1. 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 three main types of phagocytes are neutrophils, monocytes, and macrophages.
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.
Macrophages: Large-Tissue Phagocytes 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 1). 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 and drift nomadically throughout the RES. Not only are macrophages dynamic scavengers, but they also process foreign substances and prepare them for reactions with certain lymphocytes.
2. 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 with 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. The events in phagocytosis include chemotaxis, ingestion, phagolysosome formation, destruction, and elimination (figure 2).
Fig. 1. The developmental stages of monocytes and macrophages. The cells progress through maturational stages in the bone marrow and peripheral blood. Once in the tissues, a macrophage can remain nomadic or take up residence in a specific organ.
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 (TLRs) to make contact with the pathogen (figure 2). TLRs are receptors that recognize and bind the PAMP receptors of various microbes. There are about 10 different TLRs in the membranes of phagocytes. The exposed end of a receptor hooks on to 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.
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.
Fig. 2. The sequential events in phagocytosis. (1) Phagocyte is attracted to bacteria. (2) Close-up view of process showing bacteria adhering to special 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.
Phagolysosome Formation and Killing 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 (figure 2). The destructiveness 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 oxygendependent system, known as the respiratory burst or oxidative burst, elaborates products of oxygen metabolism called reactive oxygen intermediates (ROI). 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 (see case file).
Other chemicals that come into play are the 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 proteolytic and other hydrolytic enzymes complete the job. The bits of undigestible debris are released from the macrophage by exocytosis. Macrophages and dendritic cells use this system to process microbes for specific immune responses with lymphocytes.
Fig. 3. 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.
C A S E F I L E
About one in 200,000 children in the United States are diagnosed yearly with chronic granulomatous disease (CGD); of these, around 90% are males. CGD is somewhat complex in its inheritance, but there are essentially two forms. The X-linked type occurs as a defective gene on the X chromosome and is more severe. Over half of all cases are X-linked. The autosomal defect is located on a different chromosome and tends to be milder. In both types, the genetic codes carry errors that prevent phagocytes from producing certain key metabolic enzymes.
The neutrophils and macrophages of CGD patients can carry out the usual phagocytic activities such as amoeboid motion, chemotaxis, engulfment, and digestion of microbes in phagocytic vesicles, but they are unable to actively kill certain types of aerobic pathogens. They lack the enzymes to synthesize the battery of reactive oxygen intermediates (ROIs) – superoxide ion, hydrogen peroxide, and hydroxyl radical – that are essential to kill these microbes. As a result, the ineffective phagocytes harbor live microbes that can break away and continue their infectious path. In time, these phagocytes shut down, die, and build up in the infected tissue in masses (granulomas).
■ Why would children with CGD be more prone to infections from fungi and bacteria and not viruses?
■ Why is interferon an effective treatment for CGD?
This case is a striking example of how important every element of immunity can be. Although the components do overlap in some of their effects, each one carries out some essential function that cannot be totally duplicated. CGD individuals have a mostly intact immune response, but they lack a complete phagocyte defense that comes into play early in the infection process. Phagocytes (neutrophils, monocytes, and macrophages) are most active against bacteria and eukaryotic pathogens, but they also are important participants in many other immune reactions.
The swelling and inflammation of the child’s bones (osteomyelitis) were triggered by the Aspergillus hyphae growing into bone cavities. The presence of these foreign cells initiated a series of local events, beginning with infiltration of phagocytes and other white blood cells into the infected area. The white blood cells released biochemical mediators that give rise to the classic inflammatory signals of rubor, calor, tumor, and dolor. These mediators dilate blood vessels, creating rubor or redness, which also has the effect of increasing the local temperature (calor). The reactions flood the site with tissue fluid and cause swelling (tumor) and pain (dolor).
Even people with healthy phagocytes can produce granulomas, but CGD individuals produce them in pathological amounts. The granulomatous nature of this disease is triggered by chronic inflammation and buildup of dead neutrophils that could not completely finish the job of clearing the pathogen. When the backup team of macrophages enters the scene to “clean up” the dead neutrophils and infection debris, it also is unable to resolve the inflammation and leaves behind large inactive packets of cells that remain in tissues for long periods of time.
The major infectious agents that invade CGD patients are catalase-producing aerobic bacteria and fungi. These types of organisms would usually be kept in check by normal phagocytes that literally douse them in toxic oxygen intermediates such as superoxide ions, hydroxyl radicals, and hypochlorites. Because CGD phagocytes lack these intermediates, they have little or no effect on organisms such as Staphylococcus, Klebsiella, Aspergillus, and Candida. Viruses are not a significant cause of opportunistic infections in these patients because viruses are not controlled by oxygen-based chemicals. They are eliminated by other immune defenses such as interferon.
The immune stimulant gamma interferon has been beneficial in resolving infections in CGD patients. This may be due to several factors. One contribution of interferon is to activate macrophages and enhance the effectiveness of phagocytosis. Another is that it modulates T cells that can destroy infectious agents without the need for phagocyte-killing mechanisms. Interferon is often prescribed in combination with antimicrobial drugs that target the pathogen. Since this disease is genetic, it must be treated continuously with long-term prophylactic medications and other therapies. There is no cure, but several trials of gene therapy to correct the genetic defect are in progress.
TAKE NOTE: NEUTROPHILS GIVE THEIR ALL
Neutrophils are truly one of the “workhorses” of innate immunity. In addition to their phagocytic functions, a recent discovery revealed that they also have a distinct system of 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 they undergo one last aggressive act while they are dying, which is to throw out a fibrous matrix that consists of their lysed DNA, enzymes, histones, and other cell contents that works outside the cell even after it is dead. This NET works at several levels to trap bacteria and fungi, degrade their virulence factors, kill them with microbicidal chemicals, and, to top it off, immobilize microbes and prevent them from spreading.
The absence of a sufficient immune response is called an immunodeficiency, which can be either congenital or acquired.