granuloma formation. They become epithelioid cells occupying the centers of granulomas. Granulomas are the histopathologic hallmark of the cell-mediated immune response against intracellular pathogens such as tubercle bacilli. TH1 response cytokines are required for granuloma formation. Activated MNPs may also mediate destruction of these same pathogens through a contact-dependent mechanism not requiring phagocytosis. Activated MNPs may also participate in cell-mediated immune responses through more efficient antigen presentation to T cells as well as through the production of cytokines that enhance T-cell-dependent killing mechanisms. Table 2-9 lists some examples of activated MNP-dependent host defenses against microbial pathogens.

REFERENCES

Joklik WK, et al (editors): Zinsser Microbiology, 20th ed. Appleton & Lange, 1992.

Mandell GL, Bennett JE, Dolin R: Principles and Practice of Infectious Diseases, 5th ed. Churchill Livingstone, 2000.

Mims C, et al: Mims' Pathogenesis of Infectious Disease, 4th ed. Academic Press, 1995.

Paul WE (editor): Fundamental Immunology. Lippincott-Raven, 1999.

Roitt I, Brostoff J, Male D: Immunology, 5th ed. Mosby, 1998.

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