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
