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Phagocytic response component of innate defense

Filed in IMMUNOLOGY by on January 2, 2015 0 Comments

What is phagocytosis?

Phagocytosis is the process by which a cell ingests or engulfs a material and encloses the material in a vacuole within its cytoplasm. After the material (e.g. bacterial cell) has been phagocytosed, phagocytes kill engulfed bacteria by releasing lysosomal enzymes into vacuole which contains the bacteria.

A phagocyte is a cell that is able to ingest (engulf), digest, and kill invading microorganisms and particulate matter. Phagocytes play an important role in host innate defense.

They are two classes of phagocytic white bloood cells

  1. Polymorphonuclear phagocytes (neutrophils)
  2. Mononuclear phagocytes (monocytes and macrophages)

Macrophages, neutrophils, and monocytes are the main phagocytic white blood cells our body has got; they phagocytose and kill bacteria. Macrophages can even phagocytose and destroy viruses.

You should understand that eosinophils can phagocytose, but their phagocytic ability is very minimal and relatively insignificant.

Role of phagocytosis in innate host defense

Phagocytes play an important role in innate immune defense, in the sense that they phagocytose microbes (mainly bacteria) even during the first entry of the bacteria into the host. During the first few days of an infection, the body’s main defense response is to activate phagocytes which phagocytose the pathogenic cells promptly.

Of the five human pathogens (bacteria, fungi, virus, worms, and protozoa) phagocytosis is most effective against bacterial pathogens. Parasites are killed in innate defense mechanism by eosinophils; but eosinophils do not kill parasites by phagocytosis.

Neutrophils (polymorphonuclear phagocytes)

Neutrophils contain lysosomal granules in their cytoplasm; these lysosomal granules contain bioactive compounds. These bioactive compounds constitute a full complement of bactericidal and digestive enzymes that destroy engulfed microbial cells. Among the bactericidal agents contained in the lysosomal granules are lysozyme, proteases, cathepsin, phospholipase, cationic proteins, lactoferrin and peroxidase. When a neutrophil engulfs a bacterial cell, it releases the above mentioned bactericidal chemicals on the engulfed cell.

Monocytes and Macrophages (mononuclear phagocytes)

Monocytes, the largest white blood cell in human body, make up 3-7 % of total white blood count. Monocytes are active phagocytic cells. They circulate in blood for 2 days or thereabout, after which they leave blood and migrate into tissues. In the tissues, monocytes enlarge, acquire more and larger lysosomal granules in their cytoplasm and become functionally more active cells called tissue macrophages. The lysosomal granules in the cytoplasm contain several hydrolytic enzymes. Macrophages are about five to ten times larger than monocytes. The phagocytic potential of macrophages is far greater than that of monocytes.

Generally, two types of macrophages are recognized:

  1. Wandering macrophages (circulating monocytes)
  2. Fixed macrophages (tissue macrophages)

Comparing the Phagocytic ability of neutrophils with that of macrophages

Whenever an invading microbe penetrates the body, neutrophils are the first to be attracted from blood into the infected tissue. Once they arrive at the infected tissue, they gather round the invading microbes and begin to phagocytose them. But neutrophils are short-lived; they survive in the tissue for only few days, and will most likely die after few rounds of phagocytosis. Interestingly enough, once a neutrophil leaves blood into a tissue, it never returns to circulation.

On the other hand, macrophages (both fixed tissue macrophages and blood-borne macrophages) will migrate to the infected tissue several hours after the arrival of neutrophils. The principal role of macrophages is to ‘eat up’ cellular debris and clean up the mess in the infected tissue. For instance, in the combat between neutrophils and invading microbes, a lot of cellular debris must have been generated; it is the responsibility of macrophages to phagocytose the debris formed from the dead cells.

Macrophages are capable of executing several multiple rounds of phagocytosis, and some of them even survive in body tissues for decades. With regards to their phagocytic ability, macrophages are far more powerful than neutrophils. Macrophages engulf particles that are too large for neutrophils to engulf. As a matter of fact macrophages can engulf virtually anything they are unable to recognize. Because of their role in phagocytosis, neutrophils and macrophages are sometimes called professional phagocytes.

The processes of phagocytosis

Phagocytosis, and the subsequent destruction of the engulfed microbial cell, constitutes a series of cellular events that occurs as follows:

  1. Recruitment of phagocytes to the infected tissue
  2. Adherence of phagocytes to invading microbe
  3. Engulfment of invading microbe and formation of phagosome
  4. Fusion of lysosomes to the phagosome to form phagolysosome
  5. Intracellular killing of engulfed microbe: lysosomes release enzymes that kill the invading microbe
  6. Intracellular digestion of killed microbe

The diagram below illustrates the main processes in phagocytosis.

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In phagocytosis, the phagocyte recognizes the pathogenic microbe; sends out pseudopodia towards the microbe. Pseudopodia encircle the microbe and engulf it. The engulfed microbial cell is enclosed in a vesicle called a phagosome. The phagosome fuses with lysosomes to form a phagolysosome. Fused lysosomes release their hydrolytic enzymes into the phagolysosomal vacuole; lysosomal enzymes digest the microbial cell into small fragments. This marks the end the microbial cell

Recruitment of phagocytes

You should recall that neutrophils are never present in a healthy tissue; they normally circulate in blood vessels. The presence of neutrophils in a tissue indicates that the tissue is either injured or infected. When a tissue is infected, one of the things the infected tissue does is to release inflammatory substances such as prostaglandins, histamine and kinins. These inflammatory substances cause inflammatory changes on local capillaries and venules of the infected tissue. They cause the local microvessels to become more permeable to plasma and cellular components of blood.

Also, certain components of the invading bacteria, and complement-derived peptides (C3a and C5a fragments of complement) generated in the site of infection are chemotactic agents that attract neutrophils and monocytes to the site of infection. All these chemical agents attract neutrophils and monocytes to the infected tissue causing the phagocytes to gather in large amounts around the infected tissue. The response of a cell to chemical stimuli is called chemotaxis.

Both, the increase in permeability of capillary of infected tissue and the generation of chemical agents that attract neutrophils and monocytes to the infected tissue will cause neutrophils and monocytes to cross vessel wall into the infected tissue. The process whereby cells migrate across vessel walls into tissues is called diapedesis. Thus, this is the processes that recruit neutrophils and monocytes into infected tissue.

It is important for you to note that two things are central to the recruiting of neutrophils and monocytes from blood into the infected tissues. The two things are:

  1. Generation of inflammatory mediators that cause increase in permeability of capillaries of infected tissue
  2. Generation of chemical agents that attract phagocytes to the infected tissue.

The two factors above are responsible for diapedesis.

Adherence to phagocytes

Most often, bacterial cells and viral cells may become coated with an opsonin. An opsonin is an antimicrobial substance that binds to a microbial cell to make the microbe more susceptible and palatable to phagocytes. Examples of opsonins are:

  1. C3b fragment of complement
  2. Mannose
  3. Fibronectin
  4. IgG

Microbial cells that are coated with one or more of the above listed compounds are said to be opsonized. Phagocytes carry specific receptors for opsonins. Thus, the binding of an opsonin to a microbial cell surface dramatically increases the chances phagocytes to bind to the microbe, (using its receptors for the bound opsonin) and ingest the microbe. Phagocytes may adhere to non-opsonized microbial cells by non-specific attractions, but they have greater affinity for opsonized microbes, and actually adhere more strongly to microbes that are bound by opsonins. Thus, opsonins dramatically increase the chances of a phagocyte to bind to an invading microbe.

Formation of phagosome

Shortly after the adherence of phagocytes to a microbial cell, the phagocyte sends out some pseudopodia-like processes that entrap and completely encircle the microbe. After this, the microbe is delivered into the cytoplasm of the phagocyte as a membrane-bound vesicle called phagosome. I want you to see the phagosome as a vesicle that contains the engulfed microbe. You should understand that the vesicle which encloses the microbe is derived from the membrane of the phagocyte. With current knowledge, the molecular processes that underlie these changes are yet to be clearly understood.

Formation of a phagolysosome

Our phagosome remains the membrane-enclosed microbe within the cytoplasm of the phagocyte. Once the phagosome is in the cytoplasm, many lysosomes migrate towards the phagosome and eventually attach themselves to the phagosome to form a vacuole called phagolysosome. (You can go back to the illustration on this page for clarity). Lysosomes release a host of microbicidal substances into the phagolysosomal vacuole to degrade the ingested microbe.

Some of the microbicidal substances released by lysosomes of phagocytes are peroxidase, hydrolytic enzymes, cationic proteins, hydrolases, cathepsin, lysozymes, and proteases. These microbicidal substances are cytolytic and can equally kill the phagocytes if the digestion process is left uncontrolled. In order for phagocytes to spare themselves of the cytolytic effects of their own lysosomal enzymes, phagocytes ensure that all lysosomal enzymes are released into the phagolysosomal vacuole only. In other words, all lysosomal enzymes are only released into the phagolysosome, and never into the cytoplasm of the phagocyte.

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Intracellular killing of the engulfed microbe

The cytotoxic mechanisms employed by phagocytes in killing engulfed microbes are wide, complex and multi-dimensional. However, it can be grouped into two broad divisions as follows:

  1. Intracellular killing by lysosomal enzymes, also regarded as oxygen-independent mechanism
  2. Intracellular killing by metabolic products of phagocytes, also regarded as oxygen-dependent mechanism

Lysosomal enzymes (oxygen-independent mechanisms)

Lysosomal granules of neutrophils are particularly rich in lactoferrin. Lactoferrin is a powerful iron-binding compound that binds iron, and deprives bacteria of essential iron needed for their growth. Thus, one cytotoxic mechanism of phagocytes against microbes is to bind iron and deprive bacterial cells of this necessary nutrient.

Lysosomal granules of phagocytes (both neutrophils and macrophages) contain a host of extremely toxic antimicrobial proteins that strongly inactivate bacterial cells. The most common cytotoxic mechanism employed by these microbicidal proteins is to cause extreme and severe damage to components of microbial cell membrane, and this makes the microbe to be more permeable to water. This results in the loss of osmotic balance by the microbial cell. Thus, another cytotoxic mechanism of phagocytes is to cause severe damage to microbial cell membrane.

The contents of lysosomes are mainly acidic, this makes the pH of the phagolysosome to remain as low as 4.0. The resulting acidic environment created within the phagolysosomal vacuole prevents the growth of most microbes. Another powerful cytotoxic mechanism is to keep the pH of the phagolysosomal vacuole very acidic.

Metabolic products of phagocytes (oxygen-dependent mechanisms)

Whenever a phagocyte binds a microbe, the cell-membrane bound enzyme NADPH oxidase is activated. The activation of NADPH oxidase is associated with a sudden and sharp rise in oxygen consumption by phagocytes. This phenomenon is termed respiratory burst. The increase in metabolic rate and oxygen, which results from the activation of NADPH, oxidase causes the generation toxic oxygen metabolites O2-, OH, and H2O2.

O2-, OH, and H2O2 are all toxic oxidants that denatures membrane proteins and cause lipid peroxidation. The destruction of both lipid and protein components of the microbial cell membrane marks the end of the pathogenic microbe.

In addition to the toxic oxygen molecules, neutrophils also discharge the enzyme myeloperoxidase into the phagolysosomal vacuole. In the phagolysosomal vacuole, myeloperoxidase catalyzes the conversion of halide ions (Cl, Br, I) to their corresponding acids (HOCl, HOBr, etc), this reaction is termed halogenation. HOCl is the main product formed since chloride is the most abundant halide ion in body fluids. HOCl, HOBr, etc are all potent oxidant acids that cause oxidative damage to cell wall of microbial cells. These acids also contribute to keeping the pH of the interior of phagolysosomal vacuoles very acidic. Therefore, respiratory burst and halogenation remain the oxygen dependent cell-killing mechanisms employed by phagocytes.

Intracellular digestion of killed microbe and the aftermath

After phagocytosis and intracellular killing of engulfed microbes, dead microbial cells immediately undergo what is called intracellular digestion. In intracellular digestion, hydrolytic enzymes such as lysozymes, lipases, nucleases, proteases all act on, and degrade dead microbes to smaller fragments/debris. After the intracellular digestion, neutrophils and macrophages handle the products of microbial cell digestion in two distinct ways.

Neutrophils simply egest the breakdown products of the microbial cell into the surrounding tissue by exocytosis. Macrophages equally egest the debris, but they process the antigens derived from the degraded pathogenic microbe and display these antigens on a special membrane protein called Class-II Major Histocompatibility complex display foreign antigens, derived from the degraded microbe, on their membrane for CD4+ T-cells. This is why macrophages are called antigen presenting cells.

The process of displaying foreign antigens on Class-II MHC proteins is both essential and necessary in the acquired immune response. Neutrophils are unable to present antigens because they lack class II Major Histocompatibility complex (MHC) proteins on their membrane, whereas macrophages express class II MHC proteins.

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