Phagocytosis: Ingestion and Destruction by White Blood Cells
By any standard, a phagocyte represents an impressive piece of living machinery, meandering through the tissues to seek, capture, and destroy a target. The following is a summary of the major activities of phagocytes:
1. to survey the tissue compartments and target microbes, particulate matter (dust, carbon particles, antigen–antibody complexes), and injured or dead cells;
2. to ingest and eliminate these materials; and
3. to extract immunogenic information (antigens) from foreign matter.
Major Categories of Phagocytes
It is generally accepted that all cells have some capacity to engulf materials, but professional phagocytes do it for a living. The main types of phagocytes are neutrophils, monocytes, macrophages, and dendritic cells.
Neutrophils and Eosinophils As previously stated, neutrophils are general-purpose phagocytes that react early in the inflammatory response to bacteria and other foreign materials and to damaged tissue. A common sign of bacterial infection is a high neutrophil count in the blood (neutrophilia), and neutrophils are also a primary component of pus. Eosinophils are attracted to sites of parasitic infections and antigen antibody reactions, though they play only a minor phagocytic role.
Extended Functions of Neutrophils Neutrophils are truly one of the “workhorses” of innate immunity. In addition to their phagocytic functions, they have a distinct system for capturing pathogens called neutrophil extracellular traps, or NETS. Neutrophils are programmed to die after they have gone through their regular engulfment and killing of bacteria and other pathogens. But their death does not end their protective actions. Granules within the cytoplasm release an enzyme called neutrophil elastase, which migrates to the nucleus of the neutrophil and destroys the proteins needed to organize DNA into chromosomes. These dying neutrophils throw out a fibrous matrix composed of DNA, enzymes, histones, and other cell contents that stop invading microbes even after the neutrophils are completely lysed (process figure 1). The NET works at several levels to trap and immobilize bacteria and fungi, degrade their virulence factors, kill them with microbicidal chemicals, and ultimately, prevent them from spreading.
Fig1. Production of neutrophil extracellular traps (NETS). (top) (1) Granules within the cytoplasm of the neutrophil release the enzyme neutrophil elastase (NE). (2) Neutrophil elastase travels to the nucleus of the cell and cleaves proteins needed to package the cell’s DNA into chromosomes. (3) As the chromosomes decondense, DNA fills the cell. (4) The neutrophil lyses, propelling the net outward, where it traps pathogenic cells. (bottom) Salmonella cells entrapped by a neutrophil extracellular trap. (top) Source: The-scientist.com. Infographic: How NETs work; (bottom) SCIENCE PHOTO LIBRARY/Science Source
Macrophages: Dynamic Scavengers After emigrating out of the bloodstream into the tissues, monocytes are transformed by various inflammatory mediators into macrophages. This process is marked by an increase in size and by enhanced development of lysosomes and other organelles (figure 14.16). At one time macrophages were classified as either fixed (adherent to tissue) or wandering, but this terminology can be misleading. All macrophages retain the capacity to move about. Whether they reside in a specific organ or wander depends upon their stage of development and the immune stimuli they receive. Specialized macrophages called histiocytes migrate to a certain tissue and remain there during their life span. Examples are alveolar (lung) macrophages; Kupffer cells in the liver; Langerhans cells in the skin; and macrophages in the spleen, lymph nodes, bone marrow, kidney, bone, and brain. Other macrophages do not reside permanently in a particular tissue but instead drift nomadically throughout the reticuloendothelial system. Dendritic cells— another product of the monocyte line—are concentrated in skin and the linings of mucous membranes, where they process foreign substances and prepare them for reactions with selected lymphocytes.
Erythrocytes: A Role in the Immune System Recent research has indicated that red blood cells may well function as part of the immune system, an extraordinarily surprising finding. Mature erythrocytes have no nucleus, having extruded the organelle to create as much room for oxygen-carrying hemoglobin as possible. Without a nucleus, these cells cannot react to their surroundings, and they were thought to be limited to the almost mechanical role of ferrying oxygen and carbon dioxide throughout the body. However, evidence has mounted that red blood cells may be especially adept at collecting cell-free DNA present in the circulatory system, much of which belongs to pathogens. Cells carrying these small stretches of DNA are then targeted by other cells of the immune system, helping to trigger the body’s immune response.
Mechanisms of Phagocytic Recognition, Engulfment, and Killing
The term phagocytosis literally means “eating cell process.” But phagocytosis is more than just the physical act of engulfment, because phagocytes also actively attack and dismantle foreign cells using a wide array of antimicrobial substances. Phagocytosis can occur as an isolated event performed by a lone phagocytic cell responding to a minor irritant in its area or as part of the orchestrated events of inflammation described in section 14.3. The events in phagocytosis include chemotaxis, ingestion, phagolysosome formation, destruction, and elimination (process figure 2).
Fig2. The sequential events in phagocytosis. (1) Phagocyte is attracted to bacteria. (2) Close-up view of process showing bacteria adhering to phagocyte receptors by their PAMPs. (3) Vacuole is formed around bacteria during engulfment. (4) Phagosome digestive vacuole results. (5) Lysosomes fuse with phagosome, forming a phagolysosome. (6) Enzymes and toxic oxygen products kill and digest bacteria. (7) Undigested particles are released. Inset: Scanning electron micrograph of a neutrophil (blue) phagocytizing purple methicillin-resistant Staphylococcus aureus cells (10,000×). National Institute of Allergy and Infectious Diseases (NIAID)
Chemotaxis, Binding, and Ingestion Phagocytes migrate into a region of inflammation with a deliberate sense of direction, attracted by a gradient of stimulant products from the pathogen and host tissue at the site of injury.
Once a phagocyte encounters the pathogen, it uses its toll-like receptors to make contact with the pathogen (figure 2). Recall that TLRs are receptors that recognize and bind the pathogen- associated molecular pattern (PAMP) receptors of various microbes. There are about 10 different TLRs in the membranes of phagocytes. The exposed end of a receptor hooks onto a PAMP and immediately dimerizes, or joins, with a second TLR to encase the molecule (figure 3). This relays a signal into the nucleus that stimulates the intracellular phagocytic processes and the release of chemical mediators.
Fig3. Phagocyte detection and signaling with toll like receptors. Toll-like receptors (TLRs) span the membrane of phagocytes and other cells of the immune system. When a molecule such as a PAMP on a particular pathogen is recognized by this receptor, the TLRs merge and bind the foreign molecule. This induces production of chemicals and triggers engulfment.
Phagolysosome Formation On the scene of an inflammatory reaction, phagocytes often trap cells or debris against the fibrous network of connective tissue or the wall of blood and lymphatic vessels. Once the phagocyte has “caught” its prey, it extends pseudopods that enclose the cells or particles in a pocket and internalize them in a vacuole called a phagosome.
In a short time, lysosomes migrate to the scene of the phagosome and fuse with it to form a phagolysosome. Other granules containing antimicrobial chemicals are released into the phagolysosome, forming a potent brew designed to poison and then dismantle the ingested material (see process figure 2). The destructive ness of phagocytosis is evident by the death of bacteria within 30 minutes after contacting this battery of antimicrobial substances.
Destruction and Elimination Systems Destructive chemicals await the microbes in the phagolysosome. The oxygen-dependent system known as the respiratory burst, or oxidative burst, elaborates products of oxygen metabolism called reactive oxygen intermediates (ROIs). Myeloperoxidase, an enzyme found in granulocytes, forms halogen ions (OCl−) that are strong oxidizing agents. Other products of oxygen metabolism such as hydrogen peroxide, the superoxide anion (O2−), activated or singlet oxygen (1O−), and the hydroxyl free radical (HO) separately and together have formidable killing power. This series of reactive oxygen products delivers a “knockout” punch necessary to kill aerobic pathogens such as fungi and many bacteria. Other chemicals that come into play are lactic acid, lysozyme, and nitric oxide (NO), a powerful mediator that kills bacteria and inhibits viral replication. Cationic proteins that injure bacterial cell membranes and a number of hydrolytic enzymes complete the job. The bits of undigestible debris are released from the macrophage by exocytosis. As we shall see in chapter 15, macrophages and dendritic cells combine phagocytosis with further processing of microbial antigens required for specific immune responses with lymphocytes.
Interferon: Antiviral Cytokines and Immune Stimulants
Interferon (IFN) was described in chapter 12 as a small protein produced naturally by certain white blood and tissue cells. It is used in therapy against certain viral infections and cancer and can be used as an immune enhancer. Although the interferon system was originally thought to be directed exclusively against viruses, it is now known to be involved also in defenses against other microbes and in immune regulation and intercommunication. Three major types are interferon alpha, a product of lymphocytes and macrophages; interferon beta, a product of fibroblasts and epithelial cells; and interferon gamma, a product of T cells.
All three classes of interferon are produced in response to viruses, RNA, immune products, and a variety of foreign molecules. Their bio logical activities are extensive. In all cases, they bind to cell surfaces and induce changes in genetic expression, but the exact results vary. In addition to antiviral effects, discussed next, all three IFNs can inhibit the expression of cancer genes and have tumor suppressor effects. Interferon alpha and IFN beta stimulate phagocytes, and IFN gamma is an immune regulator of macrophages and T and B cells.
Characteristics of Antiviral Interferon
When a virus binds to the receptors on a host cell, a signal is sent to the nucleus that directs the cell to synthesize interferon. After transcription and translation of the interferon gene, newly synthesized interferon molecules are rapidly secreted by the cell into the extracellular space, where they bind to other host cells. The binding of interferon to a second cell induces the production of another class of proteins that inhibit viral multiplication by preventing the translation of viral proteins (figure 4). Interferon is not virus-specific, so its synthesis in response to one type of virus will also protect against other types. Because this protein is an inhibitor of viruses, it has been a valuable treatment for a number of viral infections.
Fig4. The antiviral activity of interferon. When a cell is infected by a virus, its nucleus is triggered to transcribe and translate the interferon (IFN) gene. Interferon diffuses out of the cell and binds to IFN receptors on nearby uninfected cells, where it induces production of proteins that eliminate viral genes and block viral replication. Note that the initial cell is not protected by its own IFN and that IFN does not prevent recipient cells from being invaded by viruses, but the viruses are inactivated before they can start replicating.
Other Roles of Interferon
Interferons are also important immune regulatory cytokines that activate or instruct the development of white blood cells. For example, interferon alpha produced by T lymphocytes activates a subset of cells called natural killer (NK) cells. In addition, one type of interferon beta plays a role in the maturation of B and T lymphocytes and in inflammation. Interferon gamma inhibits cancer cells, stimulates B lymphocytes, activates macrophages, and enhances the effectiveness of phagocytosis.
Complement: A Versatile Backup System
Among its many overlapping functions, the immune system has another complex and multiple-duty system called complement that is brought into play at several levels. The complement system, named for its property of “complementing” immune re actions, consists of at least 30 blood proteins released by liver cells, lymphocytes, and monocytes. Complement factors are a form of pattern recognition receptor (PRR) that works in concert with inflammation and phagocytosis to destroy a wide variety of bacteria, viruses, and parasites. This system is generally nonspecific and innate, but it can also play a role in acquired immune responses. The three primary defensive features of the complement system are (1) the membrane attack complex (MAC), which kills pathogens directly; (2) the coating of pathogens with molecules that make them more attractive to phagocytes (opsonization); and (3) the recruitment of inflammatory cells and triggering of cytokine release. Having introduced the second and third topics previously, we will focus here on the main mechanisms of the membrane attack complex.
The membrane attack function of complement is a series of reactions like that of blood clotting, in which the first substance in a chemical series activates the next sub stance, which activates the next, and so on, until a desired end product is reached. Three different versions of the complement pathways exist (process figure 5). Their main distinguishing features are how they are activated, major participating fac tors, and specificity. The end stages of all three converge at the same point and yield a similar end result, that is, destruction of a microbe or infected cell. Because the focus of damage is on the cell membrane, this portion of the complement defense is not as effective on microbes whose outer walls block access to the membrane, but is is effective on cells and viruses with exposed membranes.
Fig5. Overview of the complement pathways leading to membrane attack. (a) All three pathways have different triggers and starting points, but they all converge at the same place, C3 convertase. This enzyme begins the series of reactions (b, c, d) that give rise to the membrane attack complex characteristic of the complement “machinery.” The final result is the formation of tiny openings in the cell membrane and the destruction of the target pathogen.
The classical pathway is the most specific, activated mainly by the presence of antibody bound to microorganisms. It is this path way that links complement to reactions of acquired immunity. The other two pathways are nonspecific and triggered by common foreign molecules on the surfaces of microbes. In the lectin pathway, a host serum protein or lectin binds a sugar called mannan present in the walls of bacteria and other microbes. The alternative pathway is initiated by complement proteins that bind to certain surface molecules of microbes (PAMPS), although it may also be triggered spontaneously. Note that because the complement numbers (C1 to C9) are based on the order of their discovery, some factors are not activated in numerical order.
Stages in the Complement Cascade
In general, the complement pathways go through four stages: initiation, amplification and cascade, polymerization, and membrane attack. The starting reaction requires some type of initiator molecule such as antibodies, lectins, or microbial surface receptors (process figure 5a), depending on the pathway. The presence of this initiator on the pathogen’s membrane propels the chain of actions involving complement chemicals C1 through C4, with each step serving a dual purpose. We are omitting the fine details of the amplification part of the complement system, but the end result is a molecule, C3, that is a key factor in subsequent polymerization of the remaining complement factors in the membrane of the target cell. C3 is acted on by a convertase enzyme that splits it into factors C3a and C3b. C3b forms the recognition site and anchor for the final series of reactions to come (process figure 5b). C3b by itself is also an important factor in opsonization and thus facilitates phagocytosis. More information on opsonization can be found in chapter 15.
C3b moves the cascade reaction forward by splitting factor C5 into C5a and C5b. One of these molecules—C5b—is key to finishing up polymerization of the remaining complement factors described next. You will notice that the two cleavage products that do not continue in the cascade reaction—C3a and C5a—still have a number of immune functions (process figure 5—right side of b). Both of them are chemotactic factors that recruit white blood cells and trig ger the release of inflammatory mediators such as histamine.
The final events in the complement series involve factors C5b, C6, C7, and C8, which join together and give rise to an anchoring complex within the membrane (process figure 5c). This is followed by the insertion of several C9 molecules in the pattern of a ring-shaped formation that is the membrane attack complex (MAC) (process figure 5d). The MAC is the primary destructive force of the complement system. It effectively perforates and lyses the membranes of gram-negative bacteria, some parasitic protozoans, enveloped viruses, and virally infected host cells. Except for the classical pathway, these reactions are nonspecific and can be active against a wide spectrum of microbes.
An Outline of Major Host Defenses
At this point, it should be apparent that the body’s defenses often work in both parallel and redundant ways to fight pathogens. The result is a constant onslaught of defenses functioning at many levels. This can be illustrated by figure 6, organized by the three lines of defense that started out our discussion and that lead us naturally into chapter 15.
Fig6. Flowchart summarizing the major components of the host defenses. Defenses are classified into one of two general categories: (1) innate and nonspecific or (2) acquired and specific. These can be further subdivided into the first, second, and third lines of defense, each being characterized by a different level and type of protection. The third line of defense, the most varied, is responsible for specific immunity. It is covered in greater detail in chapter 15.
