Structural Biochemistry/Cell Signaling Pathways/Phagoptosis

=Phagoptosis=

Definition
Phagoptosis is a term proposed by Guy. C. Brown and Jonas J. Neher to describe death of working cells by phagocytosis (the engulfing of a cell by a macrophage). The word is derived from two words in ancient Greek: 'phagein' which means to devour and 'ptosis' which means to fall or die. Thus the term, phagoptosis means death by being swallowed. Phagoptosis is also known as "primary phagocytosis".

Simply stated, phagoptosis is caused by the exposure of “eat me” signals or the loss of “don’t eat me” signals on the cell surface. The exposure and loss of these signals is reversible, thus cell death can be prevented by preventing phagocytosis. .

Background
Phagoptosis is a nascent term, combining the ideas of phagocytosis and cellular apoptosis (autonomous cell death). While not yet a confirmed phenomenon in macroorganisms, phagoptosis has been proposed as an explanation for various homeostatic and pathological processes in the body that induce the engulfing of healthy or unhealthy cells by a macrophage. Phagoptosis has been proposed, therefore, to be both a contributor to cellular degenerative diseases such as Parkinson’s or Alzheimer’s, whereby healthy neurons are destroyed, and a natural aging tool used by the body to rid itself of unhealthy cells, such as the consumption of old erythrocytes.

Since the 1880s, the work of Russian biologist Ilya Ilyich Mechnikov has led us to believe that phagocytosis, a form of cell destruction, was beneficial because only dead or dying cells were targeted. However, modern-day scientists have found that sometimes healthy, viable host cells can also be marked for cell death; this process is called phagoptosis (or primary phagocytosis). .

Types of Cell Death
1. Autophage: mediated by autophagic in which the cell is eating itself to cause its own death. .

2. Apoptosis: mediated by caspases and it is a program of cell death (PCD). .

3. Necrosis: cell injuries causes cell death within high stress level of cell structures. .

4. Cornification: cell death due to conversion of proteins

5. Shedding:cell death caused by replacement in cell structures.

6. Phagoptosis: cell death caused by phagocytosis with macrophages.

 Apoptosis 

Apoptosis is sometime referred to as “programmed cell death.” It is when the cell relatively speaking commits suicide. It generally occurs in response to certain signals in the body It is better than necrosis because it follows a structured routine and is not random like necrosis. When a cell undergoes apoptosis, the protein caspases will be calld upon to break down the any cellular components necessary for survival. The enzyme DNases is also released to destroy any DNA in the nucleus of the cell, thus eliminating any existence of that cell in that area. Aside from the production of caspases and DNases, cells that goes through apoptosis will release a signal to macrophages telling them to clean up any remaining debris. The macrophages clean everything up giving no opportunity to the dead cell to damage nearby cells.

Apoptosis is actually important to human development as it gives human the fingers and toes feature that human possesses. Originally the 10 fingers are all connected to a web-like feature, but apoptosis is what causes that web to be broken down and destroyed, leaving human with 10 fingers and toes instead of four web like hands and feet. .

 Necrosis 

Necrosis is when cells and tissues die randomly, meaning it is not programmed. It is caused by inflammation, injury, cancer, toxins, etc that may harm the body. The problem with necrosis is that when cells die they do not send signals to nearby phagocytes that order them to clean up the dead cell. Therefore it is harder for the body’s immune system to react and clean up the dead cell if it goes through necrosis, causing build up of dead tissue or cell debris a that location. Cells that goes through necrosis may also release harmful chemicals that may hurt or kill nearby cells. .

 Phagoptosis 

Phagoptosis is a form of cell death when phagocytes is the primary cause of cell death, which is provoked by cells displaying “eat-me-signals” instead of “don’t eat me signals.” Initially the cell produces “find me signals,” that triggers chemotaxis of phagocytes. Upon arrival the cell will either produce “eat me signals” or “don’t eat me signals” and the will ultimately determine if the cell will live or die. The most abundant form of eat me signal is the phospholipid PS found on the inner leaflet of the plasma membrane of the cell. The eat me signal comes out of the cell as a result of calcium-activated phospholipid scramblase that causes the phospoholipid to shuffle the inner leaflet from the outer leaflet, exposing the eat-me-signal. There are different ways PS can be exposed onto the cell such as, calcium elevation, ATP depletion, oxidative stress, fusion of intracellular vesicles with plasma membrae, necrosis, and apoptosis. All of these can cause a cell to release PS signals and hence result in their removal by phagocytes.

A way a cell can protect itself from phagocytes is producing “don’t-eat-me-signals”. A type of “don’t-eat-me-signals” that a cell displays is CD47, CD200, etc. CD47 is the most abundant “don’t-eat-me-signal” expressed by a cell. Though if the “don’t-eat-me-signal” is disrupted then phagoptosis may occur. .

Problems due to Cell Death
Even though Phagoptosis support cell to shape development in cell structures, removes excess and defective cells, and protect cells from pathogens and other cancerous cells, over-excess process of Phagoptosis can cause diseases that might harm the cell replication, production and its natural cycle. Due to the malfunction and irregularity of cell death can lead to lack of important cells in our bodies. For example, excess PS exposure in brain cell structure can terminate not only old and phagocytic neurons but also important neurons that supports our brain cells. Neurons cells are phagocytised by Microglia (Microphage in brain cells) which is caused due to Inflammation Activation. Microglia can eat apoptic neurons to reduce debris or inflammation to help cell circulation and stay in good shape for brain cells .; However, it also can destroy viable neurons and neuronal processes which disintegrate the healthy process of cell, neurons in brain; thus, affecting other healthy brain cells and causing cerebral diseases or disorders like Frontotemporal Degeneration (FTD). Frontotemporal Degeneration (FTD) is heterozygous, inactivating mutation in the progranulin gene due to lack of neurons in brain cells. . It is caused when there is lack of neurons in brain cell due to excess activities of Phagocytosis by Microglia. Many neurons were phagocytised, so it caused inactivating mutation that malfunction the role of progranulin genes in DNA. This FTA disorder is also contributed to causes of Alzheimer's disease and Parkinson's disease. .

Cell Stress
Cell Stress causes several difference responses including phagoptosis, but the response depends on the amount of cell stress present. The different responses include: adaptation, phagoptosis, apoptosis, and necrosis. These lowest stress level would cause adaptation and as the stress level rises the other responses follow accordingly.

Numerous conditions may cause cell stress. Temperature shifts such as heat shock could cause proteins in the cell to denature. Heavy metals can change the conformation of the protein and therefore the function. Free radicals can cause proteins to be fragmented. Fragmentation of proteins render them ineffective. Solvents such as ethanol can cause translation errors. Cell stress response can be the upregulation of some genes and the downregulation of others. In essence, response to cell stress can either cause some proteins to be produced more and others to be produced less depending on the situation. Regulation of cell stress is different in prokaryotes and eukaryotes mainly due to codon differences.

Cell-In-Cell Phenomena: Entosis and Cannibalism
Cell-In-Cell phenomena is most likely seen in tumor cells, especially those are very strong and metastatic. Tumor cells can engulf immune cells which eventually kills tumor cells; On the other hand, tumor cells can also engulf other tumor cells for survival or protection from other harms. Some tumor cell can go through cannibalism process (Cell-In-Cell Phenomena) to protect themselves from immune cells; and Entosis process can happen to both tumor and normal cells.

Entosis
Entosis is caused when tumor cell invade each other; it is caused when detached from Extracelluar Matrix. This Entosis process can cause either survival of the cells or death of invading cells by Lysosome Digestion. Entosis mostly happen in animal cells because animal cells are mostly attached to extracellular matrix for their survival. When some cells are detached from ECM (Extracellular Matrix), they lose the adhesion and cells start to push into their neighboring cells; thus, sometimes, Entosis is considered as interaction between two neighboring cells. Interestingly, cells which are locked due to the result of Entosis are alive and they can divide themselves inside another cells. Entosis is also referred to "cell-in-cell structures" which indicates the loss of attachment from extracellular matrix. In other word, It is process by which tumor cells invade each other when detached from matrix and it is relatively common in malignant cancers. .

Cannibalism
Cannibalism is caused when cells eating up each other for survival and protection due to damages or infection. For example, Wang et al found out some tumor cell can go proceed apoptic cell death. From the bodies of patients who have Huntington's disease, immortalised lymphoblasts phagocyotes are eating up each other to survive in tough condition or to replenish and protect themselves. However, whether restriction of phagocytosis will be able to avoid cell death in cell-in-cell phenomena is still questioned. .

Types of Signaling and Known Pathways
What can cause a macrophage to consume a cell? Many intercellular interactions occur via chemical signaling; the cell displays certain compounds outside of the plasma membrane, to which other cells respond thanks to receptors on the exteriors of their plasma membranes. While some of these compounds invoke phagoptosis, others repulse it and it is the net amount of these "eat me" versus "don't eat me" signals that determine the final fate of the cell. Three main compounds have been proposed to signal macrophages to engulf a cell, each with its own signaling pathway that causes the compound to be expressed or suppressed on the plasma membrane.

Attractive Signals
1. PS (phosphidatylserine)—normally suppressed by an ATP-operated inhibitor called aminophospholipid translocase, which actively pumps PS to the inner membrane of the cell, PS has been shown to, under certain circumstances, lead to phagocytosis of so-called “activated” T cells. PS binds to the receptor Tim-4 on phagocytes, which signals the phagocyte to engulf the cell. If there is no ATP to run the translocase or if there is an overabundance of free calcium in the cytoplasm, the translocase stops running, and another membrane protein called scramblase randomizes the exposure of PS in the cell membrane, potentially exposing it to free phagocytes for endocytosis. This process can happen even in healthy cells.PS exposure can also occur due to oxidative stress which activates the scramblase and prevents translocase. Another result of PS exposure is when the intracellular vesicles fuse with the plasma membrane.

In summary, there are 5 ways that the PS “eat me” signal can be exposed: elevation in calcium levels, lack of ATP, oxidative stress (which activates scramblase and stops translocase), fusion of intracellular vesicles with plasma membrane, necrosis, or apoptosis. Another requirement for phagocytosis to be carried out is the presence of macrophages that are capable of attaching to PS.

In order for macrophages to recognize PS, different receptors must be activated. Resting macrophages express the following PS receptors: Tim4, stabilin-1, stabilin-2, and BAI1. On the other hand, macrophages that are activated express the protein MFG-E8 and its receptor, vitronectin, in addition to the MerTK receptor.

Furthermore, it is important to note that PS exposure does not always equate to cell death; the cell type and surrounding conditions also plays a role. For example, when purified PS is added to 3 different cell lines, the viable cells show an elevation of PS on their surfaces. This stimulated phagocytosis as expected, but as soon as the PS was “internalised”, phagocytosis was inhibited. In other cases such as that of lymphoma cells, PS exposure is not enough to even initiate phagocytosis because either the cells also expressed “don’t eat me” signals or they required another signal to co-stimulate phagocytosis. However, PS exposure on viable cells can be reversed. When neutrophils are activated they have galectins that induce PS on the surface of the cell. If the galectin is removed before the macrophages detect it, then phagoptosis will not occur.

2. CRT (cell-surface calreticulin)—CRT is abundant in cells, particularly in the endoplasmic reticulum. Its transport to the exterior of the cell (exocytosis) can cause it to behave as an attractive signal to phagocytes, which have the CRT receptor LRP (lipoprotein receptor-related protein). Rather than actively displaying CRT as a self-destructive signal, a stressed cell will decrease the repulsive chemical signal CD47 (discussed in the subsequent section), after which a phagocyte will become attracted to excess CRT on the external surface of the target cell. This is an especially prevalent mechanism in cancer cells, which actively attempt to display the CD47 signal. Cancer cells require strong exposure of "don't-eat-me" signals in order to prevent phagoptosis. While CRT is tied to the surface of a cell, it can also send signals to bind proteins, PS, or C1q on target cells. This will stimulate phagocytosis through the LRP, which is on the phagocyte.

An “eat me” signal similar to CRT is thrombospondin 1 (TSP). Like CRT, TSP1 activates phagocytosis with the use of lipoprotein receptor-related protein (LRP) on the phagocyte.

3. MFG-E8 (milk fat globule EFG-like factor-8)—This chemical signal binds to the phagocytotic initiator MerTK (Mer tyrosine kinase) on the phagocyte with the help of several connecting or “bridging” compounds, including Gas-6, protein S, galectin-3, tubby and Tulp 1. These can interact with PS and other receptor proteins in complicated pathways to induce phagocytosis on white blood cells called neutrophils.

Repulsive Signals
In addition to these attractive signals, there are repulsive signals that cells can display to actively prevent phagocytosis. While the attractive signals may be displayed as a result of cellular stress, repulsive signals are altogether more specific. Some repulsive signals include:

1. CD47 (Cluster of Differentiation 47: CD47 is the inhibitory membrane protein expressed on the surface of the majority of cells. It binds to signal-regulatory proteinα (SIRP-α) on the phagocyte. Examples of cells that use this protein as a 'don't eat me' signal include erythrocytes (red blood cells), cancer cells, and platelets, T-cells. In clinical studies performed on mice with cancerous growths, it has been found that blocking of this protein results both reduction of tumor size and spread throughout the body as a result of phagocytosis.

2. Sialic-Acid Derivatives: These react with a variety of receptors, such as cofactors to prevent phagocytosis. Modifications of sialic-acid on cell surfaces can stop C3b and C1q from binding and signaling nearby macrophages. Sialic acid can be removed from the cell surface by implementing neuraminidase and doing so can induce phagocytosis. Siglec-11, a receptor on brain microphages (microglia), can prevent inflammation and phagocytosis of neurons by binding to their surface. However, Siglec-11 requires polysialylated proteins to be present on the cell surface.

3. PAI-1: PAI-1 (or plasminogen activator inhibitor-1) is another major repulsive signal in cells. Neutrophil cells are an example of a cell type that mainly uses PAI-1 as its repulsive signal.

4. CD200 (Cluster of Differentiation 200): This is a protein expressed on the membrane of certain cells. In the case of myeloid cells, it can prevent phagocytosis by releasing an inhibitory signal.

How Attractive and Repulsive signals Work Together
1. However, it is important to note that the display of an “eat me” signal alone may not be sufficient to induce phagocytosis. Despite having “eat me” signals on its surface, a cell may not be attacked by phagocytes because it also displays “don’t eat me” signals. The proportion of “eat me” signals such as PS to “don’t eat me” signals such as CD47 plays a major role in determining how the cell will be recognized by macrophages. Furthermore, the type of cell as well as its environment also influences the degree of influence that signals monitoring cell death will have. Multiple cell types require a cooperative protein or PS oxidation to undergo phagocytosis.

Phagoptosis in Model Organisms
Much of the information about cell death has been obtained from model organisms, such as Caenorhabditis elegans (a species of transparent nematode) and Drosophila melanogaster (a species of fly commonly used in laboratories).

Phagocytosis in Development
In C. elegans it has been found that a combination of loss-of-function mutants in ced-1 as well as in ced-3 (two transmembrace receptors in C.elegans) led to reduced apoptosis as well as reduced phagocytosis, indicating that both are important factors in programmed cell death during development of the species. This also prevented some death from mutation and toxins. From this it was concluded that light stress on the cell (such as weak activation of caspase, which is involved in apoptosis) is not enough to cause a cell to die, but in conjunction with PS exposure could cause phagoptosis. By removing the srgp-1 gene, which prevents phagocytosis of PS-exposed cells, it was found that rates of phagocytosis in normally apoptotic cells and other stressed cells increased.



In D. melanogaster, developmental cell death is caused by 3 proteins: Hid (head involution defective), Rpr (Reaper), and Grim. All of these induce apoptosis of the cell by binding to the caspase inhibitor DIAP1. This leaves developmental phagocytosis intact, which seemed to remove most of the cells normally lost in development. This indicates that while apoptosis might not be essential for development of D. melanogaster, phagocytosis might be. One of the mechanisms through which phagoptosis occurs in D. melanogaster is the ER protein pretaporter, which is expressed externally on the surface of cells. There is also competition between cells of different genotypes within D. melanogaster through induced phagoptosis of surrounding cells.

Turnover of Cells Due to Phagoptosis
Phagoptosis is one of the main forms of cell death, causing turnovers of erythrocytes, neutrophils, T-cells and many other cells.

Erythrocytes
Erythrocytes are red blood cells. The highest rate of cell death in the body is caused erythrophagocytosis, the cell destruction of red blood cells. Two million red blood cells, or erythrocytes, are produced every second in the human body. They live for about 120 days and then are destroyed by macrophages in the spleen, liver, and bone marrow in such a way that equals its rate of production. Instead of undergoing apoptosis, red blood cells display “eat me” signals such as PS. A loss of CD47 “don’t eat me” signals in old red blood cells is enough to cause rapid phagocytosis.

Older erythrocytes tend to get phagocytized more because they contain more "eat-me" signals such as PS, phosphidatylserine. When PS is exposed on the cell’s surface, a macrophage senses this and phagocytizes the red blood cells. This can be reversed by other signals such as CD47 on the cell’s surface, binding to the macrophage’s SIRPα receptors. As erythrocytes become older, they lose CD47 signal, causing phagocytosis of erythrocytes. It was found that older erythrocytes live longer when there were depletion of macrophages, which concludes that phagoptosis was the reason for red blood cell's turnover.


 * The phagocytosis of red blood cells is mediated by “eat me” signals such as PS as well as “don’t eat me” signals such as CD47. Erythrocytes are particularly excellent for studying the regulation of phagocytosis because they are destroyed at the same rate they are produced, giving them the highest rate of cell death in the human body. Every second, about 2 million red blood cells are formed in the bone marrow. Each cell has a life span of approximately 120 days. As erythrocytes get older, they begin to display more “eat me” signals and fewer “don’t eat me” signals on their surface. Signals that prevent phagocytosis such as CD47 act on the SIRPa receptors of macrophages, preventing them from attacking viable erythrocytes. Having more “don’t eat me” signals or having fewer macrophages will prolong the survival of older erythrocytes.

Neutrophils
Neutrophils, which are the most plentiful type of white blood cell, are quite different in comparison to erythrocytes. Like red blood cells, neutrophils are also produced in bone marrow and destroyed in the spleen, liver, and bone marrow. They are created at a rate of 0.5-1 million per second in humans, with a lifespan of about 5 days. What’s peculiar about these white blood cells is that the younger cells are just as susceptible to phagocytosis as the older cells. When isolated, neutrophils undergo spontaneous apoptotic cell death. For neutrophils, the protein PAI-1 is a “don’t eat me” signal. When PAI-1 is blocked, CRT becomes the main “eat me” signal. Neutrophils are the only leukocytes to expose CRT without the need of a stimulus, which would explain their fast renewal rate in the body. They also have the ability to eat activated platelets and small cells such as bacteria.

Also, phagocytosis is not the only cause of turnovers in neutrophils like it is for erythrocytes; instead spontaneous apoptosis occurs when neutrophils are isolated.

When CRT is expressed on neutrophil’s surface, it signals the drive of phagocytosis by macrophages. In contrast, PAI-1 expresses the signal that prohibits phagocytosis, but depletion of this signal causes increasing chance of it being phagocytized. This can be reversed by adding PAI-1 proteins that binds to the white blood cell’s surface, barring phagoptosis.

Neutrophils can also act as phagocytes, but only devouring smaller bacterias.

Like erythrocytes, neutrophils are also made in the bone marrow. However, neutrophils only have a lifespan of 5 days before they are phagocytised. Another noteworthy difference between neutrophils and erythrocytes is that unlike for an erythrocyte, the phagocytosis of a neutrophil is not concurrent with its age. “Younger neutrophils are just as likely to be phagocytised as older neutrophils. (p. 328)” The protein PAI-1 acts as the primary signal preventing phagocytosis on neutrophils. When this “don’t eat me” signal is removed, viable neutrophils are attacked by macrophages. Like PS, the PAI-1 protein also exhibits reversible binding. Adding the protein back to the surface of neutrophils will decrease their rate of phagocytosis. The phagocytosis of neutrophils is also influenced by antibodies that block PAI-1. When PAI-1 is blocked, the “eat me” signal CRT can induce phagocytosis of neutrophils that are still viable leading to a decreased immune response. Although CRT acts as the primary eat me signal on neutrophils, neutrophils can also be stimulated to bring PS to their surface, encouraging attack by surrounding macrophages. The phagocytosis of red blood cells is mediated by “eat me” signals such as PS as well as “don’t eat me” signals such as CD47. Erythrocytes are particularly excellent for studying the regulation of phagocytosis because they are destroyed at the same rate they are produced, giving them the highest rate of cell death in the human body. Every second, about 2 million red blood cells are formed in the bone marrow. Each cell has a life span of approximately 120 days. As erythrocytes get older, they begin to display more “eat me” signals and fewer “don’t eat me” signals on their surface. Signals that prevent phagocytosis such as CD47 act on the SIRPa receptors of macrophages, preventing them from attacking viable erythrocytes. Having more “don’t eat me” signals or having fewer macrophages will prolong the survival of older erythrocytes.

T-Cells
T-Cells play an active role in adaptive immunity. These cells activate themselves by attaching to part of antigens and proliferate to leave memory T-cells for secondary immune responses. The activation of T-cells leads to PS exposure and recognition by the Tim-4 receptor, which leads to phagocytosis; thus, carrying out its role to destroy the body's foreign invaders. The Tim-4 receptors on phagocytes are blocked when antigens are introduced during immunization or during infection with influenza virus. This decreases the production of antigen-specific T cells which in return will increase immune responses to those antigens. Similar to erythrocytes, neutrophils, platelets, the turn over of T-cells is regulated by the CD47 signal. Once the T-cell loses that signal, it will be devoured by macrophages.

Hemophagocytosis
Phagoptosis has been linked to multiple inflammatory and immune disorders, where viable blood cells are attacked by macrophages. A reduction in red or white blood cell count is known as cytopenia. Individuals with cytopenia often have compromised immune systems and are vulnerable to infection. Inflammation can induce the increased phagocytosis of red blood cells by causing these cells to display “eat me” markers, primarily PS, on their surface. Inflammation also increases the capability of macrophages to target red blood cells. Hemophagocytosis is difficult to treat because inflammation plays a vital role in the immune response by isolating foreign substances which may potentially be harmful so they can be destroyed by phagocytes.

Opportunities for Future Research
There is still much research to be done with phagoptosis, but early experiments indicate that certain instances of cell death such as erythrocytosis, neutrophil “cannibalism,” and unnatural neurodegeneration, which were once attributed to apoptosis, may be the result of this more complicated process. Further research may be able to illicit specific solutions to problems that apoptosis models could not adequately describe, which in turn, could provide an opportunity to develop new treatments for degenerative diseases.

Neurodegeneration
While there are many different health problems that arise with unnecessary phagoptosis, one of the most serious of these comes from loss of neurons in the brain. This is particularly harmful because neurons are not able to be regenerated. Therefore, permanent damage may result from microglia, the macrophage in the brain responsible for consumption of neurons among other tasks, engulfing any neuron that exposes inflammation, whether or not that neuron is viable. It has been recently discovered that a major cause of frontotemporal degeneration (FTD) is linked to inactivation of the progranulin gene which inhibits phagocytosis. This suggests that Parkinson's disease, Alzheimers, and amyotrophic lateral sclerosis may be controlled by phagocytosis control in the brain. Controlling phagocytosis in the brain can be done, for example via PS blocking, which should stop all loss of viable neurons without having to inhibit inflammation.

Although there are problems with unnecessary phagoptosis in the brain, phagoptosis can also be potentially beneficial in the brain as well. Microglia, or brain macrophages, can also devour apoptotic neurons (neurons which have been programmed to die), reducing the debris and inflammation. There are some cases where the microglia's ability have been impaired due to inflammation, which impairs its ability to classify which neuron to devour, mistaking viable neurons for apoptotic neurons during phagocytosis.

Phagoptosis performs many beneficial functions in the body including defense against harmful pathogens and regulation of the inflammatory response. However, recent studies indicate that it may be a primary culprit of diseases associated with frontotemporal degeneration. FTD is caused by phagoptosis of viable neurons, which is normally prevented by a protein known as progranulin which regulates phagoptosis in the brain. Mutations in the gene that codes for progranulin are associated with Alzheimer’s, Parkinson’s, and other neurodegenerative diseases.

Cancer Research
It has been discovered that cancer cells contain high quantities of exposed CD47 protein which contributes to their low rate of consumption by macrophages. Potential research may find inhibitors or antibodies against CD47 that may help induce natural death of cancer cells. For example, in leukaemic cells, it has been discovered that the addition of CD47 antibodies is enough to eradicate several types of leukemia from model organisms such as mice.

Several applications are examined to illustrate the idea of phagoptosis: