Bacterial invaders can enter the blood and tissue at any time due to breaches in skin or mucosal surfaces. When this happens, the body needs a way to react quickly and in a relatively nonspecific way to those invaders, regardless of whether it has encountered them before. At the same time, the body must also have a way to distinguish the invading cells from its own cells, and cells that have been infected from those that have not. How would you design a “barcode” for bacteria? Even scientists with advanced degrees might be stumped by that question, but that is precisely what the human body has done using the cells of its immune system. The innate immune system jumps into action when the skin or mucosal barriers are breached, but it does so in a carefully controlled way—which is a good thing, because overreaction by this same defense system can cause considerable damage to the host. Finally, to ensure that all invaders are cleared from the body and that an even faster and more specific response is mounted at the next attempted invasion by that pathogen, the innate immune system prompts and primes the adaptive immune system.

Triggering Innate Immune Defenses

Skin and mucosal surfaces are highly effective in preventing pathogenic bacteria from entering the tissue and blood, but from time to time bacteria succeed in breaching these surfaces. We ended the previous chapter with a brief mention of the skin- and mucosal-associated lymphoid tissue (SALT and MALT, respectively) defense systems located at the interface between the external surface defenses and the underlying tissues and blood. When the skin and epithelial layers are breached, both SALT and MALT have critical functions in stimulating additional defenses, initially triggering the general innate immune system and then priming the more specific adaptive immune system. MALT activation also keeps microbes away from the vulnerable epithelial layer by producing secretory immunoglobulin A (sIgA) that binds to both mucin and the microbe and is then sloughed off and removed from the body. We will return to this aspect of MALT in chapter 4.

The most important function of the innate immune system is to quickly recognize and eliminate pathogens. The innate immune system is comprised of a subset of white blood cells (leukocytes) that include phagocytic cells (phagocytes), granulocytic cells (granulocytes), and natural killer (NK) cells. Because these cells are always present and ready to respond, they are the body’s first responders to pathogen invasion. Together they help clear foreign elements from the body and process them for presentation to the adaptive immune system. The innate immune system becomes activated when it recognizes components of foreign invaders. For bacteria, this recognition is through shared repeated structural components and other molecules specifically found in bacteria. These quasi “bacterial barcodes” are termed pathogen-associated molecular patterns (PAMPs) and play an important role in initiating the innate immune responses by triggering the production and release of cytokines and chemokines from infected cells and from nearby immune cells. Additional proteins and peptides found in the circulatory system, collectively referred to as the complement system, help organize and regulate the activities of the phagocytes, NK cells, and other immune cells. Along with phagocytes and NK cells, the components of the PAMP recognition system and complement system are key players in detecting invaders and triggering the innate and adaptive immune responses (major players are listed in Table 3-1).

Table 3-1. Components of the innate immune system and their activities

Innate Immune Cells That Defend Blood and Tissue

The major cell types comprising the innate immune system can be grouped into three categories based on their primary modes of action: phagocytes, granulocytes, and NK cells. Phagocytes are cells that ingest and kill foreign invaders. Granulocytes (basophils, mast cells, and eosinophils) promote the killing of invading pathogens by releasing proinflammatory cytokines and stimulating immune cells. NK cells are cytotoxic cells that act extracellularly to attack and kill host cells that are infected with bacteria or altered due to interaction with bacteria or their products. The characteristics of different phagocytes, granulocytes, and NK cells, as well as their relationships to each other and to cells of the adaptive immune defense system, are illustrated in Figure 3-1. We will begin our discussion with the phagocytes, which include polymorphonuclear leukocytes (also called PMNs or neutrophils—we will use these two terms interchangeably), monocytes, macrophages, and dendritic cells (DCs).

Figure 3-1. Characteristics and differentiation of the various types of leukocytes of the human body. Leukocytes can be divided into three groups: auxiliary cells (platelets, megakaryocytes, mast cells, and basophils); phagocytes (neutrophils, eosinophils, monocytes, macrophages, and dendritic cells); and two types of lymphocytes: B cells (antibody-producing B cells and plasma cells, memory B cells), and T cells (cytotoxic T cells [CTLs], effector T cells [helper Th1/Th2/Th17 cells, regulatory Treg cells], and memory T cells).

Neutrophils (PMNs)

Neutrophils (PMNs) are the most abundant phagocytic cell in the body. Found circulating in the bloodstream, they are the first phagocytes that arrive at a site of infection, usually within 30 minutes, and immediately engulf (phagocytose) invading microbes and release antibacterial components stored in their granules that destroy the bacteria, including: cationic peptides that poke holes in membranes; proteases, lipases, and hydrolases that degrade bacterial surface proteins, membranes, and toxins, respectively; lysozyme that hydrolyzes the peptidoglycan of bacterial cell walls; myeloperoxidase that generates toxic reactive oxygen species; and lactoferrin that sequesters iron away from the bacteria. Neutrophils secrete signaling molecules: chemokines that attract nearby monocytes and cytokines that stimulate their conversion into macrophages. They can also produce and, when stimulated, rapidly (within minutes) release neutrophil extracellular traps (NETs), networks of extracellular chromatin DNA fibers (chromosomal DNA with DNA-binding histones) that contain the proteases, elastase and cathepsin G, and myeloperoxidase. These NETs serve to trap and kill the bacteria extracellularly and prevent bacterial spread from the site of infection.

Monocytes, Macrophages, and Dendritic Cells (DCs)

Neutrophils, dendritic cells (DCs), and monocytes are derived from the same myeloid progenitor cells. In response to chemokines (chemical attractants) and other signals, monocytes and DCs leave the bloodstream and enter the site of infection. There, cytokines (stimulatory signaling molecules) released by damaged or infected cells trigger the monocytes to convert into macrophages, that are then activated by neutrophils at the site (see Table 3-2 and later in this chapter).

Table 3-2. Selected immune cytokines/chemokines and their activities

Macrophages and DCs are included here as members of the innate immune system, but they will appear again in chapter 4 as important members of the adaptive immune system. This is due to the fact that, upon first exposure to a particular pathogen, macrophages and DCs act to ingest and kill bacteria in a nonspecific way through the innate immune response, while also presenting pieces of the pathogens (antigens) to cells of the adaptive immune system. Upon subsequent exposure to a pathogen, monocytes and macrophages can also phagocytose invading bacteria that are coated with antibodies generated through the adaptive immune response. Macrophages engulf and digest cellular debris, microbes, foreign particles, infected or diseased host cells, and any other cell that does not possess the types of recognition surface molecules that indicate it is a healthy host cell.

Monocytes circulating in the blood have a half-life of about one day, but once they migrate from the circulating blood into tissues, they differentiate into different types of macrophages that become residents at the tissue sites, where they can survive for several months or years. Some examples of resident macrophages are the Kupffer cells of the liver, alveolar macrophages of the lung, and spleen macrophages. Macrophages can either stimulate the immune system and cause inflammation, or dampen the immune system and decrease inflammation through the release of specific signaling molecules (cytokines), depending on whether they are M1 macrophages or M2 macrophages, respectively. M1 macrophages metabolize the amino acid arginine into a reactive nitrogen species (nitric oxide) and stimulate inflammation, while M2 macrophages metabolize arginine into ornithine for cycling through the urea cycle to dampen the inflammatory response. M2 macrophages also release anti-inflammatory cytokines that promote repair of damaged tissue.

Dendritic cells are closely related to macrophages. Their name derives from the fact that they are covered with spiny projections that resemble the dendrites of neurons. They are instrumental in initiating and stimulating the second responder portion of the immune response, called adaptive immunity. DCs are found in tissues that are in contact with the external environment or the blood and, like macrophages, the resident forms of DCs found in the dermis or in various tissue sites develop from immature monocytes circulating in the blood. Dendritic cells, such as the specialized Langerhans cells of skin and mucosa, become activated when they recognize bacterial PAMPs and migrate through the lymphatic system to lymph nodes, where they mature and present antigens to, and stimulate the cells of, the adaptive defense system: T helper cells (Th cells), cytotoxic T cells (also called cytotoxic T lymphocytes or CTLs), and B cells. Chapter 4 will provide a more detailed description of how DCs activate T cells and B cells.

Granulocytes: Basophils, Mast Cells, and Eosinophils

Basophils, mast cells, and eosinophils are immune cells that participate in the defense against infections, but they do not act as phagocytic cells. These granulocytes contain granules loaded with histamine, a vasodilator that promotes blood flow in tissues, and heparin, an anticoagulant that prevents blood from clotting too quickly. These cells are similar in appearance and function but differ in that basophils circulate in the blood, while mast cells congregate around blood vessels in connective tissue at mucosal sites. If any foreign material is detected, these cells release the contents of the granules, causing vasodilation. This helps the neutrophils and monocytes, which are normally circulating in blood, to leave the bloodstream and move to the site of infection. Eosinophils act similarly, but, unlike basophils and mast cells, they are primarily responsible for targeting multicellular parasites and viral invaders rather than bacteria.

Transmigration—How Do Phagocytes Know When and Where to Go?

Resident macrophages and DCs tend to localize in specific areas of the body. By contrast, neutrophils (PMNs) and monocytes, which are produced in the bone marrow, migrate constantly through the bloodstream. Neutrophils are the most abundant, but also the shortest-lived of the phagocytic cells; monocytes are longer-lived, but not as numerous. All of these phagocytic cell types, as well as NK cells, are capable of significantly damaging host tissues. It is not easy to kill bacteria, and considerable firepower (in the form of toxic substances released from granules or lysosomes) has to be brought to bear to destroy them. When this firepower is released by the PMNs, macrophages, and NK cells into the surrounding tissue, as is inevitable once the battle with invading bacteria is mounted, human cells also become vulnerable targets. The body protects itself from the potentially toxic action of these host defense cells by keeping them in the bloodstream in a quiescent, immature state unless danger is sensed.

How do PMNs know when to leave the blood vessels and where to go? Only when infection triggers signaling pathways that alert these cells to prepare for battle do the PMNs, monocytes, and NK cells pass through the blood vessel walls and enter tissues through a process called transmigration (also referred to as diapedesis or extravasation) (Figure 3-2). Two groups of proinflammatory proteins alert PMNs to leave the bloodstream, and these proteins then guide the PMNs to their destination. One of these groups is the complement system, whose component C3a and C5a proteins are activated by contact with invading bacteria or by interaction with antibodies bound to foreign material. Once a PMN has moved out of the blood vessel, it follows a gradient of these complement components to the site where the bacteria have invaded (chemotaxis).

Figure 3-2. Roles of various cytokines and chemokines in directing the exit of neutrophils (PMNs) from the bloodstream at infection sites. Initially, new proteins are expressed on the surfaces of PMNs and endothelial cells, permitting a loose reversible binding. This gives PMNs a rolling motility as they flow through the blood vessel. Other cytokines cause changes in the cell surfaces resulting in tighter binding. The PMNs stop moving, flatten against the vessel wall, and force themselves across the endothelial wall. PMNs then move chemotactically along a C5a gradient. PAF, platelet-activating factor; PECAM, platelet-endothelial cell adhesion molecule.

The cytokines and chemokines are the second group of proteins (Table 3-2). Cytokines and chemokines are produced by a number of human cell types, including monocytes, macrophages, PMNs, DCs, endothelial cells, fibroblasts, and cells of the adaptive immune response (T cells and B cells). The cells of the adaptive immune system and their activities will be described in the next chapter. Complement components and cytokines/chemokines work together to orchestrate the immune response to an infection by directing the phagocytes and cytotoxic cells to the infected area and activating them through a process that is described in greater detail later in this chapter. Chemokines, like certain complement components, guide phagocytic cells to the site where an infection is occurring. Chemokines may also act as antimicrobial peptides that kill bacteria near the site of infection. Cytokines mediate the inflammatory response (redness, swelling, pain, fever) to invading microbes and other types of tissue damage.

When bacteria enter tissue, cytokines also stimulate neutrophils to become more actively phagocytic as they leave the bloodstream and migrate to an infected area but, unlike monocytes, PMNs do not develop into a different type of cell. During an infection, the transmigration process is accelerated. To compensate for this loss of PMNs from the bloodstream, the release of PMNs from bone marrow into the bloodstream is markedly increased. So, although passage of these phagocytic cells from the bloodstream into tissue is increased, the net effect is a higher concentration of innate defense cells in the blood. A high level of PMNs in blood is a useful diagnostic indicator of infection. During an infection, PMNs are so rapidly produced in bone marrow and dumped into the bloodstream that the immature forms of PMNs (called “bands” because their nuclei look like bands) are seen in the blood. The presence of bands is a clear diagnostic sign of acute infection.

Natural Killer (NK) Cells

The role of NK cells is to complement the activities of PMNs by killing human cells that are altered due to infection. Once thought to be involved primarily in controlling viral infections, NK cells are now known to be important in controlling infections by bacteria that invade and live within human cells in an attempt to evade the immune system. Unlike PMNs and other phagocytic cells, NK cells do not ingest their target, yet their mode of killing resembles that of phagocytes in many respects. Like phagocytic cells, NK cells are produced in bone marrow and circulate in the bloodstream. Also, they store their toxic substances in granules. Binding to an infected human target cell stimulates release of these granules. Instead of ingesting a bacterium or infected cell, the NK cells bombard infected cells with the toxic contents of their granules.

The granule proteins of NK cells are not the same as those of the lysosomal granules of macrophages and PMNs, but they have some similar functions. In chapter 4, we will see that these granules are very similar to those produced by cytotoxic T cells (CTLs). NK cell granules contain a protein called perforin that inserts into the membrane of an infected host cell and causes channels to form. These channels allow other granule proteins, a set of proteases called granzymes, to enter the target host cell. NK cells also secrete α-defensins that kill bacterial cells by disrupting their membranes. One effect of this assault is to force the infected host cell to initiate apoptosis (programmed cell death), a process by which the infected cell kills itself. Killing the infected host cell through apoptosis is quite different than killing through cell lysis, which can result in the release of intracellular microbes. Instead, the apoptotic process is highly regulated and controlled, producing smaller parts of the cell (called apoptotic bodies) that bleb off and are engulfed by nearby phagocytic cells before the contents can be released into the extracellular medium.

How do NK cells recognize and kill infected cells? As with many responses in the immune system, multiple signals are sensed to direct NK cells to kill infected cells, while sparing normal cells. NK cells express two types of surface receptors, activating receptors and inhibitory receptors, and they use an opposing-signals mechanism to identify infected cells (Figure 3-3). When NK cells encounter normal or infected cells, activating ligands on the surface of the target cell stimulate activating receptors on the surface of the NK cell. If left unchecked, this response will lead to activation of the NK cell, release of cytokines (mainly interferon gamma [IFN-γ]), and release of contents (granzymes and perforin) from granules, thereby killing the encountered cell. However, for uninfected host cells the activation of NK cells is counterbalanced by the presence of inhibitory receptors.

Figure 3-3. Schematic diagram indicating the complementary activities of natural killer (NK) cells and cytotoxic T cells (CTLs) in killing infected host cells. Host cells that are not infected with intracellular bacteria display self-antigens through MHC I on their surfaces. However, once they become infected, their surface molecules are altered. NK cells have inhibitory receptors on their surfaces that, in the presence of MHC I bound with self-antigen, block the activation of NK cells and prevent killing of the target host cell. CTLs (described in detail in chapter 4) recognize infected cells through those complexes and subsequently kill the infected cells through release of granule contents (perforin and granzymes) that lead to induction of apoptosis. Since intracellular pathogens can cause infected cells to express far fewer MHC I molecules on their surfaces than normal cells, NK cells complement this CTL process by recognizing and killing cells that do not have MHC I complexes on their surfaces. In these cases, the inhibitory receptors do not bind MHC I and do not block the activation of the NK cells, that then proceed to release their granules and induce apoptosis in the infected host cell.

All normal, healthy (uninfected) cells express class I major histocompatibility complex (MHC I) proteins on their surfaces, which will be discussed in more detail in the next chapter on the adaptive immune response. NK cells can distinguish between uninfected and infected cells based on the presence or absence of MHC I, respectively. The complex of MHC I bound with a self antigen (small peptides that are derived from the host cell and not from a foreign invading pathogen) is recognized by an MHC I-specific inhibitory receptor on the NK cell surface, which then sends a signal inside the NK cell that halts activation of the cytotoxic response (release of granules) stimulated by the activating ligand-receptor interactions.

By contrast, infected cells often express much less MHC I on the surface than normal cells do, and those MHC I molecules that are on the surface are more likely to have foreign antigens (rather than self antigens) bound. Complexes of MHC I bound to foreign antigens are recognized by CTLs of the adaptive immune system (see chapter 4). The absence of self antigen-bound MHC I on the surface means that the NK cell activation response is not blocked by its inhibitory receptors, and this allows the NK cell to proceed with its attack on the infected cell.

The Lymphatic System

Lymph, the fluid that moves through the lymphatic system, is also monitored and protected by phagocytes. Lymphatic vessels are tubes comprised of overlapping endothelial cells. These tubes are organized into a network similar in complexity to the circulatory system that carries blood to all parts of the body (Figure 3-4). The role of the lymphatic system is to prevent excess buildup of fluid in tissues and to recycle blood proteins. Under healthy conditions, blood fluids and proteins leak from blood vessels in the capillary beds to feed cells, donate oxygen, and remove carbon dioxide. These fluids also provide protective blood proteins such as complement components (see below), cytokines, and chemokines. Blood vessels, however, do not readily reabsorb the fluids that leak from them. Rather, these fluids are taken care of by the lymphatic vessels. Lymphatic endothelial cells are tethered to the muscle bed, so when the level of fluids in an area becomes elevated and pressure causes muscle cells to separate, the overlapping endothelial cells of the lymphatic vessels are pulled apart. This opening allows fluid from the surrounding area to enter the lymphatic vessels, where it is then returned to central holding areas such as the thoracic duct and eventually returned to the bloodstream.

Figure 3-4. The lymphatic system.

The inflammatory response to bacterial infection creates a buildup of fluid in tissue, leading to opening of the lymphatic vessels. We experience the accumulation of blood fluids as swelling and tightness that appears around infected wounds or sites of infection. In these situations, bacteria can enter the lymphatic vessels. In order to prevent these bacteria from spreading throughout the body, the lymph must first be cleansed of these bacteria before it reenters the bloodstream. This task is accomplished by lymph nodes located at strategic points along the lymphatic vessels.

Lymph nodes are major sites distributed throughout the body that are linked to lymphatic vessels and contain macrophages, DCs, T cells, and B cells. Analogous to how the liver and kidneys filter toxic substances from circulation, the lymph nodes function to filter foreign entities, such as pathogens. The spleen and tonsils are other large lymphoid tissues that function similarly to lymph nodes but filter blood instead of lymph. In chapter 4, we will also learn that macrophages and DCs in the lymph nodes act as antigen-presenting cells and stimulatory cells that potentiate the adaptive defense response against bacteria. Thus, the lymph nodes not only serve to sterilize lymph but also act as sites where the adaptive defense system is alerted.

Bacteria that enter a lymph node are usually killed by the macrophages and DCs, but there are bacteria that are able to evade this fate. Indeed, some of the most dangerous pathogens are those that can survive and multiply in the lymph nodes. An example of such a pathogen is Yersinia pestis, the cause of bubonic plague (also known as the Black Death). Y. pestis growing in lymph nodes creates an inflammatory response so intense that it causes the lymph nodes to become grossly distended (swollen), producing the so-called buboes that give bubonic plague its name. A less pronounced, but still detectable, enlargement or swelling of lymph nodes (lymphadenopathy) likewise occurs during many types of bacterial infections and serves as a diagnostic sign of infection.

How Phagocytes Recognize and Respond to Bacteria

For many years, the interaction between PMNs and invading bacteria remained mysterious. With the discovery of PAMPs and their host cell receptors, this interaction has been a focus of intense research in recent years. Much of this research has focused on components of Gram-negative bacteria, such as the cell surface component lipopolysaccharide (LPS) (see Box 1-4) and flagellin, a protein involved in bacterial cell motility. Gram-positive bacteria also trigger the same type of PMN activation through lipoteichoic acid (LTA), the anionic polymer found in the cell walls of these bacteria (see Box 1-4). Peptidoglycan (PG) fragments from both Gram-positive and Gram-negative bacteria, as well as certain types of DNA (e.g., CpG-rich DNA) and RNA, can also act as triggers.

PAMPs were discovered in an unusual way. Scientists studying the fruit fly, Drosophila melanogaster, identified a transmembrane receptor protein called Toll that was required for resistance to fungal infections. Oddly enough, only one of the Toll receptors in Drosophila is linked to a defense against infection; the others are involved in various aspects of fly development. Shortly thereafter the mammalian equivalent of Toll was discovered—hence the name “Toll-like receptor (TLR),”—and shown to be important for immunity. To date, thirteen mammalian TLR paralogs (genes found in different species that are traceable to a shared ancestry) have been described and have been shown to modulate cellular responses to pathogens. Most mammals have ten to thirteen different TLRs (humans have ten functional TLRs).

Although there are many different members of the TLR family, one common set of them is evolutionarily conserved in a wide range of organisms. This common set of diverse TLRs may reflect the fact that although all Gram-negative and Gram-positive bacteria have surface “signatures” in common, there are also numerous differences. The different TLRs in various combinations (through homo- and heterodimerization) and coupling with different downstream adaptor molecule combinations provide the flexibility to respond to differences between invading bacteria without responding specifically to each particular bacterium, as the antibody-based adaptive immune system does. For example, TLR4 is a receptor for LPS from most Gram-negative bacteria, but sometimes a complex of TLR1/TLR2 or TLR2/TLR6 is the receptor for certain pathogens that have modified LPS structures or surface lipoproteins. TLR9 responds to DNA containing stretches of unmethylated CpG sequences, which is not usually found in mammalian DNA. Double-stranded RNA activates TLR3 and TLR8, while single-stranded RNA activates TLR7, and bacterial flagellin activates TLR5. The ligand for TLR10 is not yet known. Mice have a TLR11 that recognizes parasites and uropathogenic Escherichia coli, but in humans the gene encoding TLR11 has multiple stop codons that prevent expression of a functional protein.

Figure 3-5 summarizes the different TLRs in humans and mice and where they are localized in the cell, as well as the signaling pathways that they activate. TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 are exposed on the surface of the phagocytic cell, whereas TLR3, TLR7, TLR8, and TLR9 reside within endosomes. Each TLR contains a leucine-rich repeat (LRR) domain that is extracellular for the surface TLRs or faces into the lumen of the vesicle for the endosomal TLRs. The LRR domain binds the PAMP ligand(s) and induces dimerization. The intracellular domain of each TLR has a conserved region of amino acid homology with interleukin-1 receptors (IL-1Rs), referred to as the Toll/IL-1R (TIR) domain. The TIR domain conveys the signal detected by the LRR domain binding to a PAMP ligand to the interior of the phagocytic cell through a signal transduction cascade mediated through adaptor proteins and kinases, including the myeloid differentiation primary response gene 88 protein (MyD88), IL-1R-associated kinases (IRAKs), transforming growth factor β-activated kinase 1 (TAK1), TAK1-binding protein 1 (TAB1), TAB2, tumor necrosis factor receptor-associated factor 6 (TRAF6), and others. In all cases, activation of these signaling pathways leads to expression of inflammatory genes and induction of phagocytic functions, such as maturation and secretion of cytokines and stimulation of the oxidative burst that kills opsonized bacteria.

Figure 3-5. The Toll-like receptor (TLR) family and their signaling pathways in humans and mice. TLRs mediate the activation of the innate immune system through recognition of pathogen-associated molecular patterns (PAMPs). TLR1, TLR2, and TLR6 form heterodimers on the plasma membrane that bind to microbial membrane components. TLR4 forms homodimers that bind with MD2, CD14, and LPS bound LBP on the plasma membrane (see Figure 3-6). TLR5 forms homodimers on the plasma membrane that bind bacterial flagellin. TLR3, TLR7, TLR8, and TLR9 form homodimers inside endocytic vesicles that recognize various forms of nucleic acids. The ligands for TLR10, TRL12, and TLR13 are unknown. Humans have a mutated TLR11 gene that does not express protein, and do not have genes for TLR12 or TLR13. In mice TLR11 is functional and recognizes parasites and uropathogenic bacteria. TLRs activate downstream signaling pathways that lead to expression of proinflammatory cytokines through various adaptor proteins (MyD88, TIRAP, TRAM, TRIF, IRAK, TRAF6, TRAF3, and RIP-1) that interact with their intracellular TIR domains, which have homology with the receptors for IL-1 and IL-18.

Figure 3-6. Cellular recognition of LPS through Toll-like receptor signaling.

The process by which bacterial components (PAMPs) trigger cytokine release through TLR signaling is best understood in the case of LPS (see Box 1-4). LPS that is released from outer membranes due to bacterial lysis binds to LPS-binding protein (LBP), a blood protein produced by the liver, and is delivered to CD14, a protein receptor on the surface of macrophages and other cytokine-producing cells (Figure 3-6). LPS is then transferred to the transmembrane signaling receptor TLR4 and its accessory protein MD2. This TLR4/LBP/CD14/MD2 complex triggers a cellular signal transduction pathway that leads to activation of the transcription factor NF-κB, which then moves to the nucleus and induces inflammatory cytokine gene expression.

The host cell also has a PAMP detection system for intracellular pathogens, called NOD-like receptors (NLRs). Humans have twenty-two different NLR proteins that sense a variety of PAMPs, including cell wall components of intracellular bacteria, toxins, and host-derived ligands (such as damaged membrane components). NLRs consist of three domains: a variable N-terminal domain with different sensor-like functions (depending on the type of NLR); a central nucleotide-binding domain that mediates ATP-dependent auto-oligomerization (called a NACHT domain); and a C-terminal LRR domain that functions analogously to that of TLRs to detect bacterial-derived PAMPs inside host cells (peptidoglycan components in the case of NOD1 and NOD2, flagellin in the case of NAIP and NLRC4, and anthrax toxin in the case of NLRP1). Activation of most NLRs leads to NLR oligomerization and formation of the inflammasome, a complex of multiple signaling (NLR, caspase-1) and adaptor (ASC) proteins that mediate oligomerization and self-cleavage of pro-caspase-1, a zymogen (a proenzyme that is inactive until a proteolytic cleavage event converts it into its active form) (Figure 3-7).

Figure 3-7. NOD-like receptor (NLR) sensing of intracellular pathogens. PAMP activation of TLRs and binding of the proinflammatory cytokines (IL-1β and IL-18) to their cognate receptors stimulate NF-κB signaling that leads to expression of pro-IL-1β and pro-IL-18. Infection with intracellular pathogens induces assembly of the NLRs, adaptor protein (ASC), and caspase-1 into inflammasome complexes, which lead to caspase-1 activation and subsequent cleavage and secretion of IL-1β and IL-18, as well as release of alarmin (HMGB1) and induction of pyroptosis. Unlike other NLRs, NOD1 binds the bacterial peptidoglycan derived D-Glu-meso-diaminopimelic acid (DAP), while NOD2 binds muramyl dipeptide (MDP) to form a nodosome complex, comprised of the ligand-bound NOD and a serine/threonine kinase adaptor protein, Rip2 (also called RIPK2), which in turn activates NF-κB. Adapted from Lamkanfi M, Dixit VM. 2011. J Immunol 187:597–602, with permission.

Caspase-1 signaling pathways lead to a form of programmed cell death, called pyroptosis, where cells produce cytokines, swell, and burst. Caspase-1 proteolytic activity leads to maturation of proinflammatory cytokines, especially interleukin-1β (IL-1β). Production of these cytokines leads to inflammatory responses in macrophages, such as Th1 responses and release of IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8), and activation of NK cells.

The best-described members of the NLR family are the NOD1 and NOD2 proteins, which have an N-terminal caspase activation and recruitment domain (CARD). Human NOD1 detects a part of the peptidoglycan derived from degradation of the Gram-negative cell wall, whereas NOD2 detects a degradation product derived from Gram-positive peptidoglycan. While NOD1 is expressed by many cell types, NOD2 is expressed primarily in macrophages, DCs, Paneth cells, keratinocytes, and mucosal epithelial cells. Interestingly, NOD1 and NOD2 are unlike other NRLs in that they do not appear to form inflammasomes that directly activate caspase-1, but rather they form their own signaling complex, called a nodosome. The nodosome is directed to the site of bacterial entry into the cell, where it also interacts with the receptor-interacting serine/threonine-protein kinase 2 (Rip2/RIPK2) through the CARD domains. This is followed by recruitment of a number of other signaling molecules and potent activation of the transcription factor NF-κB, which is transported into the nucleus, where it induces expression of IL-1β, IL-18, and other proinflammatory genes.

In addition to sending out cytokine signals to alert the immune system of infection by pathogens, host cells that are severely damaged as a result of pathogen invasion also produce and release other endogenous host cell molecules, called alarmins. Alarmins are released into the extracellular medium by infected cells that are lysed or die by non-apoptotic mechanisms. One such alarmin is the high mobility group box 1 (HMGB1) protein, a nuclear protein that binds to nucleosomes and promotes DNA bending. Cells that are undergoing apoptosis modify their chromatin such that HMGB1 remains tightly bound to the DNA and not released, whereas cells that are dying by necrosis or lysis release HMGB1 into the extracellular medium. In response to PAMP detection and inflammatory stimulation, cells can also actively secrete HMGB1, which then serves as a chemokine to recruit phagocytes to the site of infection.

How Phagocytes Kill Bacteria

Oxidative Burst in Phagolysosomes

The steps involved in the killing of a bacterium by a phagocyte are shown in Figure 3-8. The phagocyte first forms pseudopods that engulf the bacterium. Phagocytosis (engulfment) requires dynamic rearrangements of actin, a major component of the eukaryotic cytoskeleton. After engulfment, the bacterium is encased in an endocytic vesicle called a phagosome. The phagosomal membrane contains ATPases that pump protons into the phagosome interior, reducing the internal acidity to as low as pH 4.5. Phagocytes also carry antibacterial proteins that are as toxic to the phagocytes and surrounding tissue cells as they are to their bacterial targets. Accordingly, these toxic proteins are stored in an inactive form in lysosomes. Fusion of a lysosome with a phagosome to form a phagolysosome releases the lysosomal proteins into the phagolysosome interior, where they are activated by the low pH of the phagolysosome interior.

Figure 3-8. Steps in ingestion and killing of bacteria by phagocytes. Bacteria are first engulfed by endocytosis into a phagosome. Fusion of phagosomes and lysosomes releases toxic enzymes and proteins that kill most bacteria. Debris from dead bacteria is then released by exocytosis.

Phagocytes possess two general types of bacterial killing mechanisms mediated by the fusion of phagosomes and lysosomes: non-oxidative and oxidative. Lysosomal vesicles contain various macromolecule-degrading enzymes (proteases, lipases, glycosidases, hydrolases, nucleases, and lysozyme) that destroy bacterial components and mediate nonoxidative killing. Degrading enzymes, such as proteases, phospholipases, glycosidases, hydrolases, and lysozyme, destroy surface and membrane components of the bacteria. Nucleases hydrolyze bacterial DNA and RNA that are released during lysis. Other lysosomal proteins, such as defensins, antimicrobial peptides, and other membrane permeabilizers, insert into bacterial membranes and create pores that permeabilize the membrane, kill the bacteria, and allow the bacterial cytoplasmic components to leak into the surrounding environment.

Oxidative killing occurs through formation of toxic reactive oxygen and reactive nitrogen species. Lysosomes have another type of lysosomal protein, myeloperoxidase, that produces reactive forms of oxygen that are toxic to many bacteria (similar to lactoperoxidase). The generation of toxic forms of oxygen by phagocytes is called the oxidative (or respiratory) burst. Myeloperoxidase is only activated when it is brought into contact with an NADPH oxidase, which is located in the phagosomal membrane, and when the resulting complex is then exposed to the low pH of the phagolysosome interior. During an infection, cytokines stimulate increased production of these lysosomal enzymes, thus increasing the killing potential of the oxidative burst.

The reaction catalyzed by the myeloperoxidase complex has three steps (Figure 3-9). First, NADPH oxidase generates a superoxide radical: NADPH + 2O2—> 2O2− (superoxide radical) + NADP+ + H+. The superoxide radical is extremely reactive and is readily converted into hydrogen peroxide (H2O2) by superoxide dismutase. Myeloperoxidase catalyzes the reaction of hydrogen peroxide with chloride (Cl−) or thiocyanate (SCN−) ions (as well as bromide or iodide ions) to form hypochlorite (−OCl, the active ingredient in bleach) or hypothiocyanite (−OSCN), respectively. These reactive oxygen molecules are toxic to bacteria because they inactivate essential bacterial surface proteins through oxidization of sulfur-containing amino acids and molecules, such as cysteine, methionine, and glutathione. For example, oxidation of the thiol groups (-SH) of the amino acid side chain of cysteine in proteins results in formation of sulfenic acid, sulfonic acid, or disulfide linkages.

Figure 3-9. Oxidative burst in the phagolysosome. The phagosome contains two unique enzymes: lysosomal myeloperoxidase and phagosomal membrane-bound NADPH oxidase. Superoxide dismutase converts the superoxide radical (O2−) generated by NADPH oxidase into hydrogen peroxide (H2O2). Myeloperoxidase then catalyzes the reaction of hydrogen peroxide with chloride (Cl−), thiocyanate (SCN−), or other halide ions to form hypochlorite (−OCl), the active ingredient in bleach, or hypothiocyanite (OSCN−), both of which are extremely toxic to bacteria. Under these oxidative conditions iron ions exist predominantly in the oxidized ferric (Fe3+) state, and this free Fe3+ plus hydrogen peroxide forms hydroperoxyl radical (HOO•) via a nonenzymatic Fenton reaction, that can also damage macromolecules such as proteins and DNA.

Under these oxidative conditions iron ions exist predominantly in the oxidized ferric (Fe3+) state. Inside the body, Fe3+ ions are bound to iron-binding proteins, such as transferrin or lactoferrin, but in phagolysosomes the low pH environment causes the release of Fe3+ from the proteins. The freed Fe3+ ion plus hydrogen peroxide (H2O2) forms hydroperoxyl radical (HOO•) via the nonenzymatic Fenton reaction, which can also damage DNA.

Human monocytes and macrophages also produce another very simple but powerful antimicrobial compound, nitric oxide (NO). NO is toxic in its own right, attacking bacterial metalloenzymes, proteins, and DNA. Additionally, it can combine with superoxide to form peroxynitrite (OONO−), a very reactive molecule that oxidizes amino acids and is toxic to both bacteria and human cells. Synergistic reactions between NO and superoxide during the oxidative burst may help make the burst more toxic for bacteria. NO may also serve as a signaling molecule to regulate the functions of phagocytic cells, as well as other adaptive immune cells. NO has been implicated in so many areas of human and animal physiology that the journal Science chose it as the molecule of the year in 1992.

During an infection, cytokines induce NO synthesis by many human cell types. NO production is indicated by the presence of the stable end products of its oxidation, nitrite and nitrate, in the blood. NO may also contribute to some of the symptoms of disease, including vascular collapse and tissue injury, as mice lacking the inducible NO synthetase responsible for generating NO tolerate bacterial endotoxin with fewer toxic side effects than normal mice. On the other hand, these mice are highly susceptible to infections by intracellular bacterial and protozoan pathogens, such as Mycobacterium tuberculosis or Leishmania major, which cause tuberculosis or leishmaniasis, respectively. The human pathogen Neisseria meningitidis, which is responsible for meningococcal disease, has at least two NO detoxification enzymes that enhance survival during nasopharyngeal colonization and during phagocytosis by human macrophages.

Autophagy—Another Pathway for the Killing of Intracellular Pathogens

Some intracellular pathogens, such as Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium, are able to escape destruction through the phagolysosomal pathway by modifying the phagosome into a specialized vacuole that does not fuse with lysosomes. Others, such as Rickettsia conorii, escape the phagosome and reside within the host cytosol. In such cases of intracellular pathogenesis, another cellular pathway, called autophagy, is employed. Autophagy normally enables the breakdown and recycling of dysfunctional cellular components, such as mitochondria, by sequestering them from the rest of the cytosolic components into a double-membrane organelle called an autophagosome, which then fuses with a lysosome to destroy the contents of the resulting autophagolysosome.

Autophagosome formation is initiated by a precursor isolation membrane (phagophore), to which two ubiquitin-like conjugation complexes are recruited. One of these involves the ubiquitin-like autophagy-related protein Atg8 (also called LC3) that is conjugated with a membrane phospholipid, phosphatidylethanolamine (PE). LC3-PE mediates membrane tethering and expansion of the phagophore that associates with and encircles the intracellular pathogen or the pathogen-containing vesicle. The second ubiquitin-like complex is comprised of ubiquitin-like Atg12 conjugated with the adaptor protein Atg5 and noncovalently associated with the adaptor protein Atg16. The Atg12-Atg5-Atg16 complex oligomerizes and is recruited to the newly formed isolation membrane and controls elongation and eventual closure of the phagophore.

Intracellular pathogens can be targeted for degradation through autophagy (Figure 3-10). Bacteria that escape into the cytosol of the cell become ubiquitinated on the surface, which recruits adaptor proteins, such as NDP52 or p62, which can bind both ubiquitinated proteins on the bacterial surface and LC3-PE attached to phagophores. Another adaptor protein is optineurin (OPTN), which binds LC3 and ubiquitinated bacteria when it becomes phosphorylated by the kinase TBK1, a regulator of NF-κB signaling and interferon production.

Figure 3-10. Eliminating intracellular pathogens through autophagy. Intracellular pathogens that escape into the cytosol or invading pathogens that enter via phagosomes or reside in pathogen-specific vacuoles are targeted for lysosomal degradation by autophagy. The target cytosolic pathogen or pathogen-containing vesicle is engulfed into a specialized double-membrane organelle called an autophagosome, which then fuses with a lysosome to become an autolysosome.

In the case of phagosomes containing invasive bacteria, such as M. tuberculosis or S. Typhimurium, autophagy is triggered by the damage to the phagosomal membrane caused by the bacterium. Recently, galectin-8 (a β-galactoside-binding lectin) has been identified as a cytosolic detector protein that can initiate the autophagy response for invading pathogens. Galectin-8 recognizes galactoside residues in exposed, damaged membrane and recruits autophagy adaptor proteins, such as NDP52, which binds to the autophagy proteins LC3 and Atg12 and brings the phagophore to the altered bacterium-containing phagosome. NOD2 protein can also initiate autophagy through recruitment and activation of the autophagy protein Atg16, which controls elongation of new autophagosomal membrane, at the site of bacterial entry into the cell.

The Complement Cascade

Complement Proteins

The skin and mucosa barriers, along with the phagocytic cells, are powerful defenses against bacterial invaders. There is, however, another highly complex and equally important arm of the innate defenses—the blood proteins that mark targets for phagocytic destruction and direct the inflammatory response. These complement system proteins are produced by the liver, circulate in the blood, and enter tissues throughout the body. Their primary purpose is to help phagocytic cells and antibodies rapidly clear pathogens from the body. Along with chemokines and cytokines, complement proteins also form an important link between the innate phagocytic defense system and the adaptive immune defenses, which will be discussed further in chapter 4.

Many of the complement proteins are zymogens, which reside throughout the body as inactive precursors. Complement activation occurs during infection as the complement proteins undergo a cascade of activating proteolytic cleavages, where each activated complement zymogen cleaves and activates the next complement zymogen. In terms of nomenclature, a letter “C” is used to designate the core complement components, some of which are actually multi-protein complexes. There are nine of these core components, C1 to C9. Activated proteolytic cleavage products are indicated by an “a” or “b.” In most cases, as, for example, C3, C4, and C5, “a” designates the smaller and “b” the larger of the two proteolytic products (e.g., C3a and C3b, respectively), but this rule is not followed uniformly, notably with regard to the complement component C2. Unfortunately for us, most immunologists prefer to retain the old nomenclature for C2, where the small C2 cleavage product is designated C2b and the large one C2a. We will use this designation.

Overview of Complement Pathways and Their Function

When activated through proteolytic cleavage and complex formation, the complement system performs five important interrelated functions: (1) promotion of opsonization (engulfment) of invading bacteria by coating them with complement components (e.g., C3b); (2) enhancement of chemotaxis of phagocytes (PMNs, monocytes, macrophages, and DCs) to attract them to the site of infection by releasing chemokines (e.g., C5a); (3) enhancement of vascular permeability to promote exudation (transmigration) of phagocytes from blood vessels into tissues at the site of infection by releasing vasodilators (e.g., C3a, C4a, and C5a) that cause degranulation (release of histamine and heparin) of basophils and mast cells; (4) promotion of agglutination (clustering and clumping) of invading bacteria by binding them together and increasing the efficiency of phagocytosis and clearance from the system; and (5) direct killing of many Gram-negative bacteria by poking holes in their membranes via binding of C5b to LPS and formation of the membrane attack complex (MAC) that consists of components C5b through C9.

Complement activation can be initiated through three pathways, as shown in Figure 3-11. The first two pathways are antibody-independent and form part of the innate response to initial infection, while the third pathway is antibody-dependent and comes into play after adaptive immunity has been launched. All three pathways lead to activation of the pivotal complement component, the protease C3-convertase, that cleaves and activates C3 into C3a and C3b. C3b covalently binds to the surface of pathogens, coating them and enhancing agglutination and opsonization of the pathogen by phagocytes.

Figure 3-11. Main steps in activation of complement by the mannose-binding lectin, classical, and alternative pathways. These pathways differ only in the steps that initiate formation of C3 convertase. Important activated products are C3b (which opsonizes bacteria), C3a (which acts as a vasodilator), C5a (which acts as a vasodilator and a chemokine that attracts phagocytes to the area), and C5b-C9 (MAC [membrane attack complex], which inactivates enveloped viruses and kills Gram-negative bacteria). LPS, lipopolysaccharide.

Certain surface components of bacteria can trigger the complement cascade through the alternative pathway without the need for adaptive responses. The best-characterized complement-triggering bacterial surface molecules are LPS and LTA found in the outer and inner cell membranes of Gram-negative and Gram-positive bacteria, respectively. Complement-activating molecules of fungi, protozoa, and metazoa are not as well characterized, but they also appear to be lipid-carbohydrate complexes on the microbial cell surface. In the alternative pathway, the free hydroxyl (−OH) groups or amino (−NH2) groups of bacterial surface components react with an activated thioester group that is present in the C3b protein to form a covalent linkage that triggers the alternative pathway by recruiting factor B to the bacterial surface and initiating formation of the C3-convertase.

A more recently discovered initiation pathway that also responds to initial infection, called the lectin pathway, involves multimeric proteins called mannose-binding lectins (MBL), which are members of a family of PAMP-recognizing proteins called collectins. Collectins are calcium-dependent lectins (i.e., proteins that bind specifically to certain sugar residues in the presence of calcium). The MBLs bind mannose groups that are commonly found on the surfaces of bacteria but not on human cells. Recently, another group of PAMP-binding lectins, called ficolins (FCNs), which bind specifically to N-acetylglucosamine (GlcNAc) groups, have been shown to activate the lectin pathway in a similar manner. Collectins are produced by the liver and are part of what is called the acute phase response (or acute inflammatory response) to an infection—the initial onslaught by a variety of proteins, including cytokines and iron-binding proteins, that make it difficult for bacteria to multiply. Collectins that are bound to the surface of a bacterium not only sequester the bacteria into clumps that are then eliminated from the body by phagocytic cells, but they can also activate the complement cascade to form the C3-convertase via the lectin pathway.

Finally, antibodies generated through the adaptive defense system (to be discussed in more detail in chapter 4) can also activate complement by binding to the surface of bacteria and interacting with the C1-complex. Antibodies are blood proteins produced by B cells that bind to specific molecules on the bacterial surface called antigens. Antigen-bound antibodies can bind the C1 complex to activate it, initiating the cascade that ultimately leads to formation of the C3-convertase. Thus, both the innate and adaptive defense systems can trigger the complement cascade—yet another example of a link between the innate and adaptive defenses.

Before examining each of the pathways for complement activation in detail, it is helpful to understand the roles of the activated proteins produced by the series of proteolytic cleavages that comprise the cascade. Regardless of how the complement pathway is activated, the same key activated components are produced: C3a, C3b, C5a, and C5b (Figure 3-11).

C3a and C5a are proinflammatory molecules that stimulate granulocytes, such as basophils and mast cells, to release the vasoactive substances in their granules, thereby increasing the permeability of blood vessels and facilitating the movement of phagocytes from blood vessels into tissue (Figure 3-2). C5a also acts as a potent chemokine, signaling phagocytes to leave the bloodstream and guiding them to the infection site. Once PMNs or monocytes have left the bloodstream, they move along a gradient of C5a to find the locus of infection.

At the site of infection, C3b binds to the surface of the invading bacterium, enhancing the ability of phagocytes to ingest (engulf) the bacterium. This process of marking phagocyte targets is called opsonization, and any molecule that does this marking is referred to as an opsonin. As will be seen in the next chapter, antibodies can also perform this function. Without opsonization, phagocytes have difficulty ingesting a bacterium unless it is trapped in a small space. Because bacteria do not stick well to the phagocyte surface, the action of pseudopod encirclement can actually propel the bacterium away from the phagocytes—analogous to the way a fish can slip from your hands as you try to grab it. Phagocytes have C3b receptors on their surfaces that specifically bind C3b (Figure 3-12). Thus, by binding to the bacterial surface, complement component C3b gives the phagocyte something to grab on to as it tries to engulf the bacteria. Antibodies bound to bacterial surfaces can also act as opsonins, because a conserved portion of the heavy chain of the antibody, the Fc region, is recognized by Fc receptors on the phagocyte. The difference between these two processes is that antibodies bind to specific molecules on the surface of a bacterium, whereas C3b binds nonspecifically to glycosyl groups commonly found on bacterial surfaces. The combined effect of C3b and Fc receptors binding to their respective ligands on the bacterial surface works synergistically to enhance phagocytosis.

Figure 3-12. Opsonization of a bacterium by activated complement component C3b and antibodies. Combined opsonization by both C3b and antibodies considerably enhances the uptake of the bacterium by phagocytes. IgG, immunoglobulin G.

Activated complement components can also lead to direct killing of bacteria. Activated component C5b binds to the O-antigen of LPS of Gram-negative bacteria and recruits sequentially C6, C7, C8, and multiple C9 proteins to form a MAC in the bacterial membranes (Figure 3-13). Formation of the MAC kills bacteria by creating holes in their membranes. Bacteria that can be lysed by MAC are said to be serum sensitive, i.e., the addition of serum-containing complement components to the bacteria will kill the bacteria. Some Gram-negative bacteria have altered LPS O-antigens that do not bind C5b and therefore cannot form a MAC on their surfaces, while others have extra-long LPS O-antigens that can still bind C5b but cannot form a MAC in the bacterial membrane. Gram-negative bacteria that cannot be lysed by MAC are said to be serum resistant, i.e., they are resistant to killing by serum complement via MAC formation. Gram-positive bacteria are inherently serum resistant, because their thick cell walls prevent MAC assembly at the cytoplasmic membrane.

Figure 3-13. Formation of the membrane attack complex (MAC). (A) C5-convertase assembled on the bacterial cell surface cleaves C5 to generate C5a, which is a chemokine for phagocytes, and C5b, which binds to the O-antigen of LPS on Gram-negative bacteria and recruits C6 through C8 to initiate MAC formation and insertion into the membrane. Up to fifteen C9 proteins then oligomerize into the complex. (B) MAC pores in the membrane that then kills the bacteria. Reproduced from Janeway C, Travers P, Walport M, Shlomchik M. 2004. Immunobiology, 6/e. Garland Science, New York, NY, with permission.

Steps in Complement Activation

The steps in complement activation by each of the three pathways are shown in more detail in Figure 3-14. The classical complement pathway (called that because it was the first to be discovered) is initiated when the Fc regions of several antigen-bound IgG molecules or one pentameric IgM molecule bind to the surface of a bacterium and are subsequently cross-linked by C1, a multi-protein complex of hexameric C1q, two C1r and two C1s zymogens (C1qr2s2) (Figure 3-14A). C1q binds directly to the heavy chain of antibodies bound to the bacterial surface and activates the two C1r serine proteases, which then in turn cleave and activate the two C1s serine proteases. Activated C1s cleaves C4 into C4a and C4b. C4b, like C3b, has a reactive thioester group that enables it to covalently attach to the bacterial surface at a site near the C1qr2s2 complex. C1s also cleaves C2 into C2a and C2b. C4b binds to C2a to form the C3 convertase (C2aC4b). C3 convertase then cleaves C3 to C3a, which diffuses away from the site, and C3b, which covalently binds to the bacterial surface.

Figure 3-14. Activation of complement by the classical, lectin, and alternative pathways. (A) Classical pathway: Two IgG molecules or one IgM molecule attached to the surface of a bacterium bind complement component C1, causing an autoproteolytic event that activates it. C1, C4b, and C2a bind to each other and to the bacterium’s surface to form C3 convertase. The addition of C3b produces C5 convertase that triggers assembly of the MAC. (B) Lectin pathway: The mannose-binding lectins (MBLs) activate the complement pathway similarly to antibodies, except that they interact with C4 and C2 rather than C1. After that point, this pathway is the same as the classical pathway. (C) Alternative pathway: C3-H2O, an activated form of C3 that resembles C3b in conformation, is normally produced at low levels. If it binds a host cell surface through serum factor H, then the H-bound C3-H2O complex is targeted for destruction by serum protein I. If C3-H2O binds to the surface of a bacterium, it can form a complex with factor B, which is targeted for cleavage by factor D to generate Ba and Bb. The C3-H2O complexed with Bb then cleaves more C3 into C3a and C3b. The C3b then covalently binds to the bacterial surface, where it binds factor B, which gets cleaved by factor D to form C3bBb (C3 convertase) on the bacteria surface. Addition of more C3b produces C5 convertase. C5 convertase triggers assembly of the MAC.

The lectin pathway converges with the classical pathway in that it also stimulates cleavage of C4 and C2, with generation of C4b and C2a that form the C3 convertase complex (Figure 3-14B). The collectin MBL circulates in the blood as a multimeric complex with one or more serine proteases, called MBL-associated serine protease (MASP) proteins and small MASP-associated protein (sMAP). MASPs can also bind to ficolins (FCNs). There are three known MASPs: MASP-1, MASP-2, and MASP-3. MASP-1 and MASP-2 resemble C1r and C1s, respectively. When MBL binds mannose groups of glycoproteins found on the surface of many pathogens, the MASP-2 protein cleaves C4 and C2 and initiates formation of the C3 convertase as in the classical pathway. MASP-1 can also cleave C3 directly, whereas MASP-3 appears to associate primarily with FCN but does not appear to activate complement, and its function remains unclear.

Activation via the alternative pathway (named because it was discovered after the “classical” pathway) bypasses the need for C1, C2, C4, antibodies, or MBL/MASP, and relies instead on C3b as the initiating component. As C3 circulates in blood and tissue, it is occasionally activated into a water-bound form (C3-H2O) that can interact with the serum proteins, factor B or factor H. Tissues of the body are coated with sialic acid residues, that can bind to one end of factor H. In the absence of a bacterial surface, C3-H2O binds to the other end of factor H to form a complex that produces C3b (Figure 3-14C). This binding of C3b to factor H changes the conformation of C3b and targets it for proteolytic cleavage into iC3b by the serum protease factor I. In the absence of a bacterial surface, the iC3b bound to factor H is targeted for further destruction by factor I. However, iC3b can still covalently bind to a bacterial surface via its internal thioester group and as such can serve as an opsonin, even though iC3b can no longer form active C3 convertase.

If C3-H2O instead binds to serum protein factor B, then another serum protein factor D cleaves that B to Ba and Bb. The resulting complex of C3-H2O bound to Bb cleaves another C3 molecule to produce C3a and C3b, that then covalently binds to the bacterial surface. This membrane-bound C3b binds another factor B, and the complex C3b-B is then targeted for cleavage by factor D to form the C3 convertase C3bBb, which in turn continues to cleave more C3 to make more C3a and C3b. C3bBb, which normally has a relatively short half-life of about 90 minutes, is further stabilized by binding of the protein properdin (P), which increases the half-life of the resulting C3bBbP complex by up to ten-fold. Properdin is a soluble glycoprotein released by PMNs, monocytes, and adaptive immune cells in response to the presence of proinflammatory cytokines such as TNF-α.

Generation of large amounts of C3b has two functions. A portion of the C3b binds to the bacterial surface and acts as an opsonin to enhance uptake by phagocytes, while another portion binds to the existing C3 convertase complexes (C3bBb or C2aC4b) to form the C5 convertase complexes (C3bBbC3b or C4bC2aC3b), that then cleave C5 into C5a and C5b. C5a diffuses away from the site and acts as a chemokine to recruit phagocytes, whereas C5b binds to LPS on the surface of Gram-negative bacteria and recruits C6, C7, C8, and C9 to form the MAC. Some bacteria produce a polysaccharide surface coating, called a capsule, that preferentially binds serum factor H rather than factor B. As a result, C3b is eliminated as it deposits on the surface, effectively preventing opsonization of the bacterial surface, as well as MAC formation.

Controlling Complement Activation

In all three pathways, it is important to keep the accelerated production of C3a, C3b, C5a, and C5b under control so that overstimulation of the inflammatory response does not occur and host cells are not damaged. To this end, several mechanisms protect host cells from complement over-activation (Figure 3-15). Excess C3b molecules that bind to factor H when bound to sialic acid groups on the surface of host cells are proteolytically cleaved to produce iC3b. As mentioned previously, while iC3b is an effective opsonin (Figure 3-15A), it can no longer aid in the formation of a C3 convertase or C5 convertase. Likewise, when iC3b is bound to factor H on host cell surfaces, it is subject to further degradation by serum factor I.

Figure 3-15. Controlling complement. Multiple regulatory pathways help to dampen complement activation, returning the signaling pathways to the resting state. (A) Factor H and factor I work together to limit the amount of C3b present in the circulation. Factor H competes with factor B for binding to C3b. Formation of complex C3bH destabilizes C3b, exposing a site for the protease activity of factor I to then completely degrade C3b. C3b can also be clipped to form iC3b, which still acts as an opsonin but no longer stimulates complement. iC3b also is degraded by factor I. (B) CD35 (CR1) binds to complement component C3b (as well as C4b) and dissociates the C3 convertase complex, thereby preventing further cleavage of C3 to C3a and C3b. CD35 is also a cofactor for factor I, which clips C3b (as well as C4b) to generate iC3b (and iC4b) and further degradation products. (C) CD55 binds to C3b and C4b and causes dissociation of the C3 and C5 convertase complexes, thereby preventing further cleavage of C3 and C5 and limiting the formation of C3a, C3b, C5a, and C5b–C9 (MAC). (D) CD59 binds the C5b–C8 complex, which is inserted into the cell membrane, and thereby blocks the binding of C9 to the complex and the polymerization of C9 to form membrane pores.

Once the pathogen has been cleared from the system and the heightened immune responses are no longer needed, the host cell uses additional regulatory proteins to help inhibit the complement pathways and return them to basal levels of activity. Several proteins protect host cells from complement-mediated damage by rapidly inactivating components of the complement pathways (Figure 3-11). CD35 (complement receptor 1, CR1) is a membrane glycoprotein found on phagocytic cells that binds to opsonins on bacterial cells and mediates their phagocytosis (Figure 3-15B). CD35 thus serves as the primary pathway for processing and clearing complement-opsonized immune complexes and inhibits both classical and alternative pathways. C4b-binding protein (C4BP), a glycoprotein found in blood plasma that binds C4b (and, to a lesser extent, C3b), is an inhibitor of the classical and lectin complement pathways. C4BP serves as a cofactor of serum factor I, accelerating the degradation of C3 convertase by factor I-mediated cleavage of C4b and C3b. CD46 (membrane cofactor protein) is also a membrane protein receptor that binds factor I as a cofactor to cleave and inactivate C3b and C4b.

Several factors help protect the host cell from being damaged by complement activation by blocking MAC from forming on the host cell surface and thereby protecting the host cell membrane (Figure 3-15C and D). CD59 (MAC-inhibitory protein, MAC-IP) is a membrane-associated glycoprotein that blocks C9 polymerization and MAC formation. CD55 (complement decay-accelerating factor, DAF) is a membrane-associated glycoprotein found on the surface of many blood cells. CD55 binds to C4b and C3b and interferes with their ability to bind C2b and Bb to prevent formation of the C3 convertases (C4bC2b and C3bBb, respectively) on the host cell surface, thereby protecting the host cell membrane.

Cytokines and Chemokines—Mediators of Immune Responses

Roles of Cytokines and Chemokines in Directing Innate Immune Responses

Cytokines and chemokines play a central role in regulating the cellular activities of both the innate and adaptive defense systems (Table 3-2). These signaling molecules act as messengers by binding to receptors on the cells whose activities they direct. Cytokines are soluble glycoproteins (see Box 3-1) of 8 to 30 kDa produced by a variety of cells, including PMNs, DCs, NK cells, endothelial cells, and cells of the adaptive immune system, as well as other host cells when they are infected. Chemokines are small glycopeptides of 8 to 10 kDa that are produced by the same cells that produce cytokines. Their main function is to attract and activate phagocytes, a function similar to that of complement components C5a and C3a.

Box 3-1.

Glycoproteins

The surfaces of human cells are coated with glycoproteins that, as the name implies, contain proteins covalently linked to oligosaccharides. These glycoproteins play diverse roles in eukaryotic organisms. Shown are the structures of a typical N-glycosylation structure on human glycoproteins, that are capped with the nine-carbon monosaccharide sialic acid, called neuraminic acid. The predominant sialic acid found in mammalian cells is N-acetylneuraminic acid (core structure shown below). Not surprisingly, bacteria produce enzymes on their surfaces that can hydrolyze and release the sugars from host glycoproteins, such as the proteins NanA, BgaA, and StrH from S. pneumoniae, and provide food to the bacteria as well as opening up binding sites for bacteria to attach to the host cell surface.

Source:

King SJ, Hippe KR, Weiser JN. 2006. Deglycosylation of human glycoconjugates by the sequential activities of exoglycosidases expressed by Streptococcus pneumoniae. Mol Microbiol 59:961–974[PubMed][CrossRef].

Cytokines and chemokines recognize and bind to specific receptors on the surfaces of target immune cells, that set off signal transduction cascades that modify the functions of the immune cells. Just as complement can be activated by bacterial surfaces, cytokine release can be triggered by interaction between cytokine-producing cells and molecules on the surfaces of the invading bacterium. In the case of Gram-negative bacteria, the outer membrane LPS that activates complement is also a molecule that stimulates cytokine production. Although the surface molecules of other types of bacteria that activate complement and stimulate cytokine release have not been studied nearly as well, it appears likely that the same surface molecules on these bacteria that activate complement also stimulate cytokine production.

During an infection, phagocytes and other cells (such as endothelial cells) release a variety of different cytokines. Some appear early in the infection and are responsible for up-regulation of innate defenses. Others appear late in the infection and help to down-regulate the defense response. Among the earliest-appearing cytokines are granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3). These cytokines stimulate stem cells to produce monocytes and granulocytes (especially PMNs) and trigger their release from the bone marrow into circulation. Other early-appearing cytokines, such as TNF-α, IL-1, IFN-γ, and interleukin-8 (IL-8), stimulate the monocytes and granulocytes to leave the bloodstream and migrate to the site of infection. The steps in this process for PMNs are illustrated in Figure 3-2.

Normally, PMNs move rapidly through the blood vessels, occasionally colliding with one of the vessel walls. TNF-α, IL-1, and IFN-γ stimulate endothelial cells to produce a set of surface proteins called selectins. These selectins bind to proteins on the surface of PMNs and other phagocytic cells in the blood, causing them to loosely attach to the blood vessel endothelium. This loose attachment slows the movement of the blood cells, causing them to roll along the endothelial surface. As this occurs, other selectins will appear on the endothelial cells, while IL-8 stimulates the PMNs to produce proteins called integrins on their surfaces. The integrins bind another set of cytokine-stimulated proteins on the endothelial cell surface, the intercellular adhesion molecules (ICAMs), to generate a tighter attachment between PMNs and endothelial cells. This tight association stops the movement of the PMNs and causes them to flatten against the blood vessel wall. The slowing and stopping of the PMNs is called margination. The PMNs then force themselves between endothelial cells, a process that is assisted by a PMN protein called platelet-endothelial cell adhesion molecule (PECAM). The proinflammatory complement components C3a and C5a assist in the process of transmigration (diapedesis, or extravasation) from the bloodstream into tissue by causing mast cells to release vasoactive histamine and heparin, which dilate blood vessels and make them leakier. Dilation of blood vessels is also assisted by the cytokine platelet-activating factor (PAF). In addition to histamine and heparin, PAF triggers mast cells to produce a number of other vasoactive compounds from the membrane lipid, arachidonic acid, including various leukotrienes and prostaglandins. Once the PMNs have moved out of the blood vessel and into surrounding tissue, a gradient of soluble complement components, chemokines or bacterial peptides, leads them to the site of infection (through chemotaxis).

As PMNs move through tissue, the proinflammatory cytokines TNF-α, IL-1, IL-8, and PAF also activate the oxidative burst response in PMNs so that the PMNs arrive at the infection site with their full killing capacity in place. Monocytes and the macrophages into which they develop similarly activate as they move into an infected area. IFN-γ further stimulates the killing ability of macrophages, producing activated macrophages. Because the signals that control the activities of PMNs, cytokines, and activated complement components are at their highest concentrations near an infected area, PMNs will exit the blood vessel near the location of infection rather than in other areas of the body. Additionally, the fact that activation of the phagocytes occurs only as they are moving into the infected area minimizes the amount of collateral damage to tissues outside the infected area.

The end result of the signaling pathways just described is that a high number of PMNs and other phagocytic cells will leave the blood vessels near the site of infection. However, there are some underlying conditions that will reduce the effectiveness of this signaling system and reduce PMN transmigration, including steroid use, stress, hypoxia, and alcohol abuse. Their inhibitory effect on transmigration may explain why these underlying health conditions are frequently associated with increased susceptibility to infection.

If the phagocytes are successful in eliminating the invading bacterium, other cytokines begin to predominate. In addition to the inhibitory regulators of the complement cascade mentioned earlier, a number of anti-inflammatory cytokines, such as IL-4, IL-10, and interleukin-13 (IL-13), down-regulate production of TNF-α and reduce the killing activities of phagocytes, thereby countering the proinflammatory response and allowing the phagocyte defense system to return to its normal, relatively inactive level.

Inflammation and Collateral Damage

Inflammation, derived from the Latin word inflammare, which means “to set on fire,” is the immunologic and vascular response of the body to invasion by pathogens. Inflammation is caused by the release of proinflammatory cytokines, including C3a and C5a generated by the complement cascade; tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) released by basophils, mast cells, activated DCs, macrophages, and other phagocytes; and subsequent production of prostaglandins and leukotrienes. Other inflammatory mediators include the vasoactive peptides kallidin and bradykinin, which dilate blood vessels leading to a drop in blood pressure and increase vascular permeability at the site of infection to allow neutrophil and monocyte transmigration (extravasation).

Although inflammation serves to protect and control infections, it can also cause further tissue damage, which is manifested as the disease symptoms of redness, swelling, heat, and pain. The increased blood flow to the area due to vasodilation results in redness and elevated temperature. The increased vascular permeability causes blood fluids to leak out of the vessels as the phagocytes transmigrate, causing edema (swelling) of the surrounding tissue. The source of the pain is still not clearly understood, but it is likely caused by the combined effects of cytokines, prostaglandins, and coagulation cascade components on nerve endings in the inflamed region. Bradykinin also appears to increase sensitivity to pain. To counter the proinflammatory response, there are a number of anti-inflammatory cytokines, including IL-1 receptor antagonist, interleukin-4 (IL-4), and interleukin-10 (IL-10), that serve to regulate the immune response by inhibiting the action of the proinflammatory cytokines.

Although phagocytic cells are effective killers of bacteria and are essential for clearing the invading bacteria from an infected area, the body can pay a high price for this service. During active killing of a bacterium, lysosomal enzymes are released into the surrounding area as well as into the phagolysosome. Released lysosomal enzymes damage adjacent tissues and can be the main cause of tissue damage that results from a bacterial infection. Also, PMNs, NK cells, and macrophages kill themselves as a result of their killing activities, and lysosomal granules released by dying PMNs contribute further to tissue destruction. Pus, a common sign of infection, is composed mainly of dead PMNs and tissue cells. If pus accumulates in an enclosed tissue area, then an abscess can form. Pus is usually whitish or yellowish in color, but sometimes it can have a greenish color due to the abundant presence of myeloperoxidase. Bacteria that cause pus formation are called suppurative, purulent, or pyogenic.

Some of the other roles of the proinflammatory cytokines are listed in Table 3-2 and illustrated in Figure 3-16. These proinflammatory cytokines are responsible for common symptoms of infectious diseases other than those localized at the site of infection: fever, somnolence (drowsiness), malaise, anorexia, chills, decrease in blood iron levels, and weight loss. Cytokines IL-1 and TNF-α interact with the hypothalamus and adrenal gland to produce fever and somnolence, which the patient interprets as a feeling of malaise. Indifference to food, which can also characterize this state, contributes to the anorexia. TNF-α and other cytokines stimulate muscle cells to increase their metabolic rate and catabolize proteins to provide fuel for the mobilization of host defenses. Increased metabolism of muscle cells may be the cause of chills seen in some types of systemic infections. If the infection persists, the combination of anorexia and muscle cell breakdown of protein results in weight loss and visible loss of muscle tissue (wasting syndrome).

Figure 3-16. Overview of nonspecific responses to infection and the effects of cytokines on these responses. Proinflammatory cytokines include TNF-α, IL-1, IL-6, and IL-8, as well as the complement components C3a, C4a, C2b, and C5a. C3a, C4a, and C5a act as anaphylatoxins that increase histamine and heparin release from mast cells and basophils, smooth muscle contraction, and vascular permeability. C5a and (to a lesser extent) C3a also act as potent chemokines (enhancing chemotaxis of phagocytes to the infection site).

The cytokine IL-6 released from macrophages and T cells stimulates the liver to increase production of transferrin (to sequester iron), complement components (to regenerate complement components used up during complement activation), LPS-binding proteins (to continue stimulation of cytokine production as long as bacteria are detected in the body), and two general opsonins—MBL, discussed above in the context of complement, and C-reactive protein (CRP). CRP is a pentameric protein produced in the liver whose expression and release into blood is greatly up-regulated in response to IL-6 during inflammation. CRP is also another PAMP recognition protein in that it binds to LTA of some bacterial species as well as to phosphocholine found on the surface of dead or dying bacterial cells. Phagocytes have receptors for CRP, which allows it to act as an opsonin by enhancing phagocytosis.

Septic Shock: The Dark Side of the Innate Defenses

Inappropriately functioning or over-stimulated host defenses can sometimes lead to disastrous consequences such as septic shock. Septic shock is a form of shock caused by bacterial infection (Figure 3-17), during which a massive release of cytokines (sometimes called a cytokine storm) leads to a subsequent overwhelming inflammatory response. Other causes of shock include massive crush injuries, burns, radiation poisoning, and exposure to toxins (toxic shock). In people with septic shock, vasodilation causes a dramatic drop in vascular resistance and blood pressure (hypotension) despite normal-to-high cardiac output. The heart rate and respiratory rate increase as the body tries to compensate for decreasing blood pressure and lack of oxygen. Septic shock is a serious condition because severely reduced levels of blood flow deprive essential organs of oxygen and nutrients (ischemia), causing them to malfunction. The consequent failure of multiple organs, such as the kidneys, heart, brain, and lungs, is the usual cause of death in septic shock patients.

Figure 3-17. Triggering of proinflammatory cytokine release by Gram-negative LPS and its role in septic shock. TNF-α, IL-1, IL-6, and IL-8 are proinflammatory cytokines.

Septicemia (bacteria infecting and growing in the bloodstream) is one of the leading causes of septic shock and occurs in 500,000 to 750,000 patients per year in the United States alone. Up to half of the patients with septicemia end up dying in the hospital. Even those who survive may have long-term aftereffects, such as stroke or permanent damage to lungs, kidneys, liver, or other organs. Indeed, statistics show that those who survive an episode of septic shock have a significantly greater risk of dying during the next five-year period than people with the same underlying conditions but no previous episode of shock. Septic shock is not only serious business for the patient, but it is also a seriously expensive business, as hospital administrators and insurance executives are quick to point out, with annual hospital costs of $5 to 10 billion.

The all-inclusive term sepsis has now been defined more precisely in an effort to aid diagnosis of the various stages of disease. The first stage of shock, called systemic inflammatory response syndrome (SIRS), is characterized by a temperature over 38°C or under 36°C, elevated heart rate, elevated respiratory rate, and an abnormally high or low neutrophil count. The second stage, termed sepsis, is SIRS with a culture-documented infection (i.e., laboratory results showing the presence of bacteria in the bloodstream). The third stage is severe sepsis, characterized by organ dysfunction and very low blood pressure (hypotension). The fourth stage is septic shock, which is characterized by low blood pressure despite intravenous fluid administration.

How does a bacterial infection of the bloodstream produce such a serious condition? Steps involved in septic shock are illustrated in more detail in Figure 3-18. From previous sections of this chapter, it should be evident that the body goes to great lengths to confine the inflammatory response to specific areas of the body. Septic shock is an example of what happens when an inflammatory response is triggered throughout the body. Shock occurs when bacteria or their products reach high enough levels in the bloodstream to trigger complement activation, cytokine release, and the coagulation cascade in many parts of the body. High levels of cytokines, especially TNF-α, IL-1, IL-6, IL-8, and IFN-γ, cause increased levels of PMNs in the blood and encourage these PMNs to leave the blood vessels throughout the body. This leads to massive leakage of fluids into surrounding tissue. PMNs and macrophages activated by IFN-γ also damage blood vessels directly, resulting in loss of fluid from these vessels.

Figure 3-18. Sources of hypotension, disseminated intravascular coagulation (DIC), and internal hemorrhages seen in cases of septic shock.

Activation of complement throughout the body further increases the transmigration of phagocytes. The vasodilating action of C3a, C5a, leukotrienes, and prostaglandins contributes to leakage of fluids from blood vessels and further reduces the ability of blood vessels to maintain blood pressure. Some cytokines cause inappropriate constriction and relaxation of blood vessels, an activity that undermines the ability of the circulatory system to maintain normal blood flow and blood pressure.

Another sign that is frequently associated with septic shock is disseminated intravascular coagulation (DIC), which can be seen as blackish or reddish skin lesions (petechiae). DIC results from widespread activation of blood clotting (coagulation system) in blood capillaries throughout the body. A mediator of DIC is the release of tissue factor (TF), a glycoprotein found on the surface of many cells, such as endothelial cells, monocytes, and macrophages. TF is released into the circulation in response to proinflammatory cytokines (IL-1 and TNF-α), as well as LPS-stimulated TLR4 signaling during sepsis from Gram-negative bacteria. As clots form, blood flow to organs is impaired and ischemia ensues, leading to the tissue damage seen in DIC. On the other hand, as clotting factors are consumed during the formation of multiple clots, bleeding results in other areas where vasodilation has occurred. In addition, activation of the clotting cascade releases excess thrombin, a protease that cleaves the zymogen plasminogen to plasmin, which in turn causes fibrinolysis (the breakdown of clots) and results in hemorrhaging (bleeding), a common symptom of DIC. Hemorrhages not only contribute to hypotension but also damage vital organs.

Once septic shock enters the phase in which organs start to fail, it is extremely difficult to treat successfully. The death rate for severe septic shock is 30–50%, but DIC worsens the prognosis to 70–80%. Treatment is most likely to be effective if it is started early in the infection; however, diagnosis of septic shock in its early stages is not straightforward, as the early symptoms of shock (fever, hypotension, and tachycardia) are very nonspecific. Also, the transition from the early stages to multiple organ failure can occur with frightening rapidity. Of the hundreds of thousands of cases of septic shock that occur in the United States each year, many occur in patients hospitalized with some underlying condition other than infectious disease, which complicates the treatment options for patients.

Accordingly, a massive effort has been made to develop new techniques for treating septic shock patients more successfully, especially in cases where the disease has reached the point at which treatment with antibacterial agents is no longer sufficient to avert disaster. Early efforts to combat septic shock have centered on the administration of glucocorticoids, which down-regulate cytokine production. Physicians have long believed that administration of steroids or ibuprofen would help shock victims because these compounds dampen various aspects of the inflammatory response, but there is now general agreement that glucocorticoid treatment is not effective in treating most types of shock. Indeed, clinical trials have now shown no significant effects from either of these therapies.

More recently, attention has focused on the proinflammatory cytokines, such as TNF-α, that seem to play a central role in the pathology of shock. Antibodies or other compounds that bind and inactivate cytokines have been tested for efficacy in clinical trials. Although the outcome of early clinical trials has been disappointing, newer anti-cytokine agents now being tested appear to be more promising. Nonetheless, it is clear that this type of therapy will never be as effective as catching septic shock in its very early stages.

One impediment to early diagnosis of shock has already been mentioned: the nonspecific nature of the signs and symptoms of shock. Another impediment to early diagnosis is that so many different types of bacteria can cause septic shock. If diagnosed early enough, intensive antibiotic treatment can be effective in halting the shock process. However, until recently, no definitive microbial diagnosis could be made in about a third of patients with clinical signs of sepsis. Additionally, although bacteria are the microorganisms most frequently implicated in septic shock (approximately 80% of cases), many different species of Gram-positive and Gram-negative bacteria can cause shock. Because no single antibiotic is effective against all of these bacterial pathogens, it is important to determine the species of bacterium causing the infection. This is further complicated by the varied natures and mechanisms by which antibiotics work to perform bacterial killing, As we will further explore in chapter 15, antibiotics that result in lysis of the bacterium might cause further harm by causing release of membrane components such as the endotoxin LPS.

Fortunately, some new methods for rapid bacterial identification have been approved by the FDA and others are in late-stage development. These methods rely on highly sensitive technologies, such as mass spectroscopy and PCR-coupled high-throughput sequencing, which enable determination of protein or DNA content from samples taken directly from blood or cultured from blood for only a few hours. The protein or DNA profiles obtained are then quickly compared to library databases of hundreds of standard profiles from known bacterial pathogens. These new methods make identification of pathogens possible in less than a day, and sometimes in just a few hours, instead of the several days required by standard microbiological testing. Other research efforts are focused on determining biomarkers indicative of different causes of sepsis to guide appropriate antibiotic administration and shock treatments. We will discuss more of these methods in chapter 9.

Other Innate Defenses of the Body—Nutritional Immunity

Another innate defense that is present in blood and helps to hamper spread of the invading microbes is a group of proteins that sequester metals away from bacteria that enter the body. Transition metals, such as iron, manganese, and zinc, are essential to the survival of invading bacteria. By restricting metal availability through a group of metal-binding proteins as part of the innate defenses, the body is able to prevent the growth of invading microbes. This limitation of essential nutrient transition metals, termed nutritional immunity, is gaining recognition as a potent innate defense against pathogens.

We have already discussed the role of lactoferrin as a secreted iron-binding protein of surface defenses (milk, tears, saliva, and nasal secretions) that hampers microbial growth and colonization outside the body. Like lactoferrin, transferrin sequesters iron and prevents iron acquisition by microbes that enter the blood. Transferrin is a glycoprotein produced by the liver that circulates through blood and lymph and binds extremely tightly to the ferric form of iron (Fe3+), controlling the level of free iron available. Transferrin bound with iron is recognized by a cell surface receptor that transports the Fe3+-transferrin complex into recycling endosomes, where the iron is sequestered and removed from the transferrin, and the transferrin is then recycled to the surface, where it can scavenge more iron. In this way, the levels of iron in blood drop to an even lower level than normal, which severely limits the growth of most bacteria. Transferrin and lactoferrin, along with their receptors, are also thought to mediate cellular uptake of other divalent metal ions, such as manganese, zinc, and copper, albeit to a lesser extent.

Manganese, like iron, is required for many essential enzymes in bacteria. Given its importance for bacterial growth, the host has also devised strategies to restrict bacterial invaders from acquiring manganese. In phagosomes, the divalent-metal ion transporter NRAMP1 is an integral membrane protein that depletes manganese, iron, and other essential metals from the phagosomal compartment to effectively starve the engulfed bacteria. Genetic mutations in the gene encoding NRAMP1 are associated with susceptibility to tuberculosis, leprosy, inflammatory bowel disease, and autoimmune diseases.

Calprotectin is an abundant cytoplasmic protein that is constitutively expressed in neutrophils, but it can also be expressed in other cell types during infection. Extracellular concentrations of calprotectin can exceed 1 mg/ml at sites of infection. Calprotectin acts as a dimer of two proteins (S100A8 and S100A9) that bind and sequester the essential nutrients manganese and zinc with high affinity. The sequestration of these metal ions by the host inhibits pathogen growth and can render the invading bacteria more sensitive to the oxidative burst produced by phagocytic cells. Calcium binding enhances the affinity of calprotectin for manganese and zinc. This suggests that calcium functions as a molecular switch, keeping the protein in a low affinity state within the host cell cytoplasm, where calcium concentrations are very low, but allowing calprotectin to convert into a high affinity state in the calcium-replete extracellular medium, where it can chelate and sequester manganese and zinc once it is mobilized and released from the cells.

SELECTED READINGS

Beltrame MH, Boldt ABH, Catarino SJ, Mendes HC, Boschmann SE, Goeldner I, Messias-Reason I. 2015. MBL-associated serine proteases (MASPs) and infectious diseases. Mol Immunol 67:85–100.[PubMed][CrossRef]

Brubaker SW, Bonham KS, Zanoni I, Kagan JC. 2015. Innate immune pattern recognition: a cell biological perspective. Annu Rev Immunol 33:257–290.[PubMed][CrossRef]

Caruso R, Warner N, Inohara N, Núñez G. 2014. NOD1 and NOD2: signaling, host defense, and inflammatory disease. Immunity 41:898–908.[PubMed][CrossRef]

Creagh EM, O’Neill LA. 2006. TLRs, NLRs and RLRs: a trinity of pathogen sensors that co-operate in innate immunity. Trends Immunol 27:352–357.[PubMed][CrossRef]

Damo SM, Kehl-Fie TE, Sugitani N, Holt ME, Rathi S, Murphy WJ, Zhang Y, Betz C, Hench L, Fritz G, Skaar EP, Chazin WJ. 2013. Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens. Proc Natl Acad Sci USA 110:3841–3846.[PubMed][CrossRef]

Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, Sevransky JE, Sprung CL, Douglas IS, Jaeschke R, Osborn TM, Nunnally ME, Townsend SR, Reinhart K, Kleinpell RM, Angus DC, Deutschman CS, Machado FR, Rubenfeld GD, Webb SA, Beale RJ, Vincent JL, Moreno R, Surviving Sepsis Campaign Guidelines Committee including the Pediatric Subgroup. 2013. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med 41:580–637.[PubMed][CrossRef]

Deretic V. 2011. Autophagy in immunity and cell-autonomous defense against intracellular microbes. Immunol Rev 240:92–104.[PubMed][CrossRef]

Franchi L, Muñoz-Planillo R, Núñez G. 2012. Sensing and reacting to microbes through the inflammasomes. Nat Immunol 13:325–332.[PubMed][CrossRef]

Imlay JA. 2013. The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat Rev Microbiol 11:443–454.[PubMed][CrossRef]

Motta V, Soares F, Sun T, Philpott DJ. 2015. NOD-like receptors: versatile cytosolic sentinels. Physiol Rev 95:149–178.[PubMed][CrossRef]

Murphy K. 2011. Janeway’s Immunobiology, 8th ed. Garland Science Publishing Inc, New York, NY.

Pareja ME, Colombo MI. 2013. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms. Front Cell Infect Microbiol 3:54.[PubMed]

Rath M, Müller I, Kropf P, Closs EI, Munder M. 2014. Metabolism via arginase or nitric oxide synthase: two competing arginine pathways in macrophages. Front Immunol 5:532.[PubMed][CrossRef]

Rock KL, Kono H. 2008. The inflammatory response to cell death. Annu Rev Pathol 3:99–126.[PubMed][CrossRef]

Sellge G, Kufer TA. 2015. PRR-signaling pathways: learning from microbial tactics. Semin Immunol 27:75–84.[PubMed][CrossRef]

Weiss G, Schaible UE. 2015. Macrophage defense mechanisms against intracellular bacteria. Immunol Rev 264: 182–203.[PubMed][CrossRef]

Wessling-Resnick M. 2015. Nramp1 and other transporters involved in metal withholding during infection. J Biol Chem 290:18984–18990.[PubMed][CrossRef]

Zackular JP, Chazin WJ, Skaar EP. 2015. Nutritional immunity: S100 proteins at the host-pathogen interface. J Biol Chem 290:18991–18998.[PubMed][CrossRef]

Questions

1. What are two types of bacterial killing mediated by complement?

2. In the activation of complement by the alternative pathway, the stabilization of one particular component is essential. What is this component? How is it stabilized?

3. Define opsonization and two components of the immune system that can act as opsonins. Are they acquired or are they are innate?

4. How do the functions of various activated complement components resemble the functions of cytokines and chemokines?

5. Why does the body have such a complex system (complement plus cytokines) for directing the activities of the phagocytes? What is this system trying to achieve?

6. Why are there two pathways for activating complement?

7. What is the point of using proteolytic cleavage to activate complement? Why not just have the molecules made in their active form?

8. Why does blood pressure fall during septic shock?

9. Why are antibiotics (chemicals that kill bacteria) ineffective after a certain point in the course of septic shock, even though the causative bacterium is susceptible to them? Make an educated guess as to why so many anticytokine therapeutic agents fail.

10. What kinds of evasive action could a bacterium take to prevent it from being killed by a phagocyte? (Hint: Consider the steps in the killing process.)

11. Why do neutrophils circulate in blood? Why not have them migrate permanently into tissue, as macrophages do?

12. Inflammation near the skin is characterized by redness and swelling. What causes these symptoms?

13. What is the primary function of Toll-like receptors and NOD proteins? Where are they located? How do they stimulate immune responses?

14. What might happen to individuals with genetic defects in the following molecules?

a. C3

b. C5

c. Factor B

d. MBL

e. TLR4

f. transferrin

15. What possible advantages would there be for the intracellular Gram-positive pathogen Listeria monocytogenes to grow within the cytosol of a phagocytic cell? What possible advantages would there be for Salmonella Typhimurium to reside within its own phagosomes?

16. Name three different specific immune mechanisms that enhance a phagocyte’s ability to clear a pathogen from the body. How do they each work to help phagocytic clearing of the pathogen?

17. What are the two primary functions of the lymphatic system?

18. NK cells play important functions in the innate immune response. Provide an explanation for each of the following statements:

a. NK cells kill infected host cells nonspecifically, but not randomly.

b. NK cells can recognize infected cells directly and indirectly.

c. NK cells are similar to, but differ from, CTLs.

Comic Relief

19. How would you attract the attention of a good-looking phagocyte?

20. How would you describe a macrophage that is wearing sunglasses and a camera?

Answers to Comic Relief:

19. Try complements.

20. Phago-sightseer.

Подняться наверх