Epithelial-Derived Chemicals

The epithelial surfaces of the body secrete a complex array of proteins that destroy potential pathogens. Epithelial cells secrete small-molecular-weight antimicrobial peptides.³ These are generally positively charged polypeptides of approximately 15 to 95 amino acids and can be divided into two classes—cathelicidins and defensins—based on their 3-dimensional structures. Both classes are in very high local concentrations and are toxic to several bacteria, fungi, and viruses. Cathelicidins have a linear α-helical shape, and only one is currently known to function in humans. In contrast, about 50 different defensins have been identified thus far. All are triple-stranded β-sheet structures. Defensin molecules contain 3 intrachain disulfide bonds and can be further subdivided into α (at least 6 identified in humans) and β types (at least 10 identified, but perhaps up to 40 different molecules), depending on how the cysteine residues are connected during formation of the disulfide linkages.⁴ The α-defensins often require activation by proteolytic enzymes, whereas the β-defensins are synthesized in active forms. Bacteria have cholesterol-free cell membranes, which may allow cathelicidins to insert into and disrupt their membranes. Given the similarity in their chemical charges, defensins may kill bacteria in the same way. These same chemicals also may contribute to other means of protection because they are also produced by monocytes, macrophages, and neutrophils, which are components of the inflammatory response. Cathelicidin is stored in neutrophils, mast cells, and a variety of epithelial cells. The α-defensins are particularly rich in the granules of neutrophils and may contribute to the killing of bacteria by those cells. They are also found in Paneth cells lining the small intestine, where they protect against a variety of disease-causing microorganisms. The β-defensins are found in a variety of epithelial cells lining the respiratory, urinary, and intestinal tracts, as well as in the skin. In addition to antibacterial properties, β-defensins may also help protect epithelial surfaces from human immunodeficiency virus (HIV) infection. Both classes of antimicrobial peptides also can activate cells of innate and adaptive immunity.

The lung also produces and secretes a family of glycoproteins, collectins, which includes surfactant proteins A through D and mannose-binding lectin. The binding site of each collectin reacts with different affinities to a range of monosaccharides, enabling collectins to recognize a wide array of pathogenic microorganisms. Collectin binding facilitates recognition of the microorganism by macrophages, enhancing macrophage attachment, phagocytosis, and killing. Collectins play a major role in protection against respiratory tract infections.

Other epithelial antimicrobials include resistin-like molecule β, bactericidal/permeability-inducing protein, and antimicrobial lectins. Resistin-like molecule β is found in the intestinal goblet cells, where it appears to protect against helminth infections. Bactericidal/permeability-inducing (BPI) protein is stored in neutrophils and intestinal epithelium. BPI protein specifically reacts with lipopolysaccharide on the surface of gram-negative bacteria, resulting in bacterial lysis. Antimicrobial lectins are carbohydrates that are found in intestinal epithelium and have activity against gram-positive bacteria.

Bacteria-Derived Chemicals

The body’s surfaces are colonized with a spectrum of microorganisms, the normal microbiome. Each surface, including the skin and the mucous membranes of the eyes, upper and lower gastrointestinal tracts, urethra, and vagina, is colonized by a combination of mostly bacteria and occasionally fungi that is unique to the particular location. The microorganisms in the microbiome do not normally cause disease, and although their relationship with humans has been referred to as commensal (to the benefit of one organism without affecting the other), the relationship may be more mutualistic (to the benefit of both organisms). Using the colon for an example, at birth the lower gut is relatively sterile but colonization with bacteria begins quickly, with the number, diversity, and concentration increasing progressively during the first year of life. To the benefit of humans, many of these microorganisms help digest fatty acids, large polysaccharides, and other dietary substances; produce biotin and vitamin K; and assist in the absorption of various ions, such as calcium, iron, and magnesium.⁵

These bacteria contribute to our innate protection against pathogenic microorganisms in the colon. They compete with pathogens for nutrients and block attachment to the epithelium. Members of the normal microbiome also produce chemicals (ammonia, phenols, indoles, and other toxic materials) and toxic proteins (bacteriocins) that inhibit colonization by pathogenic microorganisms. Prolonged treatment with broad-spectrum antibiotics can alter the normal intestinal microbiome, decreasing its protective activity, and lead to an overgrowth of pathogenic microorganisms, such as the yeast Candida albicans or the bacteria Clostridium difficile (overgrowth can cause pseudomembranous colitis, an infection of the colon). Additionally, the normal microbiome of the gut help train the adaptive immune system by inducing the growth of gut-associated lymphoid tissue (where cells of the adaptive immune system reside) and the development of both local and systemic adaptive immune systems.⁶

The bacterium Lactobacillus is a major constituent of the normal vaginal microbiome in healthy women.⁷ This microorganism produces a variety of chemicals (e.g., hydrogen peroxide, lactic acid, bacteriocins) that help prevent infections of the vagina and urinary tract by other bacteria and yeast. Prolonged antibiotic treatment can diminish colonization with Lactobacillus and increase the risk for urologic or vaginal infections, such as vaginosis.

Some members of the normal bacterial microbiome are opportunistic; opportunistic microorganisms can cause disease

Complement System

The complement system consists of several plasma proteins (sometimes called complement components) that together constitute about 10% of the total circulating serum protein. The complement system is extremely important because activation of the complement cascade may destroy pathogens directly and can activate or collaborate with virtually every other component of the inflammatory response. Proteins of the complement system are among the body’s most potent defenders, particularly against bacterial infection.

Activation of the complement system can be accomplished in three different pathways, all of which converge at the third component (C3) of the pathway:

The principal routes by which the complement cascade may be activated are shown in Figure 7-5.

Activation of the classical pathway begins with the activation of complement protein C1 and is preceded by formation of a complex between an antigen and an antibody to form an antigen-antibody complex (immune complex) (discussed in Chapter 8). The antigen may be a unique chemical component of the surface of a bacterium or other microorganism. Most pathogens express multiple antigens; therefore, multiple antibodies are usually bound in the complex. The first component of the classical complement cascade, C1, has six sites that can bind to antibodies, and efficient activation of the complement cascade usually requires concurrent binding of C1 to at least two antibody molecules. The complex formed by antigen-antibody-complement binding is shown in Figure 7-5. C1 is a macromolecular complex consisting of C1q and two molecules each of C1r and C1s. A conformational change in C1 results in an enzymatically active molecule whose substrates are C4 and C2. The resultant complex formed by the interaction of C1, C4, and C2 uses C3 as a substrate, resulting in the production of C3a and C3b. A complex that has C3 as a substrate is generally referred to as a C3 convertase. The addition of C3b to the complex changes the substrate specificity to C5, resulting in the conversion of C5 to C5a and C5b. A complex that has C5 as a substrate is generally called a C5 convertase. Thus activation of C1 initiates the sequential enzymatic activation of all other components of the classical pathway, ultimately resulting in the activation of C5. The classical pathway also can be activated to a lesser degree by biologic molecules other than antibody, including heparin (a charged molecule that prevents clotting), deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and C-reactive protein, which is increased in the blood during inflammation.

Even under normal conditions small amounts of circulating C3 are spontaneously broken down into C3b and C3a by a number of naturally occurring enzymes in the blood. The rate of C3 spontaneous activation is generally very low, and C3b is readily inactivated by complement regulator proteins in the blood (e.g., factor H and factor I). However, materials produced by some infectious microorganisms (e.g., lipopolysaccharides [endotoxins] on the bacterial surface, yeast cell wall carbohydrates [zymosans]) can bind the naturally produced C3b and protect it from inactivation. This will initiate activation of the alternative complement pathway. The C3b bound to bacterial products can react with another normally occurring component, factor B. The complex of C3b and factor B is recognized by an enzyme, factor D, which activates factor B, producing factor Bb. The resultant C3b/Bb complex is very unstable unless it binds to properdin (P). The C3b/Bb/P complex is a C3 convertase that produces further C3b, resulting in a C3b/Bb/P/C3b complex that is a C5 convertase, which activates C5.

The lectin pathway is similar to the classical pathway but is antibody independent. It is activated by a plasma protein called mannose-binding lectin (MBL). MBL is similar to C1q and binds to bacterial polysaccharides containing the carbohydrate mannose. MBL-associated serine proteases (MASP-1 and MASP-2) substitute for C1r and C1s and activate C4 and C2 to create a C3 convertase.

After activation of C5, the cascade continues through the terminal components C6, C7, C8, and C9. Components C5b through C9 assemble to form complexes (membrane attack complex, or MAC) capable of creating pores in cell membranes and permitting the influx of water and ions and may ultimately result in cell lysis.

The most important result of complement activation is the production of fragments during the activation of C4, C2, C3, and C5. The fragments C4a, C2b, C3a, and C5a are soluble and of low-molecular-weight that contribute in other ways to the inflammatory response. C2b affects smooth muscle, causing vasodilation and increased vascular permeability. C3a and C5a, and to a limited extent C4a, are anaphylatoxins; that is, they induce rapid mast cell degranulation (release of granular contents) and the release of histamine (see Figure 7-11, p. 206), causing vasodilation and increased capillary permeability.⁹ C5a is the major chemotactic factor for neutrophils. C3a is approximately 100 times less potent in chemotactic and anaphylatoxic activity. A chemotactic factor is a biochemical substance that attracts leukocytes to the site of inflammation.

The dual functions of a chemotactic factor and an anaphylatoxin are not needed simultaneously or to the same degree. Anaphylatoxic activity is necessary early in inflammation and occurs close to the inflammatory site to induce local mast cell degranulation and to increase the number of soluble mediators available to enhance vascular permeability and vasodilation. Chemotactic activity, on the other hand, is required for a much longer period and occurs distal to the inflammatory site to attract leukocytes from the circulation. Thus it is beneficial to an effective inflammatory response to limit the range of anaphylatoxic activity while allowing widespread chemotactic activity. A plasma enzyme, a carboxypeptidase, removes a terminal arginine on both C3a and C5a peptides, thereby producing “C3a desArg” and “C5a desArg,” which are inactive as anaphylatoxins but retain chemotactic activity. Thus chemotactic activity is retained, while not inducing distal mast cell degranulation that would result in considerable enlargement of the inflammatory response to the detriment of surrounding healthy tissue.

C3b adheres to the surface of a pathogenic microorganism and serves as an efficient opsonin. Opsonins are molecules that “tag” microorganisms for destruction by cells of the inflammatory system (primarily neutrophils and macrophages [see pp. 208 and 209]). C3b on the cell surface also can be broken down by several enzymes in the blood into inactive fragments (e.g., iC3b), which retain opsonic activity.

In summary, the complement cascade can be activated by at least three different means, and its products have four functions: (1) anaphylatoxic activity resulting in mast cell degranulation, (2) leukocyte chemotaxis, (3) opsonization, and (4) cell lysis.

Clotting System

The clotting (coagulation) system is a group of plasma proteins that form a fibrinous meshwork at an injured or inflamed site. This (1) prevents the spread of infection to adjacent tissues, (2) traps microorganisms and foreign bodies at the site of inflammation for removal by infiltrating cells (e.g., neutrophils and macrophages), (3) forms a clot that stops bleeding, and (4) provides a framework for future repair and healing. The main substance in this fibrinous mesh is an insoluble protein called fibrin that is the end product of the coagulation cascade.

The clotting system can be activated by many substances that are released during tissue injury and infection, including collagen, proteinases, kallikrein, and plasmin, as well as by bacterial products such as endotoxins. Like the complement cascade, the coagulation cascade can be activated through different convergent pathways (see Figure 7-6). The tissue factor (extrinsic) pathway is activated by tissue factor (TF) (also called tissue thromboplastin) that is released by damaged endothelial cells in blood vessels and reacts with activated factor VII (VIIa). The contact activation (intrinsic) pathway is activated when the vessel wall is damaged and Hageman factor (factor XII) in plasma contacts negatively charged subendothelial substances. The pathways converge at factor X. Activation of factor X begins a common pathway leading to activation of fibrin that polymerizes to form a fibrin clot. The coagulation system is discussed in more detail and illustrated again in Chapter 27.

As with the complement system, activation of the clotting system produces fragments that enhance the inflammatory response. Two low-molecular-weight fibrinopeptides, A and B, are released from fibrinogen when fibrin is produced. Both fibrinopeptides (especially fibrinopeptide B) are chemotactic for neutrophils and increase vascular permeability by enhancing the effects of bradykinin (formed from the kinin system).

Kinin System

The third plasma protein system, the kinin system, augments inflammation in several ways. The primary kinin produced from the kinin system is bradykinin, which causes dilation of blood vessels, acts with prostaglandins to stimulate nerve endings and induce pain, causes smooth muscle cell contraction, increases vascular permeability, and may increase leukocyte chemotaxis (see Figure 7-2). Bradykinin induces smooth muscle contraction more slowly than histamine and, along with prostaglandins of the E series, is probably responsible for endothelial cell retraction and increased vascular permeability in the later phases of inflammation (endothelial cell retraction is shown in Figures 7-3 and 7-15).

The kinin system is activated by stimulation of the plasma kinin cascade (see Figure 7-7). The conversion of plasma prekallikrein to kallikrein is induced by prekallikrein activator, which is identical to factor XIIa (the product that results from activation of Hageman factor—factor XII) of the clotting cascade. Kallikrein then converts kininogen to bradykinin. Although the plasma kinin cascade is one pathway that leads to the production of bradykinin, tissue kallikreins in saliva, sweat, tears, urine, and feces provide other sources for this inflammatory mediator. These tissue kallikreins convert serum kininogens to kallidin, also known as Lys-bradykinin, which may be converted to bradykinin by plasma aminopeptidase. In order to limit the extent of inflammation, kinins are rapidly degraded by kininases, enzymes present in plasma and tissues.

Interactions Among the Plasma Protein Systems

The three plasma protein systems are highly interactive so that activation of one system results in the production of a large number of very potent, biologically active substances that further activate the other systems (Figure 7-8). Very tight control of these processes is essential for two reasons:

Therefore, multiple mechanisms are available to either activate or inactivate (regulate) these plasma protein systems. As an example, plasmin regulates clot formation by degrading fibrin and fibrinogen, and it can activate the complement cascade through components C1, C3, and C5. Plasmin can activate the plasma kinin cascade as well by activating Hageman factor (factor XII) and producing prekallikrein activator. This activation of Hageman factor has four effects that impact all three of the plasma protein systems:

The activity of plasmin itself is also regulated because it is synthesized as a proenzyme, plasminogen. Plasminogen is converted to plasmin by several factors, including plasminogen activator generated from the kallikrein system, thrombin generated from the clotting system, bacterial factors such as streptokinase produced by hemolytic streptococci, plasminogen activators produced by endothelial cells, and several cellular enzymes released during tissue destruction.

Many enzymes from the plasma regulate the activity of these pathways, such as carboxypeptidase that inactivates the anaphylatoxic activities of C3a and C5a, and kininases that degrade kinins. Many other natural inhibitors are present, including enzymes that degrade histamine (histaminase), activated complement components, kallikrein, and plasmin. Another example of a common regulator is C1-esterase inhibitor (C1-inh).¹⁰ C1-inh inhibits complement activation through reactivity with C1 (classical pathway), MASP-2 (lectin pathway), and C3b (alternative pathway). It is also a major inhibitor of the clotting and kinin pathways (e.g., kallikrein, activated Hageman factor XIIa). A genetic defect in C1-inh (C1-inh deficiency) results in hereditary angioedema, which is a self-limiting edema of cutaneous and mucosal layers resulting from stress, illness, or relative minor or unapparent trauma. The disease is characterized by hyperactivation of all three plasma protein systems, although excessive production of bradykinin appears to be the principal cause of increased vascular permeability.

Cellular Receptors

Cells of both innate and adaptive immunity must recognize and respond to their environment, whether to products of damaged cells or to potential pathogenic microorganisms. Each cell has receptors on the cell surface that specifically bind soluble substances (ligands) produced during tissue damage or infection. The binding of a ligand to its receptor results in activation of intracellular signaling pathways and activation of the cell. As will be discussed in Chapter 8, B and T lymphocytes of the adaptive immune system have evolved surface receptors (i.e., the T-cell receptor, or TCR, and the B-cell receptor, or BCR) that bind a large spectrum of antigens. Cells involved in innate resistance have evolved a different set of receptors that recognize a much more limited array of specific molecules. These are referred to as pattern recognition receptors (PRRs), and they recognize molecular “patterns” on infectious agents or their products (pathogen-associated molecular patterns, or PAMPs), or products of cellular damage (necrosis or apoptosis; damage-associated molecular patterns, or DAMPs). PRRs are generally found on cells at the interface of the host and environment (i.e., skin, respiratory tract, gastrointestinal tract, genitourinary tract), where they monitor for products of cellular damage and potentially infectious microorganisms. Although most PRRs are on the cell surface, some are secreted or intracellular.¹¹ An example of a secreted PRR is mannose-binding lectin of the lectin pathway of complement activation. Cellular PRRs include Toll-like receptors, complement receptors (CRs), scavenger receptors, glucan receptors, and mannose receptors. A group of intracellular protein complexes, called inflammasomes, function as PRRs within cells.¹² Inflammasomes primarily bind cellular stress–related molecules, a type of DAMPs, and control the production of interleukin-1β (IL-1β).¹³

In humans, at least 11 different Toll-like receptors (TLRs) have been described, 10 of which are functional.¹⁴ They are expressed on the surface of many cells that have direct and early contact with potential pathogenic microorganisms. These include mucosal epithelial cells, mast cells, neutrophils, macrophages, dendritic cells, and some subpopulations of lymphocytes. (Dendritic cells are found in the skin, mucosa, and lymphoid tissues, where they have developed from Langerhans cells and function as highly specialized initiators of the adaptive immune response.) TLRs recognize a large variety of PAMPs located on the microorganism’s cell wall or surface (e.g., bacterial lipopolysaccharide [LPS], peptidoglycans, and lipoproteins, yeast zymosan, viral coat proteins), other surface structures (e.g., bacterial flagellin), or microbial nucleic acid (e.g., bacterial DNA, viral double-stranded RNA). Some TLRs recognize host factors that are produced by “stressed” or damaged cells (e.g., breakdown products of extracellular matrix proteins, chromatin). Interactions between PAMPs and TLRs, with the collaboration of other cellular receptors (e.g., CD14), can result in activation of the cell and the release of soluble products (e.g., cytokines) that increase local resistance to the pathogenic microorganism. TLRs are also one of the bridges between innate resistance and the adaptive immune response through the induction of cytokines that increase the response of lymphocytes to foreign antigens on the pathogens. Genetic polymorphisms in TLRs may explain some observed differences among individuals’ resistance and susceptibility to infections. Information on each of the TLRs found in humans is shown in Table 7-2.

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