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11.4: Pathogen Recognition and Phagocytosis - Biology

11.4: Pathogen Recognition and Phagocytosis - Biology



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Skills to Develop

  • Explain how leukocytes migrate from peripheral blood into infected tissues
  • Explain the mechanisms by which leukocytes recognize pathogens
  • Explain the process of phagocytosis and the mechanisms by which phagocytes destroy and degrade pathogens

Several of the cell types discussed in the previous section can be described as phagocytes—cells whose main function is to seek, ingest, and kill pathogens. This process, called phagocytosis, was first observed in starfish in the 1880s by Nobel Prize-winning zoologist Ilya Metchnikoff (1845–1916), who made the connection to white blood cells (WBCs) in humans and other animals. At the time, Pasteur and other scientists believed that WBCs were spreading pathogens rather than killing them (which is true for some diseases, such as tuberculosis). But in most cases, phagocytes provide a strong, swift, and effective defense against a broad range of microbes, making them a critical component of innate nonspecific immunity. This section will focus on the mechanisms by which phagocytes are able to seek, recognize, and destroy pathogens.

Extravasation (Diapedesis) of Leukocytes

Some phagocytes are leukocytes (WBCs) that normally circulate in the bloodstream. To reach pathogens located in infected tissue, leukocytes must pass through the walls of small capillary blood vessels within tissues. This process, called extravasation, or diapedesis, is initiated by complement factor C5a, as well as cytokines released into the immediate vicinity by resident macrophages and tissue cells responding to the presence of the infectious agent (Figure (PageIndex{1})). Similar to C5a, many of these cytokines are proinflammatory and chemotactic, and they bind to cells of small capillary blood vessels, initiating a response in the endothelial cells lining the inside of the blood vessel walls. This response involves the upregulation and expression of various cellular adhesion molecules and receptors. Leukocytes passing through will stick slightly to the adhesion molecules, slowing down and rolling along the blood vessel walls near the infected area. When they reach a cellular junction, they will bind to even more of these adhesion molecules, flattening out and squeezing through the cellular junction in a process known as transendothelial migration. This mechanism of “rolling adhesion” allows leukocytes to exit the bloodstream and enter the infected areas, where they can begin phagocytosing the invading pathogens.

Note that extravasation does not occur in arteries or veins. These blood vessels are surrounded by thicker, multilayer protective walls, in contrast to the thin single-cell-layer walls of capillaries. Furthermore, the blood flow in arteries is too turbulent to allow for rolling adhesion. Also, some leukocytes tend to respond to an infection more quickly than others. The first to arrive typically are neutrophils, often within hours of a bacterial infection. By contract, monocytes may take several days to leave the bloodstream and differentiate into macrophages.

Figure (PageIndex{1}): Damaged cells and macrophages that have ingested pathogens release cytokines that are proinflammatory and chemotactic for leukocytes. In addition, activation of complement at the site of infection results in production of the chemotactic and proinflammatory C5a. Leukocytes exit the blood vessel and follow the chemoattractant signal of cytokines and C5a to the site of infection. Granulocytes such as neutrophils release chemicals that destroy pathogens. They are also capable of phagocytosis and intracellular killing of bacterial pathogens.

Watch the following videos on leukocyte extravasation and leukocyte rolling to learn more.

Exercise (PageIndex{1})

Explain the role of adhesion molecules in the process of extravasation.

As described in the previous section, opsonization of pathogens by antibody; complement factors C1q, C3b, and C4b; and lectins can assist phagocytic cells in recognition of pathogens and attachment to initiate phagocytosis. However, not all pathogen recognition is opsonin dependent. Phagocytes can also recognize molecular structures that are common to many groups of pathogenic microbes. Such structures are called pathogen-associated molecular patterns (PAMPs). Common PAMPs include the following:

  • peptidoglycan, found in bacterial cell walls;
  • flagellin, a protein found in bacterial flagella;
  • lipopolysaccharide (LPS) from the outer membrane of gram-negative bacteria;
  • lipopeptides, molecules expressed by most bacteria; and
  • nucleic acids such as viral DNA or RNA.

Like numerous other PAMPs, these substances are integral to the structure of broad classes of microbes.

The structures that allow phagocytic cells to detect PAMPs are called pattern recognition receptors (PRRs). One group of PRRs is the toll-like receptors (TLRs), which bind to various PAMPs and communicate with the nucleus of the phagocyte to elicit a response. Many TLRs (and other PRRs) are located on the surface of a phagocyte, but some can also be found embedded in the membranes of interior compartments and organelles (Figure (PageIndex{2})). These interior PRRs can be useful for the binding and recognition of intracellular pathogens that may have gained access to the inside of the cell before phagocytosis could take place. Viral nucleic acids, for example, might encounter an interior PRR, triggering production of the antiviral cytokine interferon.

In addition to providing the first step of pathogen recognition, the interaction between PAMPs and PRRs on macrophages provides an intracellular signal that activates the phagocyte, causing it to transition from a dormant state of readiness and slow proliferation to a state of hyperactivity, proliferation, production/secretion of cytokines, and enhanced intracellular killing. PRRs on macrophages also respond to chemical distress signals from damaged or stressed cells. This allows macrophages to extend their responses beyond protection from infectious diseases to a broader role in the inflammatory response initiated from injuries or other diseases.

Figure (PageIndex{2}): Phagocytic cells contain pattern recognition receptors (PRRs) capable of recognizing various pathogen-associated molecular patterns (PAMPs). These PRRs can be found on the plasma membrane or in internal phagosomes. When a PRR recognizes a PAMP, it sends a signal to the nucleus that activates genes involved in phagocytosis, cellular proliferation, production and secretion of antiviral interferons and proinflammatory cytokines, and enhanced intracellular killing.

Exercise (PageIndex{2})

  1. Name four pathogen-associated molecular patterns (PAMPs).
  2. Describe the process of phagocyte activation.

Once pathogen recognition and attachment occurs, the pathogen is engulfed in a vesicle and brought into the internal compartment of the phagocyte in a process called phagocytosis (Figure (PageIndex{3})). PRRs can aid in phagocytosis by first binding to the pathogen’s surface, but phagocytes are also capable of engulfing nearby items even if they are not bound to specific receptors. To engulf the pathogen, the phagocyte forms a pseudopod that wraps around the pathogen and then pinches it off into a membrane vesicle called a phagosome. Acidification of the phagosome (pH decreases to the range of 4–5) provides an important early antibacterial mechanism. The phagosome containing the pathogen fuses with one or more lysosomes, forming a phagolysosome. Formation of the phagolysosome enhances the acidification, which is essential for activation of pH-dependent digestive lysosomal enzymes and production of hydrogen peroxide and toxic reactive oxygen species. Lysosomal enzymes such as lysozyme, phospholipase, and proteases digest the pathogen. Other enzymes are involved a respiratory burst. During the respiratory burst, phagocytes will increase their uptake and consumption of oxygen, but not for energy production. The increased oxygen consumption is focused on the production of superoxide anion, hydrogen peroxide, hydroxyl radicals, and other reactive oxygen species that are antibacterial.

In addition to the reactive oxygen species produced by the respiratory burst, reactive nitrogen compounds with cytotoxic (cell-killing) potential can also form. For example, nitric oxide can react with superoxide to form peroxynitrite, a highly reactive nitrogen compound with degrading capabilities similar to those of the reactive oxygen species. Some phagocytes even contain an internal storehouse of microbicidal defensin proteins (e.g., neutrophil granules). These destructive forces can be released into the area around the cell to degrade microbes externally. Neutrophils, especially, can be quite efficient at this secondary antimicrobial mechanism.

Once degradation is complete, leftover waste products are excreted from the cell in an exocytic vesicle. However, it is important to note that not all remains of the pathogen are excreted as waste. Macrophages and dendritic cells are also antigen-presenting cells involved in the specific adaptive immune response. These cells further process the remains of the degraded pathogen and present key antigens (specific pathogen proteins) on their cellular surface. This is an important step for stimulation of some adaptive immune responses, as will be discussed in more detail in the next chapter.

Figure (PageIndex{3}): The stages of phagocytosis include the engulfment of a pathogen, the formation of a phagosome, the digestion of the pathogenic particle in the phagolysosome, and the expulsion of undigested materials from the cell.

Visit this link to view a phagocyte chasing and engulfing a pathogen.

Exercise (PageIndex{3})

What is the difference between a phagosome and a lysosome?

WHEN PHAGOCYTOSIS FAILS

Although phagocytosis successfully destroys many pathogens, some are able to survive and even exploit this defense mechanism to multiply in the body and cause widespread infection. Protozoans of the genus Leishmania are one example. These obligate intracellular parasites are flagellates transmitted to humans by the bite of a sand fly. Infections cause serious and sometimes disfiguring sores and ulcers in the skin and other tissues (Figure (PageIndex{4})). Worldwide, an estimated 1.3 million people are newly infected with leishmaniasis annually.1

Salivary peptides from the sand fly activate host macrophages at the site of their bite. The classic or alternate pathway for complement activation ensues with C3b opsonization of the parasite. Leishmania cells are phagocytosed, lose their flagella, and multiply in a form known as an amastigote (Leishman-Donovan body) within the phagolysosome. Although many other pathogens are destroyed in the phagolysosome, survival of the Leishmania amastigotes is maintained by the presence of surface lipophosphoglycan and acid phosphatase. These substances inhibit the macrophage respiratory burst and lysosomal enzymes. The parasite then multiplies inside the cell and lyses the infected macrophage, releasing the amastigotes to infect other macrophages within the same host. Should another sand fly bite an infected person, it might ingest amastigotes and then transmit them to another individual through another bite.

There are several different forms of leishmaniasis. The most common is a localized cutaneous form of the illness caused by L. tropica, which typically resolves spontaneously over time but with some significant lymphocyte infiltration and permanent scarring. A mucocutaneous form of the disease, caused by L. viannia brasilienfsis, produces lesions in the tissue of the nose and mouth and can be life threatening. A visceral form of the illness can be caused by several of the different Leishmania species. It affects various organ systems and causes abnormal enlargement of the liver and spleen. Irregular fevers, anemia, liver dysfunction, and weight loss are all signs and symptoms of visceral leishmaniasis. If left untreated, it is typically fatal.

Figure (PageIndex{4}): (a) Cutaneous leishmaniasis is a disfiguring disease caused by the intracellular flagellate Leishmania tropica, transmitted by the bite of a sand fly. (b) This light micrograph of a sample taken from a skin lesion shows a large cell, which is a macrophage infected with L. tropica amastigotes (arrows). The amastigotes have lost their flagella but their nuclei are visible. Soon the amastigotes will lyse the macrophage and be engulfed by other phagocytes, spreading the infection. (credit a: modification of work by Otis Historical Archives of “National Museum of Health & Medicine”; credit b: modification of work by Centers for Disease Control and Prevention)

Key Concepts and Summary

  • Phagocytes are cells that recognize pathogens and destroy them through phagocytosis.
  • Recognition often takes place by the use of phagocyte receptors that bind molecules commonly found on pathogens, known as pathogen-associated molecular patterns (PAMPs).
  • The receptors that bind PAMPs are called pattern recognition receptors, or PRRs. Toll-like receptors (TLRs) are one type of PRR found on phagocytes.
  • Extravasation of white blood cells from the bloodstream into infected tissue occurs through the process of transendothelial migration.
  • Phagocytes degrade pathogens through phagocytosis, which involves engulfing the pathogen, killing and digesting it within a phagolysosome, and then excreting undigested matter.

Multiple Choice

PAMPs would be found on the surface of which of the following?

A. pathogen
B. phagocyte
C. skin cell
D. blood vessel wall

A

________ on phagocytes bind to PAMPs on bacteria, which triggers the uptake and destruction of the bacterial pathogens?

A. PRRs
B. AMPs
C. PAMPs
D. PMNs

A

Which of the following best characterizes the mode of pathogen recognition for opsonin-dependent phagocytosis?

A. Opsonins produced by a pathogen attract phagocytes through chemotaxis.
B. A PAMP on the pathogen’s surface is recognized by a phagocyte’s toll-like receptors.
C. A pathogen is first coated with a molecule such as a complement protein, which allows it to be recognized by phagocytes.
D. A pathogen is coated with a molecule such as a complement protein that immediately lyses the cell.

C

Fill in the Blank

________, also known as diapedesis, refers to the exit from the bloodstream of neutrophils and other circulating leukocytes.

extravasation

Toll-like receptors are examples of ________.

pattern-recognition receptors (PRRs)

Short Answer

Briefly summarize the events leading up to and including the process of transendothelial migration.

Contributor

  • Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University) with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free at https://openstax.org/books/microbiology/pages/1-introduction)


The function of phagocytosis is to ingest solid particles into the cell. Phagocytosis is a type of endocytosis, which is when cells ingest molecules via active transport as opposed to molecules passively diffusing through a cell membrane. Only certain small molecules can pass through the cell membrane easily larger ones have to go through special channels in the cell or be ingested via endocytosis. Other types of endocytosis include pinocytosis, also called “cell drinking”, and receptor-mediated endocytosis, which is when molecules bind to specific receptors on the cell membrane that causes the cell to engulf them.

Phagocytosis is different from pinocytosis because phagocytosis involves the ingestion of solid particles while pinocytosis is the ingestion of liquid droplets. Phagocytosis is also used by cells to take in much larger particles than those that are ingested through pinocytosis. Some single-celled protists, such as amoebae, use phagocytosis to ingest food particles it is literally how they eat food. Since their entire body consists of one cell, they can ingest food particles through engulfing them, and then digest these particles by connecting with a lysosome. In pinocytosis, the particles that are engulfed do not need to be broken down by a lysosome because they are so small, and instead the vesicle empties its contents directly into the cell.


Phagocytosis Process

Living cells take in different types of material across their cell membrane. For the most part, a majority of these material/molecules such as ions, fluids, and oxygen among others easily pass through the membrane through such mechanisms as ion pumps and osmosis among others.

Some of the matter, e.g. particles like viruses, may prove too large to pass through the membrane through such mechanisms. For this reason, the cell has to engulf such matter/objects into the cell.

This process involves the invagination of the cell membrane in question which allows the cell to take in the object/particle. Depending on the cell and the mechanism used to engulf such material/objects, endocytosis is divided into phagocytosis, pinocytosis and another process known as receptor-mediated endocytosis.

What differentiates phagocytosis from pinocytosis is that phagocytes posses special surface proteins that allow them to specifically identify and bind to given particles before ingesting them. This type of endocytosis is dependent on the binding between the cell and the target object/particle.


TAM receptors are dispensable in the phagocytosis and killing of bacteria

Many receptors that are employed for the engulfment of apoptotic cells are also used for the recognition and phagocytosis of bacteria. Tyro3, Axl, and Mertk (TAM) are important in the phagocytosis of apoptotic cells by macrophages. Animals lacking these receptors are hypersensitive to bacterial products. In this report, we examine whether the TAM receptors are involved in the phagocytosis of bacteria. We found that macrophages lacking Mertk, Axl, Tyro3 or all three receptors were equally efficient in the phagocytosis of Gram-negative E. coli. Similarly, the phagocytosis of E. coli and Gram-positive S. aureus bioparticles by macrophages lacking TAM receptors was equal to wild-type. In addition, we found that Mertk did not play a role in killing of extracellular E. coli or the replication status of intracellular Francisella tularensis. Thus, while TAM receptors may regulate signal transduction to bacterial components, they are not essential for the phagocytosis and killing of bacteria.

Figures

Figure 1. TAM receptors are not required…

Figure 1. TAM receptors are not required for the phagocytosis of E. coli O111:B4 GFP…

Figure 2. Phagocytosis of E coli O…

Figure 2. Phagocytosis of E coli O 111:B4 is time dependent and Mertk independent in…

Figure 3. Phagocytosis of E. coli O…

Figure 3. Phagocytosis of E. coli O 111:B4 is similar in thioglycollate and resident peritoneal…

Figure 4. In vitro phagocytosis assay by…

Figure 4. In vitro phagocytosis assay by flow cytometry

Figure 5. Lack of affect in mertk…

Figure 5. Lack of affect in mertk −/− macrophages is not dependent on Gram status


Mechanisms of Fc Receptor and Dectin-1 Activation for Phagocytosis

Phagocytosis is a key cellular process, both during homeostasis and upon infection or tissue damage. Receptors on the surface of professional phagocytic cells bind to target particles either directly or through opsonizing ligands, and trigger actin-mediated ingestion of the particles. The process must be carefully controlled to ensure that phagocytosis is triggered efficiently and specifically, and that the antimicrobial cytotoxic responses that often accompany it are initiated only when required. In this review, we will describe and compare the molecular mechanisms that regulate phagocytosis triggered by Fcγ receptors, which mediate the uptake of immunoglobulin G-opsonized targets, and Dectin-1, which is responsible for internalization of fungi with exposed cell wall β-glucan. We will examine how these receptors detect their ligands, how signal transduction is initiated and regulated, and how internalization is instructed to achieve rapid and yet controlled uptake of their targets.


Contents

Phagocytosis was first noted by Canadian physician William Osler (1876), [1] and later studied and named by Élie Metchnikoff (1880, 1883). [2]

Phagocytosis is one main mechanisms of the innate immune defense. It is one of the first processes responding to infection, and is also one of the initiating branches of an adaptive immune response. Although most cells are capable of phagocytosis, some cell types perform it as part of their main function. These are called 'professional phagocytes.' Phagocytosis is old in evolutionary terms, being present even in invertebrates. [3]

Professional phagocytic cells Edit

Neutrophils, macrophages, monocytes, dendritic cells, osteoclasts and eosinophils can be classified as professional phagocytes. [2] The first three have the greatest role in immune response to most infections. [3]

The role of neutrophils is patrolling the bloodstream and rapid migration to the tissues in large numbers only in case of infection. [3] There they have direct microbicidal effect by phagocytosis. After ingestion, neutrophils are efficient in intracellular killing of pathogens. Neutrophils phagocytose mainly via the Fcγ receptors and complement receptors 1 and 3. The microbicidal effect of neutrophils is due to a large repertoire of molecules present in pre-formed granules. Enzymes and other molecules prepared in these granules are proteases, such as collagenase, gelatinase or serine proteases, myeloperoxidase, lactoferrin and antibiotic proteins. Degranulation of these into the phagosome, accompanied by high reactive oxygen species production (oxidative burst) is highly microbicidal. [4]

Monocytes, and the macrophages that mature from them, leave blood circulation to migrate through tissues. There they are resident cells and form a resting barrier. [3] Macrophages initiate phagocytosis by mannose receptors, scavenger receptors, Fcγ receptors and complement receptors 1, 3 and 4. Macrophages are long-lived and can continue phagocytosis by forming new lysosomes. [3] [5]

Dendritic cells also reside in tissues and ingest pathogens by phagocytosis. Their role is not killing or clearance of microbes, but rather breaking them down for antigen presentation to the cells of the adaptive immune system. [3]

Initiating receptors Edit

Receptors for phagocytosis can be divided into two categories by recognised molecules. The first, opsonic receptors, are dependent on opsonins. [6] Among these are receptors that recognise the Fc part of bound IgG antibodies, deposited complement or receptors, that recognise other opsonins of cell or plasma origin. Non-opsonic receptors include lectin-type receptors, Dectin receptor, or scavenger receptors. Some phagocytic pathways require a second signal from pattern recognition receptors (PRRs) activated by attachment to pathogen-associated molecular patterns (PAMPS), which leads to NF-κB activation. [2]

Fcγ receptors Edit

Fcγ receptors recognise IgG coated targets. The main recognised part is the Fc fragment. The molecule of the receptor contain an intracellular ITAM domain or associates with an ITAM-containing adaptor molecule. ITAM domains transduce the signal from the surface of the phagocyte to the nucleus. For example, activating receptors of human macrophages are FcγRI, FcγRIIA, and FcγRIII. [5] Fcγ receptor mediated phagocytosis includes formation of protrusions of the cell called a 'phagocytic cup' and activates an oxidative burst in neutrophils. [4]

Complement receptors Edit

These receptors recognise targets coated in C3b, C4b and C3bi from plasma complement. The extracellular domain of the receptors contains a lectin-like complement-binding domain. Recognition by complement receptors is not enough to cause internalisation without additional signals. In macrophages, the CR1, CR3 and CR4 are responsible for recognition of targets. Complement coated targets are internalised by 'sinking' into the phagocyte membrane, without any protrusions. [5]

Mannose receptors Edit

Mannose and other pathogen-associated sugars, such as fucose, are recognised by the mannose receptor. Eight lectin-like domains form the extracellular part of the receptor. The ingestion mediated by the mannose receptor is distinct in molecular mechanisms from Fcγ receptor or complement receptor mediated phagocytosis. [5]

Phagosome Edit

Engulfment of material is facilitated by the actin-myosin contractile system. The phagosome is the organelle formed by phagocytosis of material. It then moves toward the centrosome of the phagocyte and is fused with lysosomes, forming a phagolysosome and leading to degradation. Progressively, the phagolysosome is acidified, activating degradative enzymes. [2] [7]

Degradation can be oxygen-dependent or oxygen-independent.

  • Oxygen-dependent degradation depends on NADPH and the production of reactive oxygen species. Hydrogen peroxide and myeloperoxidase activate a halogenating system, which leads to the creation of hypochlorite and the destruction of bacteria. [8]
  • Oxygen-independent degradation depends on the release of granules, containing enzymes such as lysozymes, and cationic proteins such as defensins. Other antimicrobial peptides are present in these granules, including lactoferrin, which sequesters iron to provide unfavourable growth conditions for bacteria. Other enzymes like hyaluronidase, lipase, collagenase, elastase, ribonuclease, deoxyribonuclease also play an important role in preventing the spread of infection and degradation of essential microbial biomolecules leading to cell death. [4][5]

Leukocytes generate hydrogen cyanide during phagocytosis, and can kill bacteria, fungi, and other pathogens by generating several other toxic chemicals. [9] [10] [11]

Some bacteria, for example Treponema pallidum, Escheria coli and Staphylococcus aureus, are able to avoid phagocytosis by several mechanisms.

Following apoptosis, the dying cells need to be taken up into the surrounding tissues by macrophages in a process called efferocytosis. One of the features of an apoptotic cell is the presentation of a variety of intracellular molecules on the cell surface, such as calreticulin, phosphatidylserine (from the inner layer of the plasma membrane), annexin A1, oxidised LDL and altered glycans. [12] These molecules are recognised by receptors on the cell surface of the macrophage such as the phosphatidylserine receptor or by soluble (free-floating) receptors such as thrombospondin 1, GAS6, and MFGE8, which themselves then bind to other receptors on the macrophage such as CD36 and alpha-v beta-3 integrin. Defects in apoptotic cell clearance is usually associated with impaired phagocytosis of macrophages. Accumulation of apoptotic cell remnants often causes autoimmune disorders thus pharmacological potentiation of phagocytosis has a medical potential in treatment of certain forms of autoimmune disorders. [13] [14] [15] [16]

In many protists, phagocytosis is used as a means of feeding, providing part or all of their nourishment. This is called phagotrophic nutrition, distinguished from osmotrophic nutrition which takes place by absorption. [ citation needed ]

  • In some, such as amoeba, phagocytosis takes place by surrounding the target object with pseudopods, as in animal phagocytes. In humans, the amoebozoan Entamoeba histolytica can phagocytose red blood cells. also engage in phagocytosis. [17] In ciliates there is a specialized groove or chamber in the cell where phagocytosis takes place, called the cytostome or mouth.

As in phagocytic immune cells, the resulting phagosome may be merged with lysosomes(food vacuoles) containing digestive enzymes, forming a phagolysosome. The food particles will then be digested, and the released nutrients are diffused or transported into the cytosol for use in other metabolic processes. [18]

Mixotrophy can involve phagotrophic nutrition and phototrophic nutrition. [19]


Cytokine Release Effect

The binding of PRRs with PAMPs triggers the release of cytokines, which signal that a pathogen is present and needs to be destroyed along with any infected cells. A cytokine is a chemical messenger that regulates cell differentiation (form and function), proliferation (production), and gene expression to affect immune responses. At least 40 types of cytokines exist in humans that differ in terms of the cell type that produces them, the cell type that responds to them, and the changes they produce. One type cytokine, interferon, is illustrated in Figure 2.

Figure 2. Interferons are cytokines that are released by a cell infected with a virus. Response of neighboring cells to interferon helps stem the infection.

One subclass of cytokines is the interleukin (IL), so named because they mediate interactions between leukocytes (white blood cells). Interleukins are involved in bridging the innate and adaptive immune responses. In addition to being released from cells after PAMP recognition, cytokines are released by the infected cells which bind to nearby uninfected cells and induce those cells to release cytokines, which results in a cytokine burst.

A second class of early-acting cytokines is interferons, which are released by infected cells as a warning to nearby uninfected cells. One of the functions of an interferon is to inhibit viral replication. They also have other important functions, such as tumor surveillance. Interferons work by signaling neighboring uninfected cells to destroy RNA and reduce protein synthesis, signaling neighboring infected cells to undergo apoptosis (programmed cell death), and activating immune cells.

In response to interferons, uninfected cells alter their gene expression, which increases the cells’ resistance to infection. One effect of interferon-induced gene expression is a sharply reduced cellular protein synthesis. Virally infected cells produce more viruses by synthesizing large quantities of viral proteins. Thus, by reducing protein synthesis, a cell becomes resistant to viral infection.


18.5 Vaccines

For many diseases, prevention is the best form of treatment, and few strategies for disease prevention are as effective as vaccination. Vaccination is a form of artificial immunity. By artificially stimulating the adaptive immune defenses, a vaccine triggers memory cell production similar to that which would occur during a primary response . In so doing, the patient is able to mount a strong secondary response upon exposure to the pathogen—but without having to first suffer through an initial infection. In this section, we will explore several different kinds of artificial immunity along with various types of vaccines and the mechanisms by which they induce artificial immunity.

Classifications of Adaptive Immunity

All forms of adaptive immunity can be described as either active or passive. Active immunity refers to the activation of an individual’s own adaptive immune defenses, whereas passive immunity refers to the transfer of adaptive immune defenses from another individual or animal. Active and passive immunity can be further subdivided based on whether the protection is acquired naturally or artificially.

Natural active immunity is adaptive immunity that develops after natural exposure to a pathogen (Figure 18.24). Examples would include the lifelong immunity that develops after recovery from a chickenpox or measles infection (although an acute infection is not always necessary to activate adaptive immunity). The length of time that an individual is protected can vary substantially depending upon the pathogen and antigens involved. For example, activation of adaptive immunity by protein spike structures during an intracellular viral infection can activate lifelong immunity, whereas activation by carbohydrate capsule antigens during an extracellular bacterial infection may activate shorter-term immunity.

Natural passive immunity involves the natural passage of antibodies from a mother to her child before and after birth. IgG is the only antibody class that can cross the placenta from mother’s blood to the fetal blood supply. Placental transfer of IgG is an important passive immune defense for the infant, lasting up to six months after birth. Secretory IgA can also be transferred from mother to infant through breast milk .

Artificial passive immunity refers to the transfer of antibodies produced by a donor (human or animal) to another individual. This transfer of antibodies may be done as a prophylactic measure (i.e., to prevent disease after exposure to a pathogen) or as a strategy for treating an active infection. For example, artificial passive immunity is commonly used for post-exposure prophylaxis against rabies , hepatitis A, hepatitis B , and chickenpox (in high risk individuals). Active infections treated by artificial passive immunity include cytomegalovirus infections in immunocompromised patients and Ebola virus infections. In 1995, eight patients in the Democratic Republic of the Congo with active Ebola infections were treated with blood transfusions from patients who were recovering from Ebola. Only one of the eight patients died (a 12.5% mortality rate), which was much lower than the expected 80% mortality rate for Ebola in untreated patients. 2 Artificial passive immunity is also used for the treatment of diseases caused by bacterial toxins, including tetanus , botulism , and diphtheria .

Artificial active immunity is the foundation for vaccination . It involves the activation of adaptive immunity through the deliberate exposure of an individual to weakened or inactivated pathogens, or preparations consisting of key pathogen antigens.

Check Your Understanding

  • What is the difference between active and passive immunity?
  • What kind of immunity is conferred by a vaccine?

Herd Immunity

The four kinds of immunity just described result from an individual’s adaptive immune system. For any given disease, an individual may be considered immune or susceptible depending on his or her ability to mount an effective immune response upon exposure. Thus, any given population is likely to have some individuals who are immune and other individuals who are susceptible. If a population has very few susceptible individuals, even those susceptible individuals will be protected by a phenomenon called herd immunity . Herd immunity has nothing to do with an individual’s ability to mount an effective immune response rather, it occurs because there are too few susceptible individuals in a population for the disease to spread effectively.

Vaccination programs create herd immunity by greatly reducing the number of susceptible individuals in a population. Even if some individuals in the population are not vaccinated, as long as a certain percentage is immune (either naturally or artificially), the few susceptible individuals are unlikely to be exposed to the pathogen. However, because new individuals are constantly entering populations (for example, through birth or relocation), vaccination programs are necessary to maintain herd immunity.

Eye on Ethics

Vaccination: Obligation or Choice

A growing number of parents are choosing not to vaccinate their children. They are dubbed “ antivaxxers ,” and the majority of them believe that vaccines are a cause of autism (or other disease conditions), a link that has now been thoroughly disproven. Others object to vaccines on religious or moral grounds (e.g., the argument that Gardasil vaccination against HPV may promote sexual promiscuity), on personal ethical grounds (e.g., a conscientious objection to any medical intervention), or on political grounds (e.g., the notion that mandatory vaccinations are a violation of individual liberties). 3

It is believed that this growing number of unvaccinated individuals has led to new outbreaks of whooping cough and measles . We would expect that herd immunity would protect those unvaccinated in our population, but herd immunity can only be maintained if enough individuals are being vaccinated.

Vaccination is clearly beneficial for public health. But from the individual parent’s perspective the view can be murkier. Vaccines, like all medical interventions, have associated risks, and while the risks of vaccination may be extremely low compared to the risks of infection, parents may not always understand or accept the consensus of the medical community. Do such parents have a right to withhold vaccination from their children? Should they be allowed to put their children—and society at large—at risk?

Many governments insist on childhood vaccinations as a condition for entering public school, but it has become easy in most states to opt out of the requirement or to keep children out of the public system. Since the 1970s, West Virginia and Mississippi have had in place a stringent requirement for childhood vaccination, without exceptions, and neither state has had a case of measles since the early 1990s. California lawmakers recently passed a similar law in response to a measles outbreak in 2015, making it much more difficult for parents to opt out of vaccines if their children are attending public schools. Given this track record and renewed legislative efforts, should other states adopt similarly strict requirements?

What role should health-care providers play in promoting or enforcing universal vaccination? Studies have shown that many parents’ minds can be changed in response to information delivered by health-care workers, but is it the place of health-care workers to try to persuade parents to have their children vaccinated? Some health-care providers are understandably reluctant to treat unvaccinated patients. Do they have the right to refuse service to patients who decline vaccines? Do insurance companies have the right to deny coverage to antivaxxers? These are all ethical questions that policymakers may be forced to address as more parents skirt vaccination norms.

Variolation and Vaccination

Thousands of years ago, it was first recognized that individuals who survived a smallpox infection were immune to subsequent infections. The practice of inoculating individuals to actively protect them from smallpox appears to have originated in the 10 th century in China, when the practice of variolation was described (Figure 18.25). Variolation refers to the deliberate inoculation of individuals with infectious material from scabs or pustules of smallpox victims. Infectious materials were either injected into the skin or introduced through the nasal route. The infection that developed was usually milder than naturally acquired smallpox, and recovery from the milder infection provided protection against the more serious disease.

Although the majority of individuals treated by variolation developed only mild infections, the practice was not without risks. More serious and sometimes fatal infections did occur, and because smallpox was contagious, infections resulting from variolation could lead to epidemics. Even so, the practice of variolation for smallpox prevention spread to other regions, including India, Africa, and Europe.

Although variolation had been practiced for centuries, the English physician Edward Jenner (1749–1823) is generally credited with developing the modern process of vaccination. Jenner observed that milkmaids who developed cowpox , a disease similar to smallpox but milder, were immune to the more serious smallpox. This led Jenner to hypothesize that exposure to a less virulent pathogen could provide immune protection against a more virulent pathogen, providing a safer alternative to variolation. In 1796, Jenner tested his hypothesis by obtaining infectious samples from a milkmaid’s active cowpox lesion and injecting the materials into a young boy (Figure 18.26). The boy developed a mild infection that included a low-grade fever, discomfort in his axillae (armpit) and loss of appetite. When the boy was later infected with infectious samples from smallpox lesions, he did not contract smallpox. 4 This new approach was termed vaccination , a name deriving from the use of cowpox (Latin vacca meaning “cow”) to protect against smallpox. Today, we know that Jenner’s vaccine worked because the cowpox virus is genetically and antigenically related to the Variola viruses that caused smallpox. Exposure to cowpox antigens resulted in a primary response and the production of memory cells that identical or related epitopes of Variola virus upon a later exposure to smallpox.

The success of Jenner’s smallpox vaccination led other scientists to develop vaccines for other diseases. Perhaps the most notable was Louis Pasteur , who developed vaccines for rabies , cholera , and anthrax . During the 20 th and 21 st centuries, effective vaccines were developed to prevent a wide range of diseases caused by viruses (e.g., chickenpox and shingles, hepatitis, measles, mumps, polio, and yellow fever) and bacteria (e.g., diphtheria, pneumococcal pneumonia, tetanus, and whooping cough,).

Check Your Understanding

  • What is the difference between variolation and vaccination for smallpox?
  • Explain why vaccination is less risky than variolation.

Classes of Vaccines

For a vaccine to provide protection against a disease, it must expose an individual to pathogen-specific antigens that will stimulate a protective adaptive immune response. By its very nature, this entails some risk. As with any pharmaceutical drug, vaccines have the potential to cause adverse effects. However, the ideal vaccine causes no severe adverse effects and poses no risk of contracting the disease that it is intended to prevent. Various types of vaccines have been developed with these goals in mind. These different classes of vaccines are described in the next section and summarized in Table 18.3.

Live Attenuated Vaccines

Live attenuated vaccines expose an individual to a weakened strain of a pathogen with the goal of establishing a subclinical infection that will activate the adaptive immune defenses. Pathogens are attenuated to decrease their virulence using methods such as genetic manipulation (to eliminate key virulence factors) or long-term culturing in an unnatural host or environment (to promote mutations and decrease virulence).

By establishing an active infection, live attenuated vaccines stimulate a more comprehensive immune response than some other types of vaccines. Live attenuated vaccines activate both cellular and humoral immunity and stimulate the development of memory for long-lasting immunity. In some cases, vaccination of one individual with a live attenuated pathogen can even lead to natural transmission of the attenuated pathogen to other individuals. This can cause the other individuals to also develop an active, subclinical infection that activates their adaptive immune defenses.

Disadvantages associated with live attenuated vaccines include the challenges associated with long-term storage and transport as well as the potential for a patient to develop signs and symptoms of disease during the active infection (particularly in immunocompromised patients). There is also a risk of the attenuated pathogen reverting back to full virulence. Table 18.3 lists examples live attenuated vaccines.

Inactivated Vaccines

Inactivated vaccines contain whole pathogens that have been killed or inactivated with heat, chemicals, or radiation. For inactivated vaccines to be effective, the inactivation process must not affect the structure of key antigens on the pathogen.

Because the pathogen is killed or inactive, inactivated vaccines do not produce an active infection, and the resulting immune response is weaker and less comprehensive than that provoked by a live attenuated vaccine. Typically the response involves only humoral immunity, and the pathogen cannot be transmitted to other individuals. In addition, inactivated vaccines usually require higher doses and multiple boosters, possibly causing inflammatory reactions at the site of injection.

Despite these disadvantages, inactivated vaccines do have the advantages of long-term storage stability and ease of transport. Also, there is no risk of causing severe active infections. However, inactivated vaccines are not without their side effects. Table 18.3 lists examples of inactivated vaccines.

Subunit Vaccines

Whereas live attenuated and inactive vaccines expose an individual to a weakened or dead pathogen, subunit vaccines only expose the patient to the key antigens of a pathogen—not whole cells or viruses. Subunit vaccines can be produced either by chemically degrading a pathogen and isolating its key antigens or by producing the antigens through genetic engineering. Because these vaccines contain only the essential antigens of a pathogen, the risk of side effects is relatively low. Table 18.3 lists examples of subunit vaccines.

Toxoid Vaccines

Like subunit vaccines, toxoid vaccines do not introduce a whole pathogen to the patient they contain inactivated bacterial toxins , called toxoids. Toxoid vaccines are used to prevent diseases in which bacterial toxins play an important role in pathogenesis. These vaccines activate humoral immunity that neutralizes the toxins. Table 18.3 lists examples of toxoid vaccines.

Conjugate Vaccines

A conjugate vaccine is a type of subunit vaccine that consists of a protein conjugated to a capsule polysaccharide. Conjugate vaccines have been developed to enhance the efficacy of subunit vaccines against pathogens that have protective polysaccharide capsules that help them evade phagocytosis , causing invasive infections that can lead to meningitis and other serious conditions. The subunit vaccines against these pathogens introduce T-independent capsular polysaccharide antigens that result in the production of antibodies that can opsonize the capsule and thus combat the infection however, children under the age of two years do not respond effectively to these vaccines. Children do respond effectively when vaccinated with the conjugate vaccine, in which a protein with T-dependent antigens is conjugated to the capsule polysaccharide. The conjugated protein-polysaccharide antigen stimulates production of antibodies against both the protein and the capsule polysaccharide. Table 18.3 lists examples of conjugate vaccines.

Classes of Vaccines
Class Description Advantages Disadvantages Examples
Live attenuated Weakened strain of whole pathogen Cellular and humoral immunity Difficult to store and transport Chickenpox, German measles, measles, mumps, tuberculosis, typhoid fever, yellow fever
Long-lasting immunity Risk of infection in immunocompromised patients
Transmission to contacts Risk of reversion
Inactivated Whole pathogen killed or inactivated with heat, chemicals, or radiation Ease of storage and transport Weaker immunity (humoral only) Cholera, hepatitis A, influenza, plague, rabies
No risk of severe active infection Higher doses and more boosters required
Subunit Immunogenic antigens Lower risk of side effects Limited longevity Anthrax, hepatitis B, influenza, meningitis, papillomavirus, pneumococcal pneumonia, whooping cough
Multiple doses required
No protection against antigenic variation
Toxoid Inactivated bacterial toxin Humoral immunity to neutralize toxin Does not prevent infection Botulism, diphtheria, pertussis, tetanus
Conjugate Capsule polysaccharide conjugated to protein T-dependent response to capsule Costly to produce Meningitis
(Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitides)
No protection against antigenic variation
Better response in young children May interfere with other vaccines

Check Your Understanding

  • What is the risk associated with a live attenuated vaccine?
  • Why is a conjugated vaccine necessary in some cases?

Micro Connections

DNA Vaccines

DNA vaccines represent a relatively new and promising approach to vaccination. A DNA vaccine is produced by incorporating genes for antigens into a recombinant plasmid vaccine. Introduction of the DNA vaccine into a patient leads to uptake of the recombinant plasmid by some of the patient’s cells, followed by transcription and translation of antigens and presentation of these antigens with MHC I to activate adaptive immunity. This results in the stimulation of both humoral and cellular immunity without the risk of active disease associated with live attenuated vaccines.

Although most DNA vaccines for humans are still in development, it is likely that they will become more prevalent in the near future as researchers are working on engineering DNA vaccines that will activate adaptive immunity against several different pathogens at once. First-generation DNA vaccines tested in the 1990s looked promising in animal models but were disappointing when tested in human subjects. Poor cellular uptake of the DNA plasmids was one of the major problems impacting their efficacy. Trials of second-generation DNA vaccines have been more promising thanks to new techniques for enhancing cellular uptake and optimizing antigens. DNA vaccines for various cancers and viral pathogens such as HIV, HPV, and hepatitis B and C are currently in development.

Some DNA vaccines are already in use. In 2005, a DNA vaccine against West Nile virus was approved for use in horses in the United States. Canada has also approved a DNA vaccine to protect fish from infectious hematopoietic necrosis virus. 5 A DNA vaccine against Japanese encephalitis virus was approved for use in humans in 2010 in Australia. 6

Clinical Focus

Resolution

Based on Olivia’s symptoms, her physician made a preliminary diagnosis of bacterial meningitis without waiting for positive identification from the blood and CSF samples sent to the lab. Olivia was admitted to the hospital and treated with intravenous broad-spectrum antibiotics and rehydration therapy. Over the next several days, her condition began to improve, and new blood samples and lumbar puncture samples showed an absence of microbes in the blood and CSF with levels of white blood cells returning to normal. During this time, the lab produced a positive identification of Neisseria meningitidis , the causative agent of meningococcal meningitis , in her original CSF sample.

N. meningitidis produces a polysaccharide capsule that serves as a virulence factor. N. meningitidis tends to affect infants after they begin to lose the natural passive immunity provided by maternal antibodies. At one year of age, Olivia’s maternal IgG antibodies would have disappeared, and she would not have developed memory cells capable of recognizing antigens associated with the polysaccharide capsule of the N. meningitidis. As a result, her adaptive immune system was unable to produce protective antibodies to combat the infection, and without antibiotics she may not have survived. Olivia’s infection likely would have been avoided altogether had she been vaccinated. A conjugate vaccine to prevent meningococcal meningitis is available and approved for infants as young as two months of age. However, current vaccination schedules in the United States recommend that the vaccine be administered at age 11–12 with a booster at age 16.

Go back to the previous Clinical Focus box.

Link to Learning

In countries with developed public health systems, many vaccines are routinely administered to children and adults. Vaccine schedules are changed periodically, based on new information and research results gathered by public health agencies. In the United States, the CDC publishes schedules and other updated information about vaccines.

Footnotes

    K. Mupapa, M. Massamba, K. Kibadi, K. Kivula, A. Bwaka, M. Kipasa, R. Colebunders, J. J. Muyembe-Tamfum. “Treatment of Ebola Hemorrhagic Fever with Blood Transfusions from Convalescent Patients.” Journal of Infectious Diseases 179 Suppl. (1999): S18–S23. Elizabeth Yale. “Why Anti-Vaccination Movements Can Never Be Tamed.” Religion & Politics, July 22, 2014. http://religionandpolitics.org/2014/07/22/why-anti-vaccination-movements-can-never-be-tamed. N. J. Willis. “Edward Jenner and the Eradication of Smallpox.” Scottish Medical Journal 42 (1997): 118–121. M. Alonso and J. C. Leong. “Licensed DNA Vaccines Against Infectious Hematopoietic Necrosis Virus (IHNV).” Recent Patents on DNA & Gene Sequences (Discontinued) 7 no. 1 (2013): 62–65, issn 1872-2156/2212-3431. doi 10.2174/1872215611307010009. S.B. Halstead and S. J. Thomas. “New Japanese Encephalitis Vaccines: Alternatives to Production in Mouse Brain.” Expert Review of Vaccines 10 no. 3 (2011): 355–64.

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    Phagocytosis

    Phagocytosis is a specific form of endocytosis by which cells internalise solid matter, including microbial pathogens. While most cells are capable of phagocytosis, it is the professional phagocytes of the immune system, including macrophages, neutrophils and

    mmature dendritic cells, that truly excel in this process. In these cells, phagocytosis is a mechanism by which microorganisms can be contained, killed and processed for antigen presentation and represents a vital facet of the innate immune response to pathogens, and plays an essential role in initiating the adaptive immune response.

    The process of phagocytosis begins with the binding of opsonins (i.e. complement or antibody) and/or specific molecules on the pathogen surface (called pathogen-associated molecular pathogens [PAMPs]) to cell surface receptors on the phagocyte. This causes receptor clustering and triggers phagocytosis. The cell membrane then extends around the target, eventually enveloping it and pinching-off to form a discreet phagosome. This vesicle can mature and acidify through fusion with late endosomes and lysosomes to form a phagolysosome, in which degradation of the contents can occur via the action of lysosomal hydrolases.

    Figure 1. (a) Three stages of phagocytosis receptor binding and formation of a phagocytic cup, pinching-off and formation of a discreet phagosome and fusion with lysosomes. (b) Human macrophage phagocytosing Candida albicans. Arrows: phagosomes stained for actin (red) and calreticulin, an endoplasmic reticulum marker (green). (c) IFN-g-treated mouse macrophages infected with Mycobacterium bovis BCG (red) and stained for the lysosomal marker CD69 (LAMP3, green).

    Numerous receptors are involved in phagocytosis. Complement receptors and Fc receptors are particularly important for the recognition and phagocytosis of opsonised microbes and other solid matter. Other receptors, including the Toll-like receptors (TLRs), scavenger receptors (SR) and lectins (such as DC-SIGN, dectin-1 and the mannose receptor) are also important in the uptake of many pathogenic microorganisms. Phagocytosis is typically a dynamic process that requires reorganisation of the actin cytoskeleton and the involvement of actin-binding proteins and signalling molecules.

    Moreover, phagocytosis can be influenced by numerous pathogen-associated and endogenous molecules, including lipopolysaccharide (LPS) and cytokines. In particular TNFα and IFNγ drive the formation and maturation of phagosomes. This process triggers the phagocyte to produce cytokines, which act as chemoattractants to enhance migration and activation of other immune cells to the site of infection.

    Some intracellular pathogens, including Mycobacterium tuberculosis, have evolved strategies to inhibit phagosomal maturation and can survive and replicate within the immature phagosome. Other pathogens, such as Escherichia coli and Neisseria meningitides, have developed mechanisms to fix, shed and/or degrade opsonins to prevent activation of the immune response and thus evade immune surveillance and phagocytosis.


    MATERIALS AND METHODS

    Reagents

    Herbimycin A, genistein, and staurosporine were purchased from Sigma Chemical Co. (St. Louis, MO). The protein tyrosine kinase inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine] and its inactive control PP3 [4-amino-7-phenylpyrazolo(3,4-d)pyrimidine], piceatannol, calphostin C, Gö 6976, PD98059, and SB203580 were purchased from Calbiochem Novabiochem Corp. (San Diego, CA). LY294002 was purchased from Biomol (Plymouth Meeting, PA). Calphostin was light-activated before use, as recommended by the manufacturer.

    Pathogen-free C57BL/6 female mice were used in all experiments. Mice were purchased from Charles River Laboratory (Wilmington, MA) at 7–8 weeks of age and used at 8–14 weeks of age. Mice were housed in the Animal Care Facility at the Ann Arbor VA Medical Center (Ann Arbor, MI), which is fully accredited by the American Association for Accreditation of Laboratory Animal Care, where they were fed standard animal chow (Rodent Lab Chow 5001, Purina, St. Louis, MO) and chlorinated tap water ad libitum. This study complied with the NIH “Guide for the Care and Use of Laboratory Animals” (Department of Health, Education & Welfare Publication No. NIH 80–23) and followed a protocol approved by the Animal Care Committee of the local Institutional Review Board.

    Isolation and culture of Mø

    Mice were euthanized by asphyxia in a high CO2 environment, which we have shown previously does not impair the capacity of AMø to ingest apoptotic thymocytes compared with mice euthanized by exsanguination while anesthetized using pentobarbital [10]. Resident AMø and PMø were harvested and cultured as described previously in detail [10]. PMø among the peritoneal lavage cells were first enriched by negative selection using CD19- and CD90-conjugated paramagnetic beads (Miltenyi Biotec, Auburn, CA), according to the manufacturer's instructions. Mø were plated at 2 × 10 5 cells/well in sterile 8-well Lab-Tek slides (Nalge Nunc International, Naperville, IL), and after 1 h incubation at 37°C, nonadherent cells were removed by gentle washing. Mø monolayers were cultured overnight in complete medium [RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS), 25 mM HEPES, 2 mM L-glutamine, 1 mM pyruvate, 100 units/ml penicillin/streptomycin (all obtained from Gibco-BRL, Grand Island, NY), and 55 μM 2-mercaptoethanol (Sigma Chemical Co.)] in a 5% CO2 environment at 37°C before use in the phagocytosis assay.

    Isolation and apoptosis induction in thymocytes

    Thymuses were harvested from normal mice and minced to yield a single-cell suspension. To induce apoptosis, thymocytes were resuspended with RPMI 1640 containing 10% heat-inactivated FBS at the concentration of 1 × 10 6 /ml and incubated for 6 h with a final concentration of 10 − 6 M dexamethasone (Sigma Chemical Co.). This treatment yields a population with a low percentage of late apoptotic cells (11.4±1.6%, mean± sem , n=7 experiments) as judged by positivity for annexin V and staining with propidium iodide.

    Apoptosis assay

    Leukocyte apoptosis was measured by flow cytometric analysis of surface expression of phosphatidylserine (PS) and exclusion of propidium iodide (PI), a sensitive and specific measure of early apoptosis [22, 23]. For this purpose, 100 μl aliquots were stained with annexin V-fluorescein isothiocyanate (FITC Apoptosis Detection Kit, R&D Systems, Minneapolis, MN), according to the manufacturer's protocol. Cells were analyzed without fixation by flow cytometry within 1 h of staining.

    Opsonization of Ig-sheep red blood cells (SRBC)

    SRBC (Colorado Serum, Boulder 1 ml in Alsever's solution) were washed twice in 15 ml phosphate-buffered saline (PBS) without cations. SRBC were resuspended (1.6×10 7 cells in 1.6 ml final volume) in PBS containing rabbit anti-SRBC antisera (Cedarlane Laboratories Ltd., Hornby, ON, Canada 1 μg/2×10 6 SRBC) and were incubated for 20 min at 37°C. These conditions were determined to be optimal by agglutination. SRBC were washed twice in 15 ml PBS without cations and resuspended at 1 × 10 7 /ml in complete medium, and then 200 μl/well was added to the Mø monolayers.

    Phagocytosis assays

    Phagocytosis of apoptotic thymocytes in vitro was assayed by coincubation with adherent Mø monolayers in complete medium as described previously [10]. Results were expressed as percentage of Mø containing at least one ingested thymocyte (percent phagocytic Mø) and as phagocytic index, which was generated by multiplying the percent phagocytic Mø by the mean number of phagocytosed cells per Mø. Phagocytosis of Ig-SRBC was performed in exactly the same manner, except that Ig-SRBC were substituted for apoptotic thymocytes.

    Western analysis of signaling intermediaries

    Mø isolated as above were seeded at a density of 4 × 10 5 cells/well in complete medium in 24-well tissue-culture plates (Becton-Dickinson, San Jose, CA) and were purified by overnight adherence. This method results in >95% pure Mø populations as determined by morphological and surface-marker expression analysis. Apoptotic thymocytes (4×10 6 /well) were added, and cultures were incubated in 5% CO2 at 37°C for various times. Next, Mø were washed twice in Dulbecco's PBS containing 100 mM sodium orthovanadate and then were lysed in 50 μl ice-cold lysis buffer [50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetate, 2 mM ethyleneglycol-bis(β-aminoethylether)-N, N′-tetraacetic acid, 1% Triton X-100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, and 1× protease-inhibitor cocktail (Set III, Calbiochem-Novabiochem)]. Cytoplasmic lysates were electrophoresed in a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition, and proteins were transferred to a solid support membrane [polyvinylidene difluoride (PVDF), Millipore, Milford, MA] using 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid (Calbiochem-Novabiochem), pH 10.0, and 5% methanol as transfer buffer, as described previously [24].

    After incubating membranes in blocking buffer (5% protease-free, Ig-free bovine serum albumin Sigma Chemical Co.) in Tris-buffered saline/Tween 20 (TBST 100 mM Tris-HCl, pH 7.5, NaCl 145 mM, 0.05% Tween 20), primary antibodies were added, and membranes were incubated overnight at 4°C. The antibodies used were antipan ERK, antipan JNKs, and antipan p38 from Santa Cruz Biotechnology (Santa Cruz, CA) anti-IκB-α, antiphospho-IκB-α, antiphospho-ERK1/2, and antiphospho-p38 from Cell Signaling (Beverly, MA) and antiphospho-JNKs from Promega (Madison, WI). Membranes, washed twice in TBST, were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL). Chemiluminescence was developed by the addition of a peroxidase/luminol-based substrate (SuperSignal West Femto maximum-sensitivity substrate, Pierce). Signals were detected using radiographic film (X-Omat, Kodak, Rochester, NY). For reprobing, blots were washed twice in TBST and incubated for 30 min at 55°C in a buffer containing 10 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol.

    Statistical analysis

    Data were expressed as mean ± se . Statistical calculations were performed using Statview and SuperANOVA programs (Abacus Concepts, Berkeley, CA) on a Macintosh PowerPC G3 computer. Continuous ratio scale data were evaluated by unpaired Student's t-tests (for two samples) use of this parametric statistic was deemed appropriate, because phagocytosis of apoptotic thymocytes by PMø has been shown to follow a Gaussian distribution [25]. Significant differences were defined as P < 0.05. The IC50 of pharmacological inhibitors was calculated from dose-response curves using the phagocytic index as the reference variable.


    Integrin-dependent phagocytosis and tissue remodelling

    Phagocytosis is not restricted to the clearance of pathogens by professional phagocytes. It is also a mechanism that allows the removal of ACs and components of the ECM, such as the collagen fibrils that are generated during tissue remodelling (Lee et al., 1996 Ravichandran and Lorenz, 2007 ten Cate, 1972). Interestingly, several integrins are involved in these two processes.

    ΑVβ3, αVβ5 and apoptotic-cell phagocytosis

    ACs that arise during development, normal tissue turnover, inflammation and repair are quickly cleared to avoid secondary necrosis and the release of toxic materials into the environment. Moreover, AC phagocytosis is generally associated with the inhibition of inflammation (Henson, 2005). The integrins that are involved in AC phagocytosis are αVβ3 and αVβ5, which are best known for their potential roles in angiogenesis and tumour-cell metastasis (Reynolds et al., 2002 Taverna et al., 2004). Pre-treatment with RGD peptides or with αVβ3-blocking antibodies severely inhibits the ability of human macrophages to phagocytose apoptotic neutrophils (Savill et al., 1990). By contrast, DCs – phagocytes that also express αVβ3 – use αVβ5 integrin to internalise ACs (Akakura et al., 2004 Albert et al., 1998). Nonetheless, in all cases, integrin-dependent AC uptake is indirect and is mediated by MFG-E8 (milk fat globule-EGF factor 8), a protein that is secreted by macrophages and DCs. MFG-E8 binds phosphatidylserine, an `eat-me' signal that is exposed on the cell surface shortly after induction of the apoptotic programme (Akakura et al., 2004 Hanayama et al., 2002). The crucial role of MFG-E8 in mediating the recognition of ACs by phagocytes is illustrated by the fact that AC uptake is strongly impaired in MFG-E8-knockout mice (Hanayama et al., 2004). Interestingly, αVβ5-mediated MFG-E8-dependent phagocytosis is not restricted to professional phagocytes: HEK293 epithelial cells can also phagocytose ACs in an αVβ5-dependent manner (Albert et al., 2000), as can retinal pigment epithelial cells during circadian-synchronised phagocytosis of photoreceptors, a daily process that is crucial for vision (Finnemann et al., 1997 Nandrot et al., 2007 Nandrot et al., 2004).

    The signalling pathways that mediate AC uptake have been partially characterised. Interestingly, they differ from those that are mediated by the αMβ2 integrin (compare Fig. 2 and Fig. 3). Whereas αMβ2-dependent phagocytosis is mediated by RhoA activity, integrin-dependent AC uptake is dependent on Rac1. Indeed, a dominant-negative mutant of Rac1 inhibits AC phagocytosis in parallel, the levels of active Rac1 increase during AC challenge of macrophages and DCs (Kerksiek et al., 2005 Leverrier and Ridley, 2001 Nakaya et al., 2006). By contrast, a dominant-negative mutant of RhoA enhances AC phagocytosis in macrophages (Leverrier and Ridley, 2001 Tosello-Trampont et al., 2003). The implication of Rac1 in AC uptake is also suggested by the fact that, similar to the Rac1-dependent FcγR-mediated phagocytosis of IgG-coated beads, AC engulfment requires membrane ruffling (Hoffmann et al., 2001 Ogden et al., 2001).

    Significantly, the role of Rac1 during AC uptake is phylogenetically conserved and was initially unveiled in Caenorhabditis elegans. In this genetically tractable model system, a series of mutants deficient in the engulfment of dying cells and cell corpses (ced mutants) was identified. CED proteins fall into two partially redundant pathways (CED-1–CED-6–CED-7 and CED-2–CED-5–CED-12), which converge into CED-10, the C. elegans orthologue of mammalian Rac1. In the worm, local activation of CED-10 is ensured by CED-5 and CED-12, orthologues of mammalian Dock180 and Elmo, respectively. CED-5 and CED-12 are themselves thought to be recruited to the vicinity of bound ACs by CED-2, the CrkII orthologue (Brugnera et al., 2002 Ellis et al., 1991 Gumienny et al., 2001 Hedgecock et al., 1983 Kinchen et al., 2005 Lu et al., 2004 Reddien and Horvitz, 2000 Wu et al., 2001 Zhou et al., 2001a).

    Remarkably, the AC-uptake pathway that has been identified in C. elegans appears to be conserved in mammals. In the HEK293T epithelial cell line, αVβ5-dependent phagocytosis of ACs is inhibited by dominant-negative mutants of CrkII and Rac1, and ligation of αVβ5 by vitronectin stimulates the formation of a complex between p130Cas, CrkII and Dock180 (Albert et al., 2000). However, recruitment of a CrkII-Dock180 complex and activation of Rac1 could be dependent on receptors that cooperate with integrins in the recognition and/or uptake of apoptotic bodies. Indeed, macrophages display multiple recognition receptors for ACs (reviewed by Ravichandran and Lorenz, 2007). One such receptor is Mer, a tyrosine-kinase receptor. Similar to integrins, Mer binds phosphatidylserine indirectly, through the serum protein GAS6 (Nagata et al., 1996). Macrophages from Mer-knockout mice are strongly inhibited in AC phagocytosis both in vitro and in vivo (Cohen et al., 2002 Scott et al., 2001). Interestingly, Mer stimulates the formation of a p130Cas-CrkII-Dock180 ternary complex in a αVβ5-dependent manner (Wu et al., 2005). Mammalian integrins therefore probably contribute to AC uptake together with co-receptors. The importance of CrkII in the AC-uptake pathway was recently challenged, because Dock180-Elmo can activate Rac1 independently of CrkII both in mammalian cells and in C. elegans, possibly via another Rho-family protein, RhoG (deBakker et al., 2004 Tosello-Trampont et al., 2007). Another possibility is that Dock180-Elmo is directly recruited to some receptors, such as BAI1 (brain-specific angiogenesis inhibitor 1), a seven-transmembrane protein that binds phosphatidylserine directly and mediates Rac1 activation by recruiting the Dock180-Elmo complex (Park et al., 2007).

    Others phagocytic receptors, such as CD36 (Fadok et al., 1998 Greenberg et al., 2006 Lucas et al., 2006) and CD14 (Devitt et al., 1998), are engaged in the binding of ACs and contribute to AC phagocytosis both in vitro and in vivo. Whether they induce or modulate signalling downstream of αVβ5 or αVβ3 remains unknown. In particular, whether the activation of αVβ3 and αVβ5 by inside-out signalling is necessary for integrin-dependent phagocytosis of ACs and whether co-receptors control this pathway remain unclear. Surprisingly, it has recently been shown that, whereas the NPXY motif of β5 integrin is required for the adhesion of cells on vitronectin, it is not necessary for αVβ5-dependent phagocytosis (Singh et al., 2007). Unlike β1-β3, β5 does not harbour the conventional talin-head-domain binding site, which is characterised by a tryptophan residue that is located seven to eight amino acids before the NPXY motif. It is therefore possible that αVβ5 is activated in a talin-independent manner. Alternatively, integrin-dependent AC phagocytosis could be regulated at the level of integrin availability at the plasma membrane, as recently shown for αVβ5-dependent phagocytosis of photoreceptor outer segments by retinal pigment epithelial (RPE) cells. In these cells, CD81, which belongs to the tetraspanin family (membrane proteins that regulate the activity of surface receptors, including integrins), stimulates αVβ5-dependent phagocytosis by increasing the level of αVβ5 expression at the cell surface (Chang and Finnemann, 2007).


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