The Microbial World

University of Wisconsin - Madison

The Phagocytic Response of the Host

Phagocytic Defenses

When invading an pathogen penetrates the tissues, the inflammatory response is immediately brought into play. Part of this response leads to the recruitment of phagocytes at the site of inflammation. Phagocytes are a class of cells which are capable of ingestion (engulfment) and destruction of microorganisms and viruses that are responsible for inciting the inflammatory response. First to accumulate around the invaders and initiate the phagocytic process are neutrophils. Later, local and blood-borne macrophages also migrate to the tissue site and initiate phagocytosis. Neutrophils (also known as polymorphonuclear leucocytes or PMNs) and macrophages are sometimes referred to as professional phagocytes for their roles in this process.

Properties of Neutrophils

Neutrophils have their origin in multi-potential stem cells in the bone marrow. They differentiate in the marrow and are released in a mature form, containing a full complement of bactericidal agents. They are short-lived cells which constitute 30-70% of the circulating white blood cells (leukocytes).

During differentiation in the marrow (2-3 days) the nucleus of the cell becomes multilobed (hence the name polymorphonuclear leukocyte), cell division ceases, and mitochondria and endoplasmic reticulum disappear from the cytoplasm. At the same time the cell becomes motile and actively phagocytic. Cytoplasmic granules are formed from the Golgi apparatus. These membranous granules are called lysosomes and contain the various bactericidal and digestive enzymes which can destroy bacterial cells after engulfment. The contents of lysosomes include lysozyme, cationic proteins, acid hydrolases, proteases, peroxidase and lactoferrin. Neutrophils also contain large store of glycogen; since they derive most of their metabolic energy from glycolysis, they can function efficiently in anaerobic environments.

Some additional properties of neutrophils are:

-Only half the neutrophils in human circulation are detectable in the blood; the rest adhere to vessel walls.

-For every circulating neutrophil, approximately 100 near mature cells are held in reserve in the bone marrow pool.

-Once a neutrophil enters the tissues, intestinal tract or respiratory tract, it never returns to the circulation.
 

Properties of Macrophages

Macrophages (also called mononuclear phagocytes) also arise from bone marrow stem cells which give rise to promonocytes which develop into monocytes that are released into the blood stream. Monocytes make up 3-7% of the circulating white blood cells. The monocyte is actively phagocytic and and bactericidal. Within 2 days or so, the blood stream monocytes (sometimes called wondering macrophages) emigrate into the tissues where they settle down, enlarge and become fixed macrophages (tissue histiocytes), which also have phagocytic potential. Macrophages are more active in phagocytosis than monocytes and develop many more granules containing hydrolytic enzymes. New macrophages can develop by cell division under inflammatory stimuli, but most macrophages are matured blood monocytes.

The total pool of macrophages is referred to as the system of mononuclear phagocytes. The system is scattered throughout connective tissue, basement membranes of small blood vessels, liver sinusoids, the spleen, lung, bone marrow and lymph nodes. Monocytes from the blood migrate into virtually every organ in the body where they mature into fixed macrophages. In the lymph nodes, they function as scavengers to remove foreign material from the circulation.

Compared to neutrophils, macrophages are long-lived cells. As phagocytes, neutrophils play a more important role in the acute stages of an infection, while macrophages are principally involved in chronic types of infections. Neutrophils circulate in the blood stream, and during an acute inflammatory response they migrate through the endothelial cell junctions as part of the inflammatory exudate. They migrate to the focus of the infection and ingest or "phagocytose" the foreign agents. Neutrophils which have become engorged with bacteria usually die and largely make up the material of pus. Macrophages, which are also attracted to the area during an inflammatory response, are slower to arrive and become increasingly involved in chronic infections. They, too, are actively phagocytic and will engulf and destroy foreign particles such as bacteria. However, macrophages have another indispensable function in host defense: they "process" the antigenic components of infective agents and present them to lymphocytes, a process that is usually required for the initiation of the adaptive immune responses of the host. For this activity, macrophages are known as antigen-presenting cells or APC's and they are an important bridge between the innate defenses and the adaptive immune response.

The Phagocytic Process

Phagocytosis and destruction of engulfed bacteria involves the following sequence of events:

1. Delivery of phagocytic cells to the site of infection

2. Phagocytic adherence to the target

3. Ingestion or engulfment of the target particle

4. Phagolysosome formation

5. Intracellular killing

6. Intracellular digestion (and egestion, in the case of macrophages)

These steps involved in the phagocytic process in macrophages are illustrated below.


Figure 1. Phagocytosis by a Macrophage. A bacterium, which may or may not be opsonized, is engulfed by the process of endocytosis. The bacterium is ingested in a membranous vesicle called the phagosome. Digestive granules (lysosomes) merge with phagosome, release their contents, and form a structure called the phagolysosome. The killing and digestion of the bacterial cell takes place in the phagolysosome. The macrophage egests debris while processing the antigenic components of the bacterium, which it returns to its surface in association with MHC II for antigen presentation to T cells.

Delivery of phagocytic cells to the site of infection

The delivery of phagocytic cells, monocytes or neutrophils, to the site of microbial infection involves two processes:

Diapedisis: the migration of cells across vascular walls which is initiated by the mediators of inflammation (kinins, histamine, prostaglandins, etc.)

Chemotaxis. Phagocytes are motile by ameboid action. Chemotaxis is movement of the cells in response to a chemical stimulus. The eventual concentration of phagocytes at a site of injury results from chemotactic response by the phagocytes which is analogous to bacterial chemotaxis. A number of chemotactic factors (attractants) have been identified, both for neutrophils and monocytes. These include bacterial products, cell and tissue debris, and components of the inflammatory exudate such as peptides derived from complement.

Phagocytic adherence

Phagocytosis is initiated by adherence of a particle to the surface of the plasma membrane of a phagocyte. This step usually involves several types of surface receptors on the phagocyte membrane. Three major receptors on phagocytes recognize the Fc portion of IgG antibody molecules: one is for monomeric IgG and the others are for antigen-crosslinked IgGs. Another receptor binds a complement factor, C3b. Other phagocyte receptors bind fibronectin and mannose-terminated oligosaccharides. Under certain circumstances of infection, bacteria or viruses may become coated or otherwise display on their surfaces one or another of these substances (i.e., IgG, C3b, fibronectin or mannose). Such microbes are said to be opsonized and such substances as IgG or complement C3b bound to the surface of microbes are called opsonins. (Opsonin comes from a Greek word meaning "sauce" or "seasoning": they make the bacterium or virus more palatable and more easily ingested by the phagocyte.) Opsonins provide extrinsic ligands for specific receptors on the phagocyte membrane, which dramatically increases the rate of adherence and ingestion of the pathogen. Opsonized bacteria can be cleared from the blood by phagocytes; many types of non opsonized bacteria cannot be cleared.

Less firm attachments of a phagocyte to a particle can take place in the absence of opsonization. This can be thought of as nonspecific attachment which might be due to net surface charge on the phagocyte or particle and/or hydrophobicity of the particle.

Also, a phenomenon called surface phagocytosis exists: a phagocyte can simply trap an organism against a surface and initiate ingestion. Surface phagocytosis may be an important pre-antibody defense mechanism which may determine whether an infection will become a disease and how severe the disease will become.

Ingestion

After attachment of the phagocyte to its target, some sort of signal generation, which is poorly understood, results in physical or chemical changes in the cell that triggers ingestion. Ingestion is an engulfment process that involves infolding or invagination of the cell membrane enclosing the particle and ultimately releasing it into the cytoplasm of the cell within a membrane vesicle. The end result of ingestion is entry of the particle enclosed in a vesicle derived from the plasma membrane of the cell. This structure is called the phagosome.

Formation of the phagolysosome

The phagosome migrates into the cytoplasm and collides with lysosomal granules which explosively discharge their contents into the membrane-enclosed vesicle (phagosome). Membranes of the phagosome and lysosome actually fuse resulting in a digestive vacuole called the phagolysosome. Other lysosomes will fuse with the phagolysosome. It is within the phagolysosome that killing and digestion of the engulfed microbe takes place. Some of the microbicidal constituents of the lysosomes of neutrophils and macrophages include lysozyme, cationic proteins, various proteases and  hydrolyases and peroxidases. The killing processes are confined to the membranous organelles of the phagocytes (the phagolysosome) such that none of the toxic substances and lethal activities of the phagocytes are turned against themselves.

Intracellular killing of organisms

After phagolysosome formation the first detectable effect on bacterial physiology, occurring within a few minutes after engulfment, is loss of viability (ability to reproduce). The exact mechanism is unknown. Inhibition of macromolecular synthesis occurs later. By 10 to 30 minutes after ingestion many pathogenic and nonpathogenic bacteria are killed followed by lysis and digestion of the bacteria by lysosomal enzymes. The microbicidal activities of phagocytes are complex and multifarious. Metabolic products, as well as lysosomal constituents, are responsible. These activities differ to some extent in neutrophils, monocytes and macrophages.

The microbicidal activities of phagocytes are usually divided into oxygen-dependent and oxygen-independent events

Oxygen-independent activity

Lysosomal granules contain a variety of extremely basic proteins that strongly inhibit bacteria, yeasts and even some viruses. A few molecules of any one of these cationic proteins appear able to inactivate a bacterial cell by damage to their permeability barriers, but their exact modes of action are not known. The lysosomal granules of neutrophils contain lactoferrin, an extremely powerful iron-chelating agent, which withholds potential iron needed for bacterial growth. The pH of the phagolysosome may be as low as 4.0 due to accumulation of lactic acid, which is sufficiently acidic to prevent the growth of most pathogens. This acidic environment apparently optimizes the activity of many degradative lysosomal enzymes including lysozyme, glycosylases, phospholipases, and nucleases.

Oxygen-dependent activity

Liganding of Fc receptors (on neutrophils, monocytes or macrophages) and mannose receptors (on macrophages) increases their O2 uptake, called the respiratory burst or "oxygen burst". These receptors activate a membrane-bound NADPH oxidase that reduces O2 to O2- (superoxide). Superoxide can be reduced to OH. (hydroxyl radical) or dismutated to H2O2 (hydrogen peroxide) by superoxide dismutase. O2-, OH., and H2O2 are activated oxygen species that are potent oxidizing agents in biological systems which adversely affect a number of cellular structures including membranes and nucleic acids. Furthermore, at least in the case of neutrophils, these reactive oxygen intermediates can act in concert with a lysosomal enzyme called myeloperoxidase to function as the myeloperoxidase system, or MPO.

Myeloperoxidase is one of the lysosomal enzymes of neutrophils which is released into the phagocytic vacuole during fusion to form the phagolysosome. Myeloperoxidase uses H2O2 generated during the respiratory burst to catalyze halogenation (mainly chlorination) of microbes contained within the phagolysosome. Such halogenations are a potent mechanism for killing cells.

When the NADPH oxidase and myeloperoxidase systems are operating in concert, a series of reactions leading to lethal oxygenation and halogenation of engulfed microbes occurs.

Intracellular digestion

Dead microbes are rapidly degraded in phagolysosomes to low molecular-weight components. Various hydrolytic enzymes are involved including lysozyme, proteases, lipases, nucleases, and glycosylases. Neutrophils die and lyse after extended phagocytosis, killing, and digestion of bacterial cells. This makes up the characteristic properties of pus.

Macrophages egest digested debris and allow insertion of microbial antigenic components into the plasma membrane for presentation to lymphocytes in the immunological response.



Figure 2. Phagocytosis of Streptococcus pyogenes by a macrophage. CELLS alive!


Written and Edited by Kenneth Todar   University of Wisconsin-Madison   Department of Bacteriology. All rights reserved.