I'd heard that a coronavirus has the same exact type of outer cell membrane, as our own cells - with about the same width (approximately 5-10 nanometers, according to the wikibooks
page on lipid bilayers).
In order for this theory about baking soda and lime juice' effectiveness against the coronavirus to be correct, there would have to be some reason why the surfactant properties of this concoction would be able to deactivate a coronavirus, but not do harm to our own cells (at least, not enough harm to affect us in a really bad way).
I had to wonder: If the lipid bilayer surrounding our own cells is the same width and has the same properties as the cells around a coronavirus, why is it considered safe to drink Alka Seltzer, which has surfactant properties? Wouldn't that be dangerous if a surfactant could actually damage our own cells, too?
I've done a lot of digging to try to figure this one out, and I believe the reason our own bodies can withstand the consumption of a weak surfactant, is due of one or more of these factors:
1) Our own cells appear to have the ability to repair or replace themselves, while viruses are more simple and do not have this same ability. See the article I've pasted further down the page, called "Plasma Membrane Repair: A Central Process for Maintaining Cellular Homeostasis."
(UPDATE: I no longer believe it's likely that a surfactant or detergent could leave much room for repair of a human cell. It might, but I think it is a lot more likely that our human cells would simply be replaced with new cells).
Our bodies are built pretty darn tough, to be able to withstand and recover from a LOT of damage and cell loss. Your mody makes about 100 million new red blood cells every minute, and that's just your blood cells. We have about 200 different types of cells in the human body, including white blood cells, plasma cells, endothelial cells, stem cells, bone cells and more.
(We lose about 300 million cells PER MINUTE, and this is just a normal process.
1) A human cell is much larger than a coronavirus. Typically at least 100x larger.
We have over a quadrillion mitochondria.
How Does Your Body Create Energy? "Fat For Fuel" with Dr. Joseph Mercola
https://www.verywellhealth.com/what-are-mucous-membranes-1191862
What Mucous Membranes Do in Your Body
Mucous membranes protect the inside parts of your body that are exposed to air, in a similar fashion to how your skin protects your external body. Mucous membranes are rich with mucous glands that secrete mucus to help keep the membranes moist.
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Examples of mucous membranes include lips, mouth, nasal passages, middle ear, and the eustachian tube. Other mucous membranes include the lining of the digestive tract, the lining of the urogenital tract (including the urethra and vagina), the lining of the respiratory tract, and your eyes (conjunctival membranes).1
The human body has four types of tissue with which our organs, bones, cartilage, and other parts of the body are made. One of the types, epithelium, is subdivided into two categories: mucous membranes, and serous membranes. Mucous membranes are made up of epithelial cells that usually covers and protects underlying connective tissue (fibrous and elastic tissue built for supporting other structures of the body).
Mucous Membranes of the Ears, Nose, and Throat
Because they are exposed to the outside world, mucous membranes are found in your ears, nose, and throat.
Oral mucous membranes are reddish-pink and line the inside of the mouth. The oral mucosa continues outside the mouth to form the lips. Because mucous membranes are prone to becoming dry when not adequately hydrated, the lips frequently can become dry. Under normal circumstances, your saliva helps to keep your lips moist.
Nasal mucous membranes are lined with small blood vessels that help to warm and humidify the air you breathe.2 The mucous membranes are also lined with cilia, tiny hair-like structures, that help to trap the debris that you breathe in. The cilia then move the debris either towards the front of your nose or towards the back of the throat. This is an important function of your immune system and functions to keep harmful germs out of the body.
Mucous membranes of the ears are the first line of defense for the middle ear, which is normally bacteria-free. Like the nasal mucous membranes, mucosa in the ears have cilia which move any debris towards the opening of the auditory tube. The auditory tube likewise has mucous membranes with cilia to transport the debris toward the back of the throat to be swallowed. The middle ear can be affected by allergies and infections, and can fill up with fluid as a result. The fluid can be sterile or infected, and often contains secreted IgA antibodies and white blood cells.
Esophageal mucous membranes work in conjunction with a muscular portion to allow for peristalsis, which is the process of moving food toward the stomach. Peristalsis works in a wave-like motion to assist food movement. The mucous membranes in the esophagus also contain minor salivary glands that secrete bicarbonate in high concentrations.3 The bicarbonate helps to neutralize any refluxed stomach acid.
Aging and Your Mucous Membranes
Unlike tissue (skin) on the outside of your body, mucous membranes are relatively sheltered from ultraviolet radiation and exposure to the weather. This helps the mucous membranes remain relatively unchanged throughout the aging process. Mucous membranes also replace themselves quite quickly. However, studies have found that oral mucosa becomes increasingly thin with age.4
Oral Mucous Membranes are the Mirror of the Body
The oral cavity is often referred to as the "mirror of the body" because the mucous membranes in your mouth change depending on many different diseases.5
A thorough oral exam may help tip your doctor off as to what problem you may be having.
Changes can be seen in the following types of disorders:
- Hematologic: iron-deficiency anemia
- Autoimmune: Sjögren's disease
- Pulmonary (lung) diseases: sarcoidosis
- Endocrine: diabetes, thyroid disorders, Cushing syndrome, or Addison's disease
- Gastrointestinal: Crohn's disease
Caring for Your Mucous Membranes
Dry mucous membranes are a sign of dehydration and can cause various health problems.6 For example, dry mucous membranes in the lining of the nose can cause frequent bloody noses. You can help keep your mucous membranes moist by drinking plenty of water. You can also use a humidifier, preferably a cool mist humidifier.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2826788/
Do viruses use vectors to penetrate mucus barriers?
Viral-bacterial associations produce disease
A growing body of evidence suggests that many infectious diseases result from close cooperation between viruses, bacteria and fungi [1]. One thoroughly studied example of a mixed viral-bacterial infection is influenza, commonly known as the flu. This disease spreads across the globe each year in seasonal epidemics, killing hundreds of thousands and sometimes millions of people. The initial disease is triggered by the influenza virus; however, most deaths occur due to secondary infections caused by three bacteria, Streptococcus pneumoniae, Staphylococcus aureus, and Hemophilus influenzae. The effects of bacteria and viruses are so closely associated that the bacterium Hemophilus influenzae was initially thought to be the causative agent for influenza [1].
One possible explanation for the association of influenza and bacterial infections is that influenza viral attack weakens the host, and enables the bacteria to infect. However, it is suspicious that certain bacterial strains, but not others, can take advantage of influenza infections. This suggests that the bacteria are more than just opportunistic organisms infecting a weakened host. Rather, it may indicate that viruses and bacteria cooperate to cause disease. Viruses may rely on specific bacteria to take hold of a host, just as much as bacteria may rely on viruses to provide a window of opportunity for host infection. One main obstacle to fighting diseases of this kind is the lack of knowledge about how viruses and bacteria interact to increase the morbidity and mortality of infection.
The influenza virus is transmitted to humans through aerosols that contain the virus. To achieve infection, however, the virus must overcome the body’s first line of defense, a thick mucus layer in the nose, throat, and lung, which protects the underlying cells from contact with noxious agents and pathogens. Viruses have no capacity for active movement, but they could potentially spread through mucus by passive diffusion. However, a typical mucus gel is 50–700 μm thick and represents a crowded environment with protein concentrations up to 300 mg/ml [2–4], which is similar to protein density in the cytoplasm. Assuming that molecular crowding imposes similar constraints on free diffusion in mucus as it does in cytoplasm, the passive diffusion of even relatively small viruses through a typical mucus gel should take several days. This presents a problem for viruses, since mucus is cleared and replenished by the basal epithelium several times each day. It is therefore unlikely that viruses can diffuse through the mucus gel more quickly than mucus bulk flow can sweep them out of the body.
In cytoplasm, viruses employ many mechanisms to travel long distances relative to their own size. Some viruses hitchhike on motor proteins, which mediate transport along microtubules between the periphery and center of cells [5]. Other viruses rely on transport by the actin system, either by newly polymerized actin laments that push a particle, or by myosins that move along actin laments [5]. Viruses also use cells and organisms as vehicles; for example, it has been suggested that Epstein–Barr virus (EBV), an orally transmitted herpesvirus, uses B cells as a transfer vehicle to reach epithelial cells [6]. Other well-studied examples include dengue and yellow fever viruses, which use insects as vectors to bridge travel distances as far as many kilometers [7,8]. I hypothesize that viruses have also evolved strategies to facilitate their dispersal and passage through mucus gels.
A new model of two-way cooperation between viruses and bacteria
In contrast to viruses, numerous bacteria are well adapted to life in mucus and co-exist with a healthy host. Bacteria use various forms of motility, including flagellar propulsion and gliding, to cross mucus barriers at rates much higher than diffusion would allow. By attaching to motile bacteria, viruses may reduce the time it takes them to cross mucus barriers from days to minutes. In addition to efficiently moving through mucus, some bacteria are able to reside within it and resist clearance from the host. For example, bacteria associated with flu attach to mucosal surfaces of the nose and throat [1,9–11] and grow into colonies that protrude into the mucus gel and withstand expulsion. I propose that viruses attach to bacteria that inhabit mucus, and that this initial adsorption to bacteria sets the stage for viral infection (Fig. 1).
1. Bacteria (blue) associated with the flu attach to mucosal cell surfaces of nose and throat and grow colonies. Immune cells (yellow) keep the density of bacteria at innocuous levels. 2. Viruses (red) adsorb to the sessile bacteria, setting the stage for viral infection. Viruses reciprocate the improved access to the host by inactivating the immune cells (grey) that chase the bacteria. 3. Bacteria exploit this temporary lack of immunological control to increase their populations to levels that result in pathogenic invasion.
What benefits may bacteria reap from such a direct interaction with viruses? Viruses may reciprocate their improved access to epithelial cells by locally inactivating the immune cells that chase the bacteria. A highly efficient immune system continuously contacts and consumes bacteria in order to maintain their density at innocuous levels. Influenza virions can combat the policing activity of a host’s immune system by inactivating immune cells; the viral virus NS1 protein, for example, can modulate immune cell activity in many different ways [12]. Bacteria could then exploit this temporary lack of immunological control and expand their populations to densities that result in pathogenic invasion.
According to this model, the onset of flu depends on the physical adsorption of viruses to bacterial cells inhabiting mucus. Once this has occurred, the viruses can invade and thereby pave the way for a virulent bacterial infection. A direct interaction between viruses and bacteria has not been suggested in the context of human disease, but it is a prominent phenomenon in natural waters where viruses concentrate within bacterial communities [13].
Sexually transmitted viruses might derive motility from sperm cells
A different, but conceptually related, strategy may be used by viruses that infect epithelia in the female genital tract, such as the human immunodeficiency virus (HIV), herpes simplex virus (HSV), and human papillomavirus (HPV). All these viruses are primarily transmitted through sexual intercourse, yet the exact mechanism by which sexual contact promotes their infection remains unclear. In the female genital tract, viruses face the same problem as that encountered in the lung: to infect the epithelium, they must penetrate a thick mucus gel, which is probably difficult to achieve by simple diffusion. Here too, viruses could exploit exogenous sources of motility (Fig. 2).
Numerous bacteria (blue) are well adapted to life in mucus and co-exist with a healthy host. Bacteria can cross mucus barriers at rates much higher than diffusion by using various forms of motility such as flagellar propulsion and gliding. In contrast, viruses (red) are immotile particles with no ability for directed movement. By attaching to motile bacteria, viruses would be able to cross mucus barriers much faster than diffusion would allow. Similarly, viruses that infect epithelia in the female genital tract may have evolved to exploit sperm cells (grey) as vehicles for dispersal and mucus penetration.
In contrast to viruses, sperm cells associated with sexual contact are highly motile and well adapted to passage through mucus [14]. Sexually transmitted viruses may have evolved to exploit sperm cells as vehicles for dispersal and mucus penetration within the female genital tract. Consistent with this hypothesis, HPV16 capsids, HIV, and HSV have been found associated with human sperm cells [15–21].
The mechanism of interaction between sperm and virus is not well understood; however, one study suggests that glycosaminoglycans or molecules of similar structure on the surface of sperm enable binding of viruses [22]. Many sexually transmitted viruses, including HPVs, HIV, and HSV, adsorb to epithelial cell-surface glycosaminoglycans (GAGs), particularly heparan sulfate proteoglycans (reviewed in [23]). Thus, the mechanism by which viruses interact with sperm may be similar to the manner in which they adsorb to epithelial cell surface GAGs during early stages of infection. How viruses transfer from their motile vehicles to their target cells is not yet clear. One possibility is that sperm cells have a shorter lifespan than epithelial cells in the female genital tract. Hitch-hiking viruses will likely dissociate from disintegrating sperm and become free to infect the epithelium.
It is noteworthy that a direct interaction with live sperm cells has been documented for the vertically transmitted fish rhabdovirus, which causes infectious hematopoietic necrosis in salmonids [24]. Salmonid fish are oviparous species with external fertilization. The rhabdovirus adsorbs to sperm and transfers with them through the open water toward eggs. Mechanistically similar interactions may occur between sexually transmitted viruses and sperm in humans, and promote virus dispersal and mucosal penetration in the female genital tract [22].
According to this hypothesis, viral infections could be compared to classical vector-borne diseases such as malaria, in which Plasmodium spp. use highly motile insect carriers to reach host targets over large distances and bypass their first line of defense, the skin.
I emphasize that the proposed hypothesis does not exclude alternative pathways for viral infection, for example through wounding or otherwise compromised mucosal epithelia. However, these mechanisms are less reliable and will likely not be as efficient as hitchhiking on cells that are optimized for translocation through mucus.
Testing the hypothesis
Influenza is an ideal model to test whether viruses cooperate with bacteria to overcome mucus barriers. This hypothesis makes two testable predictions: 1. The influenza virus physically binds to bacteria associated with flu morbidity and mortality. 2. The influenza virus and the bacteria that it binds will not cause disease on their own, but only in combination with each other.
Two parallel approaches can be taken to test these predictions. First, one may ask if the influenza virus directly associates with bacteria in vitro or in vivo. Protocols to produce and isolate influenza particles are established, and the cultivation of S. pneumoniae, S. aureus, and H. influenzae is routine in many laboratories. These protocols could be the basis for a test of the proposed model’s first prediction. If viral particles do attach to bacteria, one may ask if a combination of influenza virus and bacteria is necessary to cause infection. Mice are well-established models for studying human disease, and germfree mice would be especially useful in testing the second prediction of the proposed model because they are natively devoid of microbes and hence can be infected with defined combinations and quantities of viruses and bacteria. One would infect germfree mice with the influenza virus, individual bacteria, or combinations of both, and test whether viruses and bacteria indeed depend on each other to establish infection.
If we find that viruses and bacteria indeed cooperate directly to manifest the flu, it is possible that cooperative interactions of this kind are also the basis of other polymicrobial infectious diseases such as AIDS, chickenpox, or measles. With germfree mice and the proposed biochemistry experiments, we have a good stage on which to dissect the underlying microbial interactions of these diseases as well.
Implications: new strategies for viral disease prevention and treatment
The potential importance of virus-vehicle association for virus dispersal and mucosal penetration suggests that blocking viruses from binding to bacteria or sperm may be a new strategy to prevent viral diseases [22]. Good candidates to achieve this are peptides derived from the respective virus or benign intact viruses that shield binding sites on the surface of bacteria or sperm. Such tools may offer a critical advantage over vaccination with antigens, as is undertaken for Influenza or HPV: Viral populations can rapidly escape neutralization by specific antibodies by evolving new surface properties. But to bypass inhibitors of virus-vehicle attachment, the viral populations need to evolve new mechanisms that exploit novel binding sites on their vehicles, which are presumably limited. Hence, blocking viral attachment to its vehicle may be a more sustainable method of disease prevention than conventional vaccination.
It has become standard practice to treat certain viral infections, for example of the upper respiratory tract, with antibiotics. The rationale is that these infections might be of bacterial origin, or that the antibiotics will prevent secondary infections. In light of the hypothesis presented here, a specific antibiotic would slow or stop a viral infection by killing bacteria within the mucosa and thereby eliminating the foundation for successful viral entry into our bodies.
Acknowledgments
I wish to thank Otger Campas for discussions, and Andrew Murray, Sebastian Ulbert, and Carey Nadell for comments on the manuscript. This work was supported by NIH grant number P50 GM068763-06 to the FAS Center for Systems Biology, Harvard University.
Footnotes
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Very dry air in the offices
When mucous membranes are open to viruses
Up to 10,000 litres of air flow through the nose, mouth and lungs every day. Considering that office workers spend up to 90 % of their working time indoors, the importance of the air quality that is inhaled quickly becomes clear. As part of the "Healthy air – Healthy eye" campaign, measurements were carried out in 100 offices - with alarming results. "High levels of CO2, air that is too dry and too warm as well as fine dust from printers & co – the air quality, especially in offices, leaves much to be desired" stresses Thomas Schlatte from the MeineRaumluft.at platform. In every fourth office, the humidity was below 40 percent and the temperature was up to 26 °C. This leads to the mucous membranes opening and becoming more susceptible to viruses. While an optimum air humidity of more than 40 % is fatal for aerosol flu and cold viruses within minutes, they remain infectious for hours at values below that level. The eyes also suffer from dry air and screen work aggravates the situation. No less than 86 % of all those asked complained about health problems such as dry and burning eyes and headaches. Excessively dry ambient air can also irritate the vocal chords and impair well-being and performance.
What’s more, disorders of the respiratory system and the vocal apparatus are among the most common causes of lost work time. However, our air humidification systems ensure a healthy indoor climate. With the compact AIR-C60, we also have an efficient appliance in our product line for small offices that does a great job as an air purifier, air washer and humidifier.
Sources and further links:MeineRaumluft.athttps://www.asu-arbeitsmedizin.com/article-799743-30010/neu-whitepaper-dry-building-syndrom-.htmlhttps://www.asu-arbeitsmedizin.com/article-801387-30010/font-size2anzeigefontbr-dry-building-syndrom-macht-krank-.htmlPeople also ask
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4630197/
Plasma Membrane Repair: A Central Process for Maintaining Cellular Homeostasis
A eukaryotic cell is separated from the extracellular environment by a plasma membrane composed of a phospholipid bilayer containing proteins that regulate transit of molecules into and out of the cell. Loss of this barrier function can lead to compromised cellular homeostasis and death of the cell. Most cells are subjected to mechanical or chemical stresses that can disrupt the plasma membrane; thus there is strong selective pressure to ensure the integrity of this membrane. The inherent nature of phospholipids and early work with lysosomes indicated that the plasma membrane would thermodynamically reseal after disruption (112). While this is true of simple lipid bilayers or small membrane disruptions (FIGURE 1A), the plasma membrane contains integral proteins that interact with the cytoskeleton and extracellular matrix to support numerous cellular functions. These interactions create mechanical tension on the plasma membrane that holds the membrane open after disruption (25, 114). Such disruptions allow intracellular components to escape the cell and potentially permit toxic levels of Ca2+, oxidants, and other components of the extracellular milieu to enter the cell. Thus, if these disruptions are not closed rapidly, it may lead to the death of the cell. As a result, cells have evolved active methods to reseal plasma membrane disruptions in which normal cellular responses are repurposed to mend the broken membrane (70, 106, 130) through a process called membrane repair.
Models of the plasma membrane repair process
A: thermodynamic resealing occurs spontaneously due to tension produced by the disordered arrangement of the membrane phospholipids at the open edge of the break. This process is the most likely route of resealing for membrane breaks of ≤1 μm in diameter. B: exocytosis can contribute by trafficking intracellular vesicles to the wounded area where they can fuse with each other and the injured membrane to form a repair patch. C: wound constriction is mediated by caveolae. During this process, caveolae cluster and fuse around larger wounds, leading to wound constriction and intracellular fusion of caveolar endosomes. D: budding/blebbing of the membrane portion containing the wound site with release of the newly formed vesicles into the extracellular space also involves exocytosis. E: exocytosis of an intracellular patch and fusion to the wound site could result in the extracellular release or “shedding” of the wound site. F: endocytosis of wounds occurs via invagination of caveolar vesicles and subsequent intracellular fusion of caveolae.
The idea of facilitated membrane repair was supported by earlier work (23, 59) before the concept was formally presented by McNeil and colleagues, who initially showed that plasma membrane disruptions and repair occur in vivo and that damaged cells reseal by recruitment of intracellular vesicles to form a repair patch in an extracellular Ca2+-dependent manner (FIGURE 1B) (12, 96–98, 106, 126). Membrane repair can also involve fusion of vesicles at the injury site or into the proximal plasma membrane. Constriction of the membrane around disruptions can also contribute to membrane repair (FIGURE 1C) (9). Endocytotic mechanisms may be involved in resealing of larger membrane disruptions, whereas smaller disruptions of <100 nm reseal through budding and exocytosis (FIGURE 1D). Repair through budding involves pinching the membrane at the injured site and shedding the injured membrane into the extracellular space (FIGURE 1E) (4, 74, 79). Endocytosis is also thought to contribute to membrane repair by internalization of the injured membrane (FIGURE 1F) (70, 74). How and under what specific conditions these mechanisms contribute to membrane repair is still an area of investigation. These non-exclusive mechanisms could be relevant in a cell-type and context-dependent fashion. It is clear that compromised membrane repair contributes to pathophysiology in a number of different tissues and that it is linked to muscular dystrophy, heart failure, neurodegeneration, and other diseases (5, 7, 24, 27, 50, 72, 133, 140). Despite the importance of membrane repair in cellular function, the field has only recently begun to expand with the discovery of more proteins linked to resealing damaged cell membranes. The focus of this review is to identify several proteins currently linked to membrane repair and to describe some of the key findings on their functions.
Why do detergents break apart the cellular membrane but leave DNA in tact?
Because detergents have the same chemical properties as membranes. Membranes are comprised of lipids, and detergents are made up of fatty acids, which can dissolve lipids. Membranes are held together by hydrophobic forces, not covalent forces, so detergents, which have large regions of hydrophobicity can disrupt the hydrophobic forces that hold membranes together.
DNA isn’t affected because its structure doesn’t depend on hydrophobic forces in a substantial way.
https://science.sciencemag.org/content/310/5753/1451
Photo from this page:
https://www.slideshare.net/MissWander/42-parts-of-a-eukaryotic-cell-presentation
I learned that once a coronavirus hijacks a human cell, that one cell can produce hundreds of coronaviruses.
From this page:
https://www.latimes.com/projects/how-coronavirus-invade-cells-replicates/
Once inside, the coronavirus enlists the infected cell to produce the parts it needs: RNA and proteins. First, the virus commandeers the cell’s machinery into making tools that can copy coronavirus RNA in bulk. The copying process occurs in a double-membrane compartment that keeps the virus hidden from the cell.
Some of the RNA copies are packaged into the next generation of viruses. Other copies are used to tell the cell how to make viral proteins, including the ones that will encase the RNA.
Keep up-to-date on the COVID-19 discussion through the Coronavirus Space. For health and prevention information, visit the Centers for Disease Control and Prevention at CDC.gov
The safety effect of washing your hands with soap lasts until you touch something contaminated with the coronavirus (SARS-CoV-2) that causes COVID-9. Now you have coronavirus on your hands again and you should wash them again as soon as possible.
Soap essentially makes the dirt slippery, so it doesn’t stick to things (like your hands) and can be more effectively rinsed off them. Soap also usually contains ingredients that break up grease, again making it easier to rinse away. In general, washing is about sanitizing (removing dirt that might bear viruses or bacteria) rather than sterilizing (destroying viruses and bacteria). Sanitizing is a lot easier than sterilizing.
But the coronavirus (specifically the SARS-CoV-2 coronavirus) is a special case in that the soap does more than make it easier to rinse away coronavirus contamination, it actually disrupts the structure of the virus itself. The RNA payload inside the virus is surrounded by a “lipid shell”. For “lipid” think “grease”. Soap and hot water breaks up grease, so washing your hands strips away that lipid shell. Without the lipid shell, the RNA payload inside the virus is deactivated (falls apart) fairly quickly.
Note, this is part of why the advice from the CDC, etc, is to wash your hands for a good, solid 20 seconds, so the soap has time to break up the lipid shell.
Now we get into weird questions like, is it better to leave your hands soapy or not? In general the answer is no — in fact I’ve read that most soaps make a great “agar” (a technical term for an environment for bacteria, viruses, etc, to grow in). Obviously with this whole breaking-up-the-grease aspect of the coronavirus, it’s a bit more ambiguous. But I suspect most medical types would say it’s better to rinse away the soap and any remnants of the coronavirus.
Also note, the coronavirus is not a bloodborn virus, it’s respiratory. It can’t infect you just from being on your hands. The problem with getting some coronavirus on your hands is that you’re likely to unconsciously touch your face (most people have no idea how often they touch their face) and get it into your mouth, nose or eyes, which are all connected to your respiratory system. So if you think you might have contaminated your hands, be careful about touching your face and wash your hands as soon as possible.
Note, technically it’s not “killing” the virus but deactivating it. Scientists have some picky definitions of what’s “alive” and what’s not. But don’t worry, nobody outside of science circles really cares about that distinction right now.
Washing your hands is really, really effective for deactivating coronavirus, but you should mainly wash your hands when you might have come into contact with coronavirus, like after you go grocery shopping.
You should also routinely wash “high touch” surfaces, like the doorknobs and light switches in your house, to decrease the risk that you might have touched them after being contaminated, before you washed your hands.
You don’t need hand sanitizer, 70% alcohol, bleach or peroxide, though these can all be useful in some circumstances. Hand sanitizer is easier to use if you don’t have easy access to soap and water; some things are easier to spray down with alcohol/bleach/peroxide than to wash with soap and water, etc.
Disclaimer: I’m not medically trained, etc. I just pay attention to what the doctors and scientists tell me.
https://www.latimes.com/projects/how-coronavirus-invade-cells-replicates/
https://www.sciencedirect.com/science/article/pii/S0092867410001297
Autoimmunity and the Clearance of Dead Cells
Main Text
Introduction
In the Japanese movie Departures (Okuribito in Japanese), which won the 2009 Oscar for best foreign language film, death is regarded as a gate. The deceased are gently washed, dressed, and placed in a coffin for departure into the next life. Similarly, when the cells in our bodies die, an elaborate process takes place to remove them and to give them a new life by using their components.
Many extra cells are generated and die during animal development. In human adults, billions of cells die every day as part of the body's natural processes. Cells that become damaged by microbial infection or mechanical stress also die. The cell death that occurs in the physiological setting is programmed, and is therefore called programmed cell death (Lockshin and Zakeri, 2001). Apoptosis is the major death process, but necrosis and autophagic cell death have also been proposed to play roles in programmed cell death (Kroemer et al., 2009). Dying cells secrete a “find me” signal, and they expose an “eat me” signal on their surface. In response to the “find me” signal, macrophages approach the dead cells; they then recognize the “eat me” signal (Ravichandran and Lorenz, 2007). Using sophisticated cell machinery, the phagocytes ingest the dead cells, direct them to lysosomes, and degrade their cellular components into basic biochemical building blocks: amino acids, nucleotides, fatty acids, and monosaccharides. These molecules will be released from the lysosomes and reused to make new macromolecules. In definitive erythropoiesis, the process by which red blood cells are generated, the nuclei are extruded from erythroid precursor cells at the final differentiation stage and are engulfed by macrophages (Chasis and Mohandas, 2008). The machinery used for the engulfment and degradation of the extruded nuclei appears similar to that used for the removal of apoptotic cells.
Mice deficient in the engulfment of apoptotic cells develop systemic lupus erythematosus (SLE)-type autoimmune diseases (Hanayama et al., 2004). A defect in the degradation of the chromosomal DNA from engulfed cells in mice activates macrophages, leading to lethal anemia in embryos and chronic arthritis in adults (Kawane et al., 2001, Kawane et al., 2006). These observations indicate that dead cells and the nuclei expelled from erythroid precursor cells need to be swiftly cleared for animals to maintain homeostasis.
Programmed Cell Death
Based on morphological and biochemical criteria, four different cell-death processes (apoptosis, cornification, necrosis, and autophagy) have been officially proposed (Kroemer et al., 2009). In apoptosis, the cell and nuclei condense and become fragmented and are engulfed by phagocytes (Kerr et al., 1972). Apoptosis is regulated by gene products, and programmed cell death has often been used synonymously with apoptosis. However, necrosis is also regulated by gene products (Cho et al., 2009, He et al., 2009), and it may be preferable to use the term programmed cell death in only its more general sense, that is, to refer to any cell-death process that is programmed into animal development.
It is unclear to what extent the other proposed forms of cell death can be classified as programmed cell death. Of them, autophagy, in which organelles and macromolecules are trapped by the cell's own membranes and degraded in its lysosomes, is a process by which cells survive in starvation conditions (Ohsumi, 2001). Autophagy has been proposed as a cell-death process because cells undergoing severe or prolonged autophagy may die, and dying cells often show a characteristic, autophagic morphology (Tsujimoto and Shimizu, 2005). However, there are no convincing data supporting the notion that autophagy kills the cells, and hence the term autophagic cell death may be misleading (Kroemer and Levine, 2008). Cornification, a cell-differentiation process, describes the cell death that occurs at the final step in the natural differentiation of skin cells (Lippens et al., 2005). Similarly, the differentiation of the lens epithelial cells of the eye to fiber cells is accompanied by the degradation of nuclei, mitochondria, and endoplasmic reticulum (Bassnett, 2002), which can also be regarded as a cell-death process. However, it may not be appropriate to classify this cell-differentiation process as programmed cell death. In addition, although necrosis is mediated by gene products, it occurs only when apoptosis is blocked or when cells receive strong death signals under pathological conditions. Thus, we believe that apoptosis accounts for most of the physiological cell death during animal development and in the cell turnover that occurs daily.
Apoptosis
Apoptosis is activated by two pathways, the intrinsic and extrinsic pathways (Ow et al., 2008). In the intrinsic pathway, which operates in developmentally controlled and genotoxic agent-mediated apoptosis, BH3-only members of the Bcl-2 family are transcriptionally upregulated and stimulate the release of cytochrome C from the mitochondria. Together with Apaf-1, cytochrome C activates caspase 9, which leads to the activation of downstream caspases, including caspases 3 and 7. The antiapoptotic members of the Bcl-2 family inhibit the release of cytochrome C from mitochondria by a mechanism that has not been well elucidated. This intrinsic pathway is thought to be well conserved in metazoans, but its key step, the release of cytochrome C from mitochondria, is not observed in the nematode C. elegans or in the fruit fly Drosophila (Oberst et al., 2008).
Fas ligand (FasL), tumor necrosis factor (TNF), and TRAIL (TNF-related apoptosis-inducing ligand) are type II membrane proteins that can activate the extrinsic death pathway (Krammer, 2000, Nagata, 1997, Strasser et al., 2009). The binding of FasL to its receptor (Fas) induces the formation of the death-inducing signaling complex (DISC), consisting of Fas, an adaptor protein (FADD), and procaspase 8. Formation of the DISC leads to the processing and activation of caspase 8. Depending on the cell type, there are two pathways that can be activated downstream of caspase 8. In type I cells (for example, thymocytes), caspase 8 directly activates caspase 3 to kill the cells; in type II cells (hepatocytes), caspase 8 cleaves Bid, a BH3-only member of the Bcl-2 family, and the cleaved Bid (tBid) induces the release of cytochrome C from the mitochondria, which leads to the activation of the caspase 9-caspase 3 pathway.
In both the intrinsic and extrinsic pathways, apoptosis is completed by the cleavage of a set of cellular proteins (more than 500 substrates) by effector caspases (caspases 3 and 7) (Lüthi and Martin, 2007, Timmer and Salvesen, 2007) (http://cutdb.burnham.org/; http://bioinf.gen.tcd.ie/casbah/). The massive protein cleavage is probably responsible for the morphological and biochemical changes that occur during apoptosis, and for killing the cells. However, with a few exceptions (see below), the physiological meaning of the specific cleavage events is not clear. In addition to killing the cells, caspase activation is involved in the cells' production of the “find me” and “eat me” signals sensed by phagocytes.
Apoptotic DNA Degradation and Membrane Blebbing
One of notable hallmarks of apoptosis is DNA fragmentation, the cleavage of chromosomal DNA into 180 bp nucleosomal units (Wyllie, 1980). This process, accomplished by CAD (caspase-activated DNase), also called DFF-40 (DNA fragmentation factor 40), is the most representative example of how caspase activation causes a characteristic feature of apoptosis (Enari et al., 1998, Liu et al., 1997). In healthy cells, CAD is complexed with its inhibitor, ICAD (inhibitor of CAD), also called DFF-45 (Enari et al., 1998, Liu et al., 1997, Sakahira et al., 1998), which also acts as a chaperone for CAD to ensure its correct folding (Sakahira et al., 2000). Caspase 3 cleaves ICAD at two positions (Sakahira et al., 1998), which allows CAD to form a homodimer that has a scissor-like structure (Woo et al., 2004). CAD carries a nuclear-localization signal and cleaves DNA in the nucleus via specific histidine residues (Sakahira et al., 2001) located in the deep cleft between the “blades” of the “scissors.” This structure prevents CAD from accessing the DNA on nucleosomes, but allows it access to DNA in the spacer regions between them, which explains why the chromosomal DNA is degraded into nucleosomal units during apoptotic cell death. CAD generates DNA fragments with a 3′-hydroxyl group. This group is identified by TUNEL (terminal transferase-mediated dUTP nick end labeling) staining, which is widely used to detect apoptotic cells in vitro and in vivo. At the early stage of apoptosis, DNA is degraded into relatively large pieces (50–200 kb). Endonucleases other than CAD have been postulated to perform this cleavage (Samejima et al., 2001, Susin et al., 2000). However, at least in our hands, no DNA degradation (whether to high-molecular-weight fragments or nucleosomal units) can be observed in cells lacking CAD (Kawane et al., 2003), indicating that other nucleases are not involved or play only a limited role.
Caspase cleavage also explains another hallmark of apoptotis, membrane blebbing. ROCK1 (Rho-associated kinase 1), a substrate of caspase 3, phosphorylates various cytoskeletal proteins, including myosin light chain, and regulates the actin cytoskeleton. ROCK1 is normally regulated by Rho GTPase, but its cleavage by caspase 3 removes its regulatory domain and renders it constitutively active (Coleman et al., 2001, Sebbagh et al., 2001). This aberrantly activated ROCK1 intensively phosphorylates myosin light chain, leading to membrane blebbing.
The microinjection of active CAD into cells causes DNA fragmentation and quickly kills the cells (Susin et al., 2000). In contrast, CAD-deficient cells are efficiently killed by apoptotic stimuli without DNA degradation (Kawane et al., 2003). As described above, given that many proteins and enzymes essential for cell survival are cleaved and inactivated by caspases, it is likely that once caspases are activated by apoptotic stimuli, there are many ways to kill the cells.
Engulfment of Apoptotic Cells
When apoptotic cells are left on a Petri dish for a long time, their plasma membrane ruptures and cellular contents are released, in a process called secondary necrosis (Kerr et al., 1972). On the other hand, apoptotic cells in vivo are quickly recognized by phagocytes and engulfed to prevent the release of their intracellular materials, which can be immunogenic. For the specific and efficient engulfment of apoptotic cells, the dead cells discharge molecules to recruit phagocytes (“find me” signals), and they expose on their surface molecules that are recognized by phagocytes (“eat me” signals) (Figure 1).
Figure 1. Engulfment of Apoptotic Cells by Macrophages
When cells undergo apoptosis, they release “find me” signals to recruit macrophages, and expose “eat me” signals on their surface. In response to the “find me” signal, macrophages approach the dead cells, and they engulf them by recognizing the “eat me” signal. The engulfed dead cells are transferred to lysosomes, where all their components are degraded into amino acids, nucleotides, fatty acids, and monosaccharides by lysosomal enzymes.
“Find Me” Signals
By assaying the ability of the culture supernatant from apoptotic cells to trigger the chemotaxis of macrophages, Lauber et al. (2003) identified lysophosphatidylcholine (LPC) as a “find me” signal (Figure 2). It is released from apoptotic cells by the caspase-3-dependent activation of phospholipase A2, which converts phosphatidylcholine to LPC. The binding of LPC to G2A (G2 accumulation protein or G protein-coupled receptor 132) activates macrophages to undergo chemotaxis (Peter et al., 2008). This model is attractive, but the concentration of LPC required to cause the chemotaxis of phagocytes is rather high (20–30 μM) and may not be reached physiologically.
Figure 2. Proposed “Find Me” Signals
As “find me” signals, ATP/UTP, lysophosphatidylcholine (LPC), sphingosine-1-phosphate (S1P), and fractalkine CX3CL1 have been proposed. These molecules bind specific receptors on macrophages, all of which are G protein-coupled seven-transmembrane receptors, and activate them for chemotaxis.
Two other molecules, sphingosine-1-phosphate (S1P) and CX3CL1/fractalkine, have also been proposed to act as “find me” signals (Gude et al., 2008, Truman et al., 2008). S1P is produced by sphingosine kinase in a caspase-dependent manner and secreted from apoptotic cells; it stimulates the chemotaxis of macrophages by binding its specific receptor, S1P-R. Fractalkine, CX3CL1, is synthesized as a membrane-associated protein, rapidly processed, and released from apoptotic neurons or B cells. It activates microglia and macrophages to undergo chemotaxis by binding to its receptor, CX3CR. In addition, ATP and UTP released from apoptotic cells in a caspase-dependent manner have recently been shown to act as “find me” signals for apoptotic cells (Elliott et al., 2009). Whether these proposed “find me” signals are redundant, additive, or synergistic remains to be studied.
Apoptotic cells appear to mostly (or exclusively) recruit macrophages (Truman et al., 2004). Yet, the proposed molecules (LPC, S1P, and ATP/UTP) activate not only macrophages but also neutrophils and lymphocytes (Florey and Haskard, 2009, Lecut et al., 2009). Bournazou et al. (2009) propose that lactoferrin is synthesized in apoptotic cells, secreted, and inhibits the migration of neutrophils. However, this may not be consistent with the quick killing of the death-factor-induced apoptosis that does not require protein synthesis. An involvement of lactoferrin in the “find me” process should be clarified with the lactoferrin-deficient mice (Ward et al., 2003).
“Eat Me” Signals
Macrophages engulf dead cells but not healthy ones, indicating either that dying cells expose “eat me” signals recognized by phagocytes or that healthy cells display “don't eat me” signals. The best-studied “eat me” signal is phosphatidylserine, a component of the cell plasma membrane that is kept exclusively on the inner leaflet of the lipid bilayer in healthy cells (Balasubramanian and Schroit, 2003). Phosphatidylserine is exposed on the cell surface when cells undergo apoptosis (Fadok et al., 1992). Moreover, when phosphatidylserine is inserted into the plasma membrane of erythrocytes, they are recognized and engulfed by macrophages (Tanaka and Schroit, 1983). Furthermore, the masking of phosphatidylserine inhibits the engulfment of apoptotic cells in vitro and in vivo (Asano et al., 2004, Krahling et al., 1999). These results strongly point to phosphatidylserine as the most likely candidate for the “eat me” signal.
The exposure of phosphatidylserine on the surface of apoptotic cells is found not only in mammals but also in Drosophila and C. elegans (van den Eijnde et al., 1998, Venegas and Zhou, 2007). This process is caspase dependent (Martin et al., 1996), but how caspase activity leads to the cell-surface exposure of phosphatidylserine remains unsettled (Schlegel and Williamson, 2007). In one model, ATP-dependent translocases that maintain phosphatidylserine at the inner leaflet of the plasma membrane are inactivated in apoptotic cells, but Ca2+-dependent phospholipid scamblase is activated, causing randomization of the membrane leaflet components (Balasubramanian and Schroit, 2003, Sahu et al., 2007). This model has been examined in mammals and C. elegans, but with controversial results (Darland-Ransom et al., 2008, Züllig et al., 2007).
CD47, also called integrin-associated protein (IAP), is a membrane protein with five membrane-spanning regions. When CD47-deficient red blood cells are injected into mice, they are more rapidly cleared by macrophages in the spleen than are CD47-positive cells. Oldenborg et al. (2000) therefore proposed that CD47 serves as a “don't eat me” signal. However, when CD47-positive red blood cells are loaded with phosphatidylserine, they are efficiently engulfed by macrophages (Tanaka and Schroit, 1983). Thymocytes express abundant CD47. When they undergo apoptosis, their CD47 expression is not lost, yet the apoptotic thymocytes are still efficiently engulfed by macrophages (Tada et al., 2003). These observations indicate that the “eat me” signal can overcome the “don't eat me” signal.
Bridging Molecules that Recognize Phosphatidylserine
Several secreted proteins have been identified as molecules that recognize the phosphatidylserine on apoptotic cells and promote their engulfment (Figure 3). Milk fat globule EGF factor 8 (MFG-E8), originally found associated with milk fat globules in mammary glands, is a secreted protein present on a subset of phagocytes that actively engulf apoptotic cells (Hanayama et al., 2002). It is expressed by macrophages and immature dendritic cells, including tingible-body macrophages and follicular dendritic cells at the germinal centers in the spleen and lymph nodes, thioglycollate-elicited peritoneal macrophages, granulocyte-macrophage colony stimulating factor (GM-CSF)-induced bone marrow-derived immature dendritic cells, and Langerhans cells in the skin (Hanayama et al., 2004, Kranich et al., 2008, Miyasaka et al., 2004). MFG-E8 contains one (human) or two (mouse) epidermal growth factor (EGF) domains in its N-terminal half, with the human and second mouse EGF domain carrying an RGD (Arg-Gly-Asp) motif. It has two factor-VIII-homologous domains (C1 and C2) in its C-terminal region. MFG-E8 associates with αvβ3 or αvβ5 integrin on phagocytes via its RGD motif (Yamaguchi et al., 2008), binds tightly to phosphatidylserine through its C1 and C2 domains, and stimulates the engulfment of apoptotic cells (Hanayama et al., 2002).
Figure 3. Molecules Proposed to Recognize Phosphatidylserine
The most likely “eat me” signal is phosphatidylserine. MFG-E8 and Gas6 are secreted proteins that bind phosphatidylserine and work as bridging molecules between apoptotic cells and macrophages. Tim-4, BAI1, and Stabilin-2 are type I-membrane proteins that are proposed phosphatidylserine receptors. Molecules that activate Rac1 (CrkII, Dock180, Elmo, and GULP) are involved in the engulfment of apoptotic cells.
Two related proteins, growth arrest-specific 6 (Gas6) and protein S, which are abundant in plasma, bind phosphatidylserine (Nakano et al., 1997). TAM family members (Tyro3, Axl, and Mer), which are tyrosine-kinase receptors, are the receptors for Gas6 and protein S. Gas6 and protein S are involved in the vitamin K-dependent clotting system, and a deficiency in Gas6 or its receptor causes platelet dysfunction (Angelillo-Scherrer et al., 2005, Angelillo-Scherrer et al., 2001). On the other hand, mice expressing a kinase-dead mutant of Mer (MerKD) develop SLE-like autoimmunity (Scott et al., 2001), and the Gas6-TAM system has been proposed to play a role in the engulfment of apoptotic cells, particularly in the testis and retina (Prasad et al., 2006, Xiong et al., 2008). A recent report indicates that TAM receptors negatively regulate the innate immune reaction, and a lack of TAM receptors causes dendritic cells to overproduce interleukin-6 (IL-6), interferon (IFN), and TNFα (Rothlin et al., 2007). Given that SLE-type autoimmunity is regulated by cytokines, this overproduction of cytokines by dendritic cells might be responsible for the SLE-like autoimmunity found in the MERKD mice (Scott et al., 2001).
Phosphatidylserine Receptors
Whether macrophages directly recognize apoptotic cells has been difficult to elucidate. A protein initially identified by Fadok et al. (2000) as a phosphatidylserine receptor (and consequently named PSR) is now reported to have a different function. Fadok et al. identified PSR by screening a phage-display library with a monoclonal antibody that inhibits the engulfment of apoptotic cells. Several groups subsequently reported that the deficiency of PSR causes the impaired engulfment of apoptotic cells, resulting in embryonic lethality in the mouse (Kunisaki et al., 2004, Li et al., 2003) and delayed engulfment of apoptotic cells in C. elegans (Wang et al., 2003). In contrast, Böse et al. (2004) who independently established PSR knockout mice, reported that PSR is not the protein recognized by the monoclonal antibody used by Fadok et al., and that PSR null macrophages have no defect in the engulfment of apoptotic cells. Subsequent reports have indicated that PSR is a chromatin-remodeling factor called Jumonji domain-containing 6 protein (JMJD6), which is present in the nucleus (Chang et al., 2007). Hence, the increased number of unengulfed apoptotic cells in the animals lacking PSR may be due to increased cell death caused by the lack of JMJD6's chromatin remodeling function.
In investigating the mechanism by which macrophages that do not express MFG-E8 engulf apoptotic cells, we reported that type I membrane proteins called T cell immunoglobulin- and mucin-domain-containing molecule 4 (Tim-4) and Tim-1 serve as phosphatidylserine receptors (Miyanishi et al., 2007) (Figure 3). Tim-1 and Tim-4 consist of a signal sequence, an immunoglobulin V (IgV) domain, a mucin-like domain, a transmembrane domain, and a cytoplasmic region. They specifically bind phosphatidylserine with high affinity via their IgV domain. When Tim-1 or Tim-4 is expressed in mouse fibroblasts (NIH 3T3), which do not normally express Tim family members, the transformants efficiently engulf apoptotic cells. The short cytoplasmic region of Tim-4 is dispensable for the engulfment (Park et al., 2009) (M. Murai, M. Miyanishi, and S.N., unpublished data), indicating that Tim-4 associates with endogenous molecules on the fibroblast membrane to activate the engulfment signal. Among other Tim family members, Tim-3 also binds phosphatidylserine and stimulates the engulfment of apoptotic cells, although with less efficiency than Tim-1 or Tim-4 (Nakayama et al., 2009).
Tim-4 is expressed by macrophages and dendritic cells in the spleen, lymph nodes, thymus, and tonsils (Shakhov et al., 2004), and Tim-3 is expressed in CD8+ dendritic cells in the spleen (Nakayama et al., 2009). These macrophages and dendritic cells are responsible for the engulfment of apoptotic cells and for the presentation of dead cell-associated antigens (Miyake et al., 2007). Tim-1, also called kidney injury molecule 1 (Kim-1), is expressed in kidney epithelial cells after ischemic injury (Ichimura et al., 2008) and in Th2 cells (Umetsu et al., 2005). In the kidney, Tim-1 is likely to be responsible for engulfing the damaged apoptotic or necrotic cell debris generated during ischemic injury, but the role of Tim-1 in Th2 cells is not clear. The Tim family genes are clustered on human chromosome 5q33.2 and mouse chromosome 11B1.1, which is the susceptible gene locus for the development of atopy (allergic hypersensitivity) and asthma (Kuchroo et al., 2003). Whether the newly identified function of Tim-1, Tim-3, and Tim-4 in apoptotic cell engulfment or the originally proposed function of Tim-1 and Tim-4 in the costimulation of T cells (Kuchroo et al., 2003) is responsible for this phenotype remains to be studied.
Park et al. (2007) report that brain-specific angiogenesis inhibitor 1 (BAI1), a member of the secretin/vasoactive intestinal polypeptide (VIP) receptor family with 7-transmembrane regions, is another potential phosphatidylserine receptor for apoptotic cells. BAI1 binds via thrombospondin type 1 repeats (TSPs) to phosphatidylserine, as well as to cardiolipin and other phospholipids. Its cytoplasmic region can interact with the signal transducer ELMO (see below) (Park et al., 2007). However, BAI1's possible function as a phosphatidylserine receptor for apoptotic cells seems to conflict with its neuron-specific expression in the brain (Mori et al., 2002). Another candidate phosphatidylserine receptor is stabilin-2, also called HARE (hyaluronic acid receptor for endocytosis), a type I membrane protein that carries a large extracellular region with seven fasciclin domains and fifteen EGF-like domains (Park et al., 2008). It is expressed by the sinusoidal endothelial cells of the spleen, lymph nodes, and bone marrow (Nonaka et al., 2007) and functions as a receptor for hyaluronic acids and heparin to regulate blood viscosity (Harris et al., 2008). How stabilin-2 accomplishes two jobs, as a phosphatidylserine receptor for apoptotic cells and as a scavenger receptor for hyaluronic acids, would be an interesting topic for study.
Signaling Pathways for Engulfment
Genetic analyses in C. elegans identified seven genes that mediate the recognition and engulfment of apoptotic cells in two parallel and partially redundant signaling pathways (the CED-1/-6/-7 and CED-2/-5/-10/-12 pathways) (Table 1) (Reddien and Horvitz, 2004). CED-1 is a transmembrane receptor that has multiple EGF-like domains in its extracellular region; it has high homology with mammalian multiple EGF-like-domains 10 (MEGF10) (Hamon et al., 2006) and may recognize phosphatidylserine on apoptotic cells (Venegas and Zhou, 2007). CED-6 is an ortholog of mammalian GULP (PTB [phosphotyrosine-binding] domain-containing engulfment adaptor protein) and binds to the intracellular domain of CED-1 (Su et al., 2002). CED-7 is homologous to the ABC transporters that actively transport a variety of substances across the plasma membrane and was originally suggested to be responsible for exposing the “eat me” signal on apoptotic cells. However, the ABC transporter CED-7 interacts with MEGF10 (CED-1) (Hamon et al., 2006), indicating that it functions in the engulfment process in phagocytes.
Table 1. Molecules Involved in the Engulfment of Apoptotic Cells
| C. elegans | Mammalian | Properties |
|---|---|---|
| CED-1 | MEGF10 | Type I membrane protein with multiple epidermal growth factor (EGF)-like domains in the extracellular region |
| CED-2 | CrkII | Cytoplasmic protein with a Src homology 2 (SH2) and an SH3 domain, that functions as an adaptor for signal transduction |
| CED-5 | Dock180 | Cytoplasmic protein containing an SH3 domain; it associates with CrkII and ELMO, and activates the Rho family GTPase Rac1 as a guanine exchange factor |
| CED-6 | GULP | Cytoplasmic protein with a phosphotyrosine-binding domain (PTB) and four SH3-binding motifs; it binds to CED-1 and functions upstream of CED-10 |
| CED-7 | ABC transporter | Protein with two homologous repeats, each harboring six transmembrane segments and one ATP-binding site |
| CED-10 | Rac1 | Small GTP-binding protein of the Ras superfamily |
| CED-12 | ELMO | Cytoplasmic protein; it associates with CrkII and Dock180, and activates Rac1 |
CED-2, -5, -10, and -12 correspond to mammalian CrkII, Dock180, Rac1, and ELMO1, respectively. CED-2/CrkII associates with CED-5/Dock180, a guanine-nucleotide exchange factor for CED-10/Rac1, and this interaction is positively regulated by CED-12/ELMO1 (Côté and Vuori, 2007). This pathway regulates actin polymerization and is involved not only in apoptotic-cell engulfment (Figure 3) but also in cell migration, neurite growth, and myoblast fusion. Integrin family members may act upstream of this pathway, but how apoptotic cells activate this pathway remains to be determined.
The engulfment of apoptotic cells is regulated by Rho family GTPases (Rac1, RhoA, Rab5, etc.) (Nakaya et al., 2006) and can be monitored at the molecular level by imaging using an actin-green fluorescent protein (GFP) fusion protein (Nakaya et al., 2008). This type of analysis indicates that the engulfment of apoptotic cells appears to occur at a limited number of portals in the phagocyte lamellipodia. A fluorescence resonance energy transfer (FRET) analysis for Rac1 indicates that the activation and deactivation of Rac1, controlled by “engulfment synapses,” must be regulated with specific timing for the efficient engulfment of apoptotic cells. That is, when a phagocyte starts to engulf an apoptotic cell, activated Rac1 and integrin are recruited to the portal and induce the formation of phagocytic cups consisting of an actin patch. As soon as the dead cell sinks into the phagocyte through one of these cups, Rac1 is inactivated and the actin is depolymerized. Subsequently, Rab5 regulates the transfer of the dead-cell cargoes into lysosomes (Kitano et al., 2008).
The uptake of apoptotic cells by phagocytes induces the expression of transforming growth factor β (TGFβ) and IL-10 (Fadok et al., 2001) (Figure 4), which may inhibit the further recruitment of macrophages to the dying cells. On the other hand, if the dead cells persist in tissues, either because of impaired engulfment or because the number of apoptotic cells overwhelms the capacity of the phagocytes, the apoptotic cells undergo necrosis. When necrotic cells interact with or are engulfed by macrophages, the macrophages produce inflammatory cytokines (Fadok et al., 2001), which may recruit more macrophages as reinforcements. The activation of different cytokine genes upon their engulfment of apoptotic and necrotic cells suggests that the signal transduction pathways induced by these dead cells are different.
Figure 4. The Engulfment of Apoptotic versus Necrotic Cells
Macrophages engulfing apoptotic cells produce transforming growth factor β (TGFβ) and prostaglandin E2 (PGE2), which function as anti-inflammatory agents to inhibit the further recruitment of macrophages. When dead cells undergo secondary necrosis, the necrotic cells may activate macrophages through Fc receptor (FcR) and Toll-like receptors (TLRs) to produce inflammatory cytokines, such as tumor necrosis factor α (TNFα) and interleukin 8 (IL-8), which act to recruit more macrophages.
Autoimmune Disease Caused by the Inefficient Engulfment of Dead Cells
Systemic Lupus Erythematosus is a chronic autoimmune disease that causes a broad spectrum of clinical manifestations affecting the skin, kidney, lungs, blood vessels, and nervous system (D'Cruz et al., 2007). Patients with SLE have autoantibodies in their sera against nuclear components (anti-ribonucleoprotein and anti-DNA antibodies) and sometimes exhibit circulating DNA or nucleosomes (Rumore and Steinman, 1990). As unengulfed apoptotic cells are present in the germinal centers of the lymph nodes of some SLE patients and macrophages from these patients often show a reduced ability to engulf apoptotic cells, a deficiency in the clearance of apoptotic cells is proposed to be one of the causes of SLE (Gaipl et al., 2006).
MFG-E8-deficient female mice, particularly of the B6/129-mixed background, develop an age-dependent SLE type of autoimmune disease (Hanayama et al., 2004). These mice produce high concentrations of anti-double-stranded DNA and anti-nuclear antibodies and suffer from glomerular nephritis. When MFG-E8-deficient mice are immunized with keyhole limpet hemocyanin (KLH) to activate B lymphocytes, many apoptotic cells are left unengulfed on the tingible-body macrophages in the germinal centers, confirming that MFG-E8 has a nonredundant role in vivo in the engulfment of apoptotic cells by the tingible-body macrophages. It is likely that the unengulfed dead cells in MFG-E8-deficient mice undergo a secondary necrosis and release cellular components that activate the immune system to produce autoantibodies (Figure 5A). Like Fas-deficient lpr mice, in which autoreactive B cells are activated in a T cell-independent but Toll-like receptor (TLR)- and B cell receptor (BCR)-dependent mechanism (Herlands et al., 2008), the released cellular components may activate autoreactive B cells in a BCR- and TLR-dependent manner. This activation of autoreactive B cells may be further enhanced by cytokines produced by macrophages in response to stimulation by the necrotic cells. In any case, the MFG-E8-deficient mice provide a good model system for studying the molecular mechanisms by which endogenous cellular components activate the immune system extracellularly.
Figure 5. Immune System Activation by the Defective Engulfment of Apoptotic Cells
(A) Extracellular activation. If apoptotic cells are not swiftly engulfed, they undergo secondary necrosis, in which the plasma membrane is disintegrated, and the cellular components are released. Immunoglobulins and complement proteins bind to these cellular components and activate macrophages and B lymphocytes. In addition to FcR and B cell receptors (BCRs), Toll-like receptors (TLRs) appear to be involved in recognizing the cellular components and activating macrophages and B cells. The activated macrophages produce cytokines that will stimulate B cells to produce autoantibodies.
(B) Intracellular activation. After being engulfed by macrophages, dead cells are transferred to lysosomes and degraded. If the degradation does not occur properly, dead cell components accumulate in the lysosomes, leading to the intracellular activation of the innate immune system to produce proinflammatory cytokines such as interferon β (IFNβ) and tumor necrosis factor α (TNFα).
As described above, apoptotic cells are rapidly recognized and engulfed by macrophages at the early stage of their death process, mostly in a phosphatidylserine-dependent manner. On the other hand, how necrotic cells are recognized and engulfed by macrophages is not well elucidated. One likely system for clearing necrotic cells is the complement system (Trouw et al., 2008). C1q binds to dead cells at the later stages of apoptosis in an IgM-dependent manner (Ogden et al., 2005), and one of the signals on the dead cells for IgM-binding is lysophosphatidylcholine (Kim et al., 2002). Notably, in humans, almost all individuals deficient in the C1q gene develop severe SLE (Botto and Walport, 2002). In C1q-deficient mice, unengulfed dead cells persist in the glomeruli of the kidneys, and the mice develop an SLE-like phenotype; this is particularly evident in the MRL/MP strain, which has a defect in the clearance of apoptotic cells (Potter et al., 2003). In this regard, the C1q-mediated engulfment may be a backup system for clearing dead cells, and it may be informative to cross C1q-deficient mice with MFG-E8-deficient mice.
Degradation of Apoptotic Cells in Macrophages
Activation of the Innate Immunity by Undigested DNA Left in Lysosomes
After apoptotic cells are engulfed by phagocytes, all of their components are degraded into amino acids, nucleotides, fatty acids, and monosaccharides in lysosomes. As described above, the chromosomal DNA of the apoptotic cells is degraded cell autonomously into 180 bp nucleosomal units by CAD and then further degraded in the lysosomes of macrophages. The enzyme that degrades the DNA of apoptotic cells in lysosomes is DNase II (Kawane et al., 2003), which functions under acidic conditions (Evans and Aguilera, 2003). DNase II is ubiquitously expressed in various tissues, particularly in macrophages. A lack of DNase II causes the accumulation of 180 bp fragmented DNA in macrophages (Kawane et al., 2001) and activates the macrophages to produce various cytokines. One of the cytokines produced by these macrophages is IFNβ, which is cytotoxic to erythroblasts and lymphocytes (Yoshida et al., 2005b) (Figure 5B).
Lethal Anemia and Polyarthritis Resulting from a Defect in DNA Degradation
Mice lacking DNase II die late in embryogenesis because of severe anemia (Kawane et al., 2001). Many TUNEL-positive erythroblasts can be found in the liver of mouse embryos lacking DNase II, and a deficiency in the IFN-type I receptor gene rescues their lethality (Yoshida et al., 2005b), indicating that the erythroblasts are killed by the action of IFNβ. Mice with a double deficiency for DNase II and IFN-type I receptor, or mice in which the DNase II gene is deleted after birth via an inducible conditional knockout strategy, develop polyarthritis as they age (Kawane et al., 2006). Their swollen joints show severe synovitis with aggressive pannus formation. The pannus carries osteoclasts at its leading edge, fills the joint cavity, erodes the cartilage, and destroys the bone. As in the joints of human rheumatoid arthritis patients, the genes for inflammatory cytokines (IL-1β, IL-6, and TNFα) are strongly activated in the affected joints. Human patients with rheumatoid arthritis are successfully treated with reagents that antagonize TNFα or IL-6 (Feldmann, 2002, Yokota et al., 2008). Similarly, the administration of an anti-TNFα antibody significantly improves the clinical score for the polyarthritis developed by the DNase II null mice (Kawane et al., 2006).
What triggers the rheumatoid arthritis in humans is unknown. In the DNase II null mice, macrophages carrying undigested DNA express TNFα mRNA (Kawane et al., 2006), and a low, but significant, level of TNFα is found in the serum before the joints show any abnormality. Given that TNF-transgenic mice, which constitutively produce a low level of TNFα, develop polyarthritis (Keffer et al., 1991), it is likely that the TNFα produced by the macrophages carrying undigested DNA is responsible for the development of the polyarthritis. Synovial cells respond to TNFα with high sensitivity to produce IL-1β and IL-6, which in turn stimulate the expression of the TNFα gene (Taberner et al., 2005, Zhang et al., 2004), causing a “cytokine storm” in the joint. This leads to the growth of synovial cells, pannus formation, and the development of polyarthritis (Migita et al., 2001).
The pathologies (anemia and polyarthritis) caused by a deficiency of DNase II are examples of lysosomal storage diseases, which are diseases caused by the inactivation or malfunction of lysosomal enzymes, including proteases, glycosidases, and lipases (Neufeld, 1991). Proteins, polysaccharides, DNA, and RNA of bacterial or viral origin activate the innate immunity to produce various cytokines (Uematsu and Akira, 2007). The results from the DNase II null mice indicate that mammalian DNA that accumulates in the lysosomes of macrophages also activates the innate immune response. Other cellular components that escape degradation in the lysosomes may also activate the TNFα and IFNβ genes. The fact that cytokines are constitutively secreted by macrophages lacking lysosomal acid lipase (Lian et al., 2004), and by fibroblasts derived from patients with Niemann-Pick Disease Type C (Suzuki et al., 2007), an inherited lipid storage disorder, may support this notion. Some patients with rheumatoid arthritis can be cured by bone marrow transplantation (Ikehara, 2002), suggesting that these patients have a defect(s) in bone-marrow-derived cells. Determining whether these patients have lysosomal enzyme defects will be useful for improving their treatment.
Signaling from DNA to Cytokine Gene Expression
Cells that are infected by viruses or bacteria normally produce IFNβ and TNFα (Honda et al., 2006). There are two pathways by which pathogens activate the cytokine genes. In one, TLR recognizes pathogens extracellularly and transduces signals via the adaptor proteins MyD88 and TRIF to activate the transcription factors IFN-regulatory factor (IRF)3/IRF7 and NF-κB, which induce IFNβ and TNFα. In the other pathway, RIG-I/MDA5 recognizes intracellular pathogens and activates IRF3/IRF7 and NF-κB via an adaptor called IPS-1. The expression of the IFNβ and TNFα genes in the macrophages lacking DNase II is not blocked by a deficiency in the TLR system, indicating that the mammalian DNA that accumulates in the lysosomes activates the innate immune system in a TLR-independent manner (Okabe et al., 2005). We recently found that mammalian DNA in lysosomes activates TNFα and IFNβ gene expression through the system for intracellular pathogens and that this system can be regulated by Janus phosphatases called Eyes absent (Eya) (Okabe et al., 2009). Eya binds to IPS-1 and is involved not only in the mammalian DNA-mediated innate immune reaction but also in the virus-induced one, indicating that endogenous DNA and viruses intracellularly activate the innate immune response using a similar mechanism. Identification of the targets of Eya's phosphatase will contribute to the understanding how the intracellular pathogens activate innate immunity.
Engulfment and Degradation of Nuclei from Erythroid Precursors
Early in mammalian embryogenesis, red blood cells are produced in the yolk sac in a process called primitive erythropoiesis. Erythropoiesis then takes place in the fetal liver at later stages of embryogenesis and in the bone marrow after birth, and this process is called definitive erythropoiesis. Unlike the nucleated erythroid cells produced in the yolk sac, those produced in the fetal liver and bone marrow are enucleated. The definitive erythropoiesis in both the bone marrow and fetal liver takes place in anatomical units called erythroblastic islands. At the center of each island, there is a macrophage that supports the proliferation and differentiation of the erythroid precursor cells (Chasis and Mohandas, 2008). At the final stage of erythropoiesis, the erythroid cells autonomously undergo enucleation, and the expelled nuclei are engulfed by the central macrophage, suggesting that the expelled nuclei also expose an “eat me” signal on their surface (Figure 6).
Figure 6. Engulfment of the Nuclei Expelled from Erythroid Precursor Cells
At the final stage of definitive erythropoiesis, an erythroblast undergoes unequal division into a reticulocyte and a nucleus surrounded by plasma membrane. Like apoptotic cells, the plasma membrane surrounding the nucleus exposes phosphatidylserine as an “eat me” signal and is engulfed by macrophages.
The engulfment of expelled nuclei by macrophages has been shown to be phosphatidylserine dependent in experiments using nuclei collected from cultured erythroid precursor cells that spontaneously undergo enucleation (Yoshida et al., 2005a). Immediately after a nucleus is separated from its reticulocyte, phosphatidylserine is exposed on the outer leaflet of the plasma membrane surrounding the nucleus. It is likely that the plasma membrane cannot maintain its integrity because of a lack of ATP, because once separated from the reticulocyte, the nucleus loses its sources of new ATP (mitochondria and glycolysis). It is not yet known what molecules in the macrophages of the fetal liver and bone marrow are involved in recognizing the phosphatidylserine on the nuclei and engulfing them.
Every day, 2 × 1011 new red blood cells are produced in a human adult, meaning that this number of nuclei needs to be phagocytosed. This is at least ten times the number of dead cells. If nuclei, which are highly immunogenic, are released into the circulation because of inefficient engulfment, they will activate the immune system. In DNase II-deficient mice, the macrophages at the erythroblastic islands in the fetal liver and bone marrow carry a number of undigested nuclei in their lysosomes, indicating that DNase II is responsible for degrading the DNA from the engulfed erythroblast nuclei (Kawane et al., 2001). This means that, each day, the DNA from 2 × 1011 erythroblasts in a human adult, corresponding to about 1.0 g, is degraded by a single enzyme, DNase II, in macrophages. As described above for the DNA of dead cells, the inefficient digestion of the nuclear DNA from erythroid precursors can also cause severe inflammation.
Future Prospects
To combat bacterial and viral infection, mammals have developed a sophisticated immune system, which includes the acquired and innate immune systems. In the innate immune system, macrophages and dendritic cells recognize pathogens extracellularly and intracellularly and produce various cytokines such as IFNβ, IL-1β, and TNFα to contend with the pathogens directly. These cytokines also activate the acquired immune response to produce antibodies and cytotoxicity to combat the pathogens. Endogenous components derived from dead cells can also activate both the acquired and innate immune systems. Undigested DNA that accumulates in the lysosomes of macrophages can activate the intracellular signaling pathway for the innate immune reaction, while cellular components released from dead cells appear to activate the immune reaction extracellularly, through the BCR and TLR system. The immune reaction triggered by bacterial and viral pathogens is transient; that is, when the bacteria or viruses are removed by the action of IFN or TNF, the immune reaction ceases. In contrast, if a defect in the engulfment or digestion of dead cells or erythroid nuclei is not repaired, the immune system will be chronically activated, which may be responsible for SLE-type autoimmune disease and polyarthritis, two major autoimmune diseases in humans. Our knowledge about how dead cells and the expelled erythroid nuclei are recognized, engulfed, transferred to lysosomes, and degraded is still very primitive. The elucidation of these processes will help us understand the pathophysiology of various human diseases, especially autoimmune diseases, and will lead to the development of new therapeutic strategies for treating them.
Apoptotic-cell engulfment is phosphatidylserine dependent. Other examples of phosphatidylserine-dependent processes are the involution of mammary glands (Hanayama and Nagata, 2005) and the axon pruning that takes place during the development of neural circuits (Awasaki et al., 2006). In the involution of mammary glands, milk fat globules remaining in the mammary glands are re-absorbed by mammary epithelial cells in a phosphatidylserine-dependent manner (Hanayama and Nagata, 2005). MFG-E8, which is secreted from mammary epithelial cells, promotes the reabsorption of milk fat globules. The failure of this process induces mastitis, another case in which the inefficient clearance of unnecessary cellular components can lead to inflammation. In axon pruning, unnecessary or extra axons degenerate, expose phosphatidylserine, and are engulfed by glia. However, how the glial phagocytes recognize the phosphatidylserine on the pruned axons remains to be elucidated.
Finally, the main reason for the engulfment of dead cells is to degrade their intracellular materials before their cellular contents are released, which could activate the immune system. However, in some cases, phagocytes actively induce programmed cell death. For example, macrophages within the developing eye induce the programmed cell death of the vascular endothelial cells via Wnt ligand (Lobov et al., 2005), and phagocyte-induced programmed cell death serves as a backup death-inducing system in C. elegans (Reddien and Horvitz, 2004). How macrophages are induced to engulf apparently healthy cells is a mystery. The elucidation of this mechanism may help explain hemophagocytic syndrome, in which activated macrophages engulf apparently normal red blood cells and other cells.
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Gainesville, FL, USA
The canine and feline stomach suffers from a wide spectrum of primary and secondary disease. Most, if not all of these can cause vomiting and abdominal pain and are associated with some degree of damage to the gastric mucosa. The purpose of this paper is to review the mechanisms that facilitate or impair gastric mucosal protection in order to permit better understanding and treatment of gastric disease.
Why doesn't the stomach digest itself? Physicians, physiologists and many others have been puzzled by this question since René Antonoine Ferchault de Réamur, the 18th century man of many sciences, showed that juice secreted by the stomach could digest meat. One answer of course is that it sometimes does. Under some circumstances gastric juice can produce ulcers and even destroy most of the stomach lining. Normally, however, the stomach wall staunchly resists attack; as Claude Bernard observed, it behaves as if it was made of porcelain.
Gastric juice contains hydrochloric acid, one of the most corrosive acids known. At the concentration secreted by the gastric mucosa this acid is capable of dissolving zinc and is deadly to cells. Yet in the stomach it ordinarily acts only to perform the useful actions of killing ingested bacteria, softening fibrous foods and promoting pepsin formation. In the normal stomach this corrosive juice is prevented from contacting and damaging the stomach wall by a complex and interrelated series of physical and chemical processes that are only now beginning to be fully understood. Collectively these processes are called the gastric mucosal barrier and comprise the structural and functional protection of the stomach against its own secreted acid and pepsin as well as against the ravages of refluxed bile and pancreatic enzymes and ingested abrasive or toxic materials. There are five interrelated components: 1) the gastricepithelial cells; 2) gastric mucosal blood flow and local acid base balance; 3) gastric mucus; 4) mucosal prostaglandins and associated cytoprotection; and 5) the epithelial cell basement membrane.
The Gastric Epithelial Cells
The epithelial cells of the gastric mucosa form a formidable barrier against penetration by luminal contents, including hydrogen ions. The cells have very tight junctions, a lipid-rich, hydrophobic, acid-repelling mucosal surface, and secrete bicarbonate and mucus.
The gastric mucosa is routinely exposed to trauma, indeed, focal destruction of the mucosal barrier is a normal physiologic event, occurring for example, during intragastric digestion of a meal, after thermal, mechanical or osmolar damage and after ingestion of several different types of drug. Normally, however, the mucosa can repair or minimize this damage almost as soon as it occurs by a process of epithelial cell migration called restitution. This is a physiological event in which the epithelial cells at the mouth of the gastric glands adjacent to a damaged area flatten themselves and project finger like processes called lamellipodia. These extend over the underlying basal membrane and eventually fuse to form a new intact epithelial barrier.
Gastric Mucosal Blood Flow
The gastric mucosal blood supply has been called the mainstay of the gastric mucosal defense mechanisms that sustain a healthy gastric mucosa. Mucosal blood flow is achieved in a variety of ways, foremost of which is a unique vascular supply that maximizes mucosal oxygenation, bicarbonate delivery and buffering. Bicarbonate, a byproduct of acid production, is carried up to the mucosal surface by capillaries that surround each gastric gland. This bicarbonate escapes from the capillaries into the submucosa underneath the mucosa from where it is taken up by the epithelial cells and secreted into the surface mucus layer. The bicarbonate is trapped here and neutralizes any hydrogen ions that may diffuse into the mucus layer. As a result, the surface of the mucosa is maintained at a pH of about 7.2 while the pH of the lumen may be as low as 1.5. The bicarbonate-rich mucosal blood flow also maintains intramucosal acid base neutrality such that any hydrogen ions that may leak into the submucosa through a damaged epithelium are quickly neutralised. A decrease in mucosal blood flow however, will result in an increase in mucosal hydrogen ion concentration with subsequent tissue damage.
Mucus Secretion
Mucus neck cells produce the bulk of gastric mucus. This forms a viscid unstirred protective layer of varying thickness over the surface mucosa that traps secreted bicarbonate and lubricates the lining of the stomach. When damaged the epithelial cells themselves rupture and release copious quantities of protective mucus that forms a bicarbonate-rich protective mucus cap over the denuded area. Surrounding epithelial cells can migrate over the basal membrane and under this protective mucus cap.
Prostaglandin Secretion and Cytoprotection
Cytoprotection refers to the ability of certain substances to maintain tissue or cellular integrity in the face of mucosal damage. The term was originally coined to describe the ability of prostaglandins to reduce or eliminate hemorrhagic damage in mucosa exposed to injury. The term has been extended however, to describe a similar protective ability for a large number of endogenous and exogenously administered substances with anti-ulcerogenic effects. Cytoprotection is based upon the ability of these substances to inhibit acid secretion, stimulate mucus and bicarbonate secretion, and increase mucosal cell turnover and blood flow. Prostaglandins PGE2 and PGF2aare perhaps the best known examples of cytoprotectants. However, IL-1 and TNF released during inflammation increase mucosal blood flow and cytoprotection, as do exogenously administered sulfhydryl compounds such as acetylcysteine, glutathione and penicillamine. Epidermal growth factor which is found in saliva as well as in the gastric mucosa plays an important role in maintaining and stimulating mucosal cell turnover.
The Basal Membrane
This, the last defensive barrier is important for the process of restitution. The basal membrane is permeable to fluid and electrolytes and when disrupted allows an inrush of hydrogen ions and proteases from the gastric lumen. These invoke an inflammatory response and mark the changeover from physiologic to pathologic (i.e., inflammatory) repair of the mucosa.
BARRIER DISRUPTION
Pathologic and inflammatory barrier disruption occurs in virtually every type of gastric disease as well as in a variety of less well appreciated circumstances such as stress, brain and spinal cord injury, hypotension, hypoadrenocorticism, sepsis, uremia, liver disease, hypoproteinemia and protein-calorie malnutrition (Table 1). Most instances of barrier disruption associated with this diverse group of disorders can be attributed to a decrease in gastric mucosal blood flow, except for the hypoproteinemic or cachectic patient in which decreased cell turnover also plays a role.
Table 1. Disorders associated with a disrupted mucosal barrier
Gastric Disease
Hypoadrenocorticism
Liver disease
Acidosis
Sepsis (esp. peritonitis)
Shock (septic > hypovolemic > traumatic)
"Stress"
Protein-calorie malnutrition
Hypoproteinemia
Enteritis (with vomiting)
Uremia
Hypoxemia
NSAID therapy
CNS and spinal cord injury
In humans and some other species, infection with the Gram negative gastric bacteria Helicobacter is associated with gastric and duodenal injury. Mucosal damage from infection with H. pylori in humans is associated with the cytotoxic effects of a number of bacterial metabolites including ammonia, endotoxin, and a variety of inflammatory peptides. The organism also inhibits somatostatin secretion allowing an increase in acid secretion. Strangely, these properties do not appear to be present in the Helicobacter species (e.g., H. felis, H. bizzozeroni) that infect the dog and cat. Gastric structure and function for example, are no different between infected and uninfected dogs. The exact role of Helicobacter infection, if any, in the genesis of gastric lesions in the dog and cat therefore remains to be elucidated.
Virtually every critically ill patient has some degree of gastric mucosal damage that is evidenced by gastric erosions or ulceration with associated hemorrhage. This sometimes "hidden" injury very often prolongs patient morbidity, and if unappreciated or untreated may progress to a serious or life-threatening condition. An appreciation of the processes of barrier disruption and repair, together with an understanding of current methods for facilitating or enhancing the repair process are therefore important for most clinicians.
THE RESPONSE TO INJURY
A break in the gastric mucosal barrier allows hydrogen ions and pepsin to diffuse into the mucosa from the lumen and sodium ions to diffuse in the opposite direction. Back-diffusion of acid and pepsin into the tissues stimulates further acid and pepsin secretion, decreases mucosal blood flow and decreases gastric motility. The acid also damages connective tissue and submucosal capillaries to cause focal mucosal hemorrhage and microulceration. If sufficiently severe and prolonged overt gastric ulceration may occur.
Mucosal integrity, however, is rapidly reestablished if the inciting cause is removed or if appropriate and prompt treatment is given. Of concern to the clinician are factors that may delay or impair epithelial cell restitution and the repair process, such as hypoxia, sepsis or concomitant drug therapy. Corticosteroids for example, decrease gastric epithelial cell renewal and while relatively harmless in the healthy animal may delay repair or exacerbate mucosal injury in the sick one. It should be noted however that methyl prednisolone sodium succinate at a dose of 30mg/kg/day for two days induced gastric hemorrhage in 100% of healthy normal dogs and that the synthetic prostaglandin misoprostol did not prevent the hemorrhage. Nonsteroidal anti-inflammatory drugs (NSAIDs) are another group of compounds that exacerbate underlying disease and delay repair, primarily through their inhibition of cyclooxygenase and a decrease in mucosal prostaglandin concentration. Recent attention has focused on the fact that there are two types of cyclooxygenase (cyclooxygenase 1 and 2 or COX-1 and COX-2). COX-1 is constitutively expressed in most cells and tissues, notably platelets, endothelial cells, stomach and kidney. COX-1 plays a key role in the synthesis of prostaglandins responsible for mucosal cytoprotection. COX-2 on the other hand is typically undetectable in normal tissue but is induced in inflammatory conditions by cytokines or lipopolysaccharides. NSAIDs exert their antiinflammatory effect through the inhibition of COX-2 whereas many of their adverse effects are due primarily to the inhibition of COX-1. Several COX-2 specific antiinflammatory drugs have recently been introduced and purportedly reduce inflammation while sparing the gastric mucosa. Aspirin inhibits both cyclooxygenases but in addition, when the intragastric pH is less than 4.0, it undergoes a change in lipid permeability and is absorbed directly into the gastric epithelial cell where it disrupts cellular function. The drug is also purported to facilitate bile reflux in the dog. The canine stomach is particularly sensitive to NSAIDs and none of these drugs should be considered "safe" in this species.
FACILITATION OF MUCOSAL REPAIR
The most important concept in dealing with the disrupted mucosal barrier is to recognize that disruption is widespread and that it can occur in such a wide variety of diseases (Table 1). Barrier disruption occurs routinely for example in hypoadrenocorticism, peritonitis, pyometra, pneumonia, liver disease, hypoproteinemia, severe trauma, uremia and in most if not all primary gastric diseases. The most important remedy in all these disorders is to treat the underlying disease. All other actions are of secondary, but nevertheless still considerable importance.
Drug therapy, however, remains the mainstay of treatment. These fall into two main groups 1) antisecretory drugs and 2) cytoprotectants that are usually combined to achieve an optimum effect (Table 2).
Table 2. Drugs that augment the mucosal barrier
Antisecretory | Cytoprotectants |
Cimetidine 5mg/kg q8h | Misoprostol 3-4μg/kg q12h |
Ranitidine 2mg/kg q12h | Sucralfate 0.25-1.0g/patient q8-12h |
Famotidine 1mg/kg q24h | Aluminum ions to effect |
Omeprazole 0.7mg/kg q24h | Bismuth subsalts to effect |
Antisecretory Drugs
A variety of compounds have been used to reduce acid secretion but most widely used are the H2 receptor antagonists such as cimetidine, ranitidine and famotidine.
Cimetidine interferes with the cytochrome p450 system in the liver and can influence the action of some drugs (e.g., ketoconazole, theophylline, propranolol, quinidine and metronidazole) and the absorption or effect of others (e.g., metoclopramide and sucralfate). Cimetidine, however, is ineffective against aspirin-induced mucosal injury in dogs. Ranitidine binds much more strongly to the H2 receptor on the parietal cell and therefore requires a lower frequency of administration as well as indirectly increasing gastric motility. Cimetidine also has additional protective effects on the mucosal barrier (increased cell turnover, mucus production, mucosal blood flow, bicarbonate secretion and cellular integrity). Famotidine inhibits stress-induced decreases in gastric mucosal blood flow and has the benefit of once daily dosage which makes it attractive to many clients. The prophylactic use of H2 receptor antagonists should therefore be routine in critically ill patients.
Omeprazole, is another very effective antisecretory drug. Cimetidine, famotidine and ranitidine block only histamine-induced acid secretion but omeprazole blocks all acid secretion by inhibiting H+K+ATP'ase at the luminal surface of the parietal cell. While a major portion of the therapeutic efficacy of omeprazole involves inhibition of gastric acid secretion, some of its cytoprotective properties are probably associated with its action on the mucosal vasculature. Oral omeprazole for example, maintains mucosal blood flow in the face of mucosal damage and decreases mucosal production of the vasoconstrictor phospholipid platelet activating factor (PAF).
Cytoprotective Drugs
These include synthetic prostaglandins, sucralfate, antacids containing aluminum, and the bismuth sub-salts. Sucralfate is a complex polymer of sucrose with multiple substitutions of sulfate and aluminum salts. At a pH <4.0 it undergoes a change in chemical configuration, developing a positive charge which binds electrochemically with the negative charge in serum protein to form a protective layer over ulcerated areas which protects the mucosa against further injury by acid, pepsin and bile salts. The drug also stimulates the synthesis and release of prostaglandins, epidermal growth factor and nitric oxide as well as augmenting other aspects of the mucosal barrier such as gastric mucosal blood flow, bicarbonate secretion and mucus production. Sucralfate also stimulates angiogenesis in injured gastric mucosa.
The bismuth subsalts and aluminum containing antacids at doses less than those required to neutralize acid also exert beneficial effects on the mucosal barrier through augmented prostaglandin synthesis.
CONCLUSION
Gastric mucosal damage and repair are ongoing processes in the normal stomach with repair, primarily through epithelial cell restitution, rapidly restoring the ravages of normal wear and tear. The repair process however, may be impaired in gastric disease as well as in a variety of other diseases that weaken gastric defenses. If unrecognized and untreated this will increase patient morbidity with the likelihood of overt ulceration. Treatment with H2 receptor antagonists and cytoprotective drugs is critical to patient well being.
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Colin F. Burrows, BVetMed, PhD, MRCVS, DACVIM
University of Florida, College of Veterinary Medicine
Gainesville, FL
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