We have looked at the seven major cells involved in the innate immune response: mast cells, macrophages, neutrophils, dendritic cells, basophils, eosinophils and natural killer cells. There are two useful classifications we can use when discussing these cells: granulocyte and phagocyte. Some of the cells are both. A granulocyte is a cell which contains granules of molecules which aid the immune response. Some of these molecules are involved in signalling and chemotaxis of immunocompetent cells, some are involved in directly damaging the pathogen and generally they initiate and/or mediate the immune response. Phagocytes stretch themselves around pathogens, engulf them, destroy them and may then recover antigens, which they present to cells of the adaptive immune system.
Mast cells are phagocytes and granulocytes. Their primary function is to degranulate, when stimulated to do so, and thereby instigate the inflammatory response. This may be achieved by damage to the mast cell, by activation by complement and by means of antibodies. These antibodies will be produced in response to particular pathogens and will bind to mast cells. Should the relevant pathogen then bind to two or more receptors (via the antibodies), a signalling cascade will be initiated leading to the degranulation of the mast cell. Degranulation is achieved by transporting the granule to the cell membrane, where it fuses with the membrane and so expels its contents.
Macrophages are large phagocytes. They mature from monocytes and are involved in the release of cytokines, in cleaning up debris left by neutrophils and in tissue repair. Also, of course, they phagocytose. So-called M1 macrophages are particularly aggressive hunters of pathogens, while M2 macrophages are more focused on cleaning up and repair.
Neutrophils are highly effective phagocytes and are also granulocytes and are responsible, in part, for releasing inflammatory cytokines. As well as this, they form neutrophil extracellular traps (NETs), which trap invading pathogens and destroy them by means of the proteins which make up the NETs.
Dendritic cells are "sentinels." They are prolific phagocytes and even "nibble" on perfectly healthy host cells when looking for pathogens. When they find one they phagocytose it and present antigens from it to cells of the adaptive immune system. Dendritic cells are perhaps the most effective antigen presenting cells.
Basophils are useful granulocytes. Their granules contain a number of important molecules, including histamine and heparin, as well as IL-4. Notably, basophil degranulation can be mediated by IgE antibodies.
Eosinophils are also granulocytes but, as well as inflammatory mediators, eosinophils are particularly noteworthy for releasing highly destructive chemicals. Eosinophils, then, are particularly involved with the actual killing.
Natural killer cells attack cells which have been infected by viruses, or which are otherwise "stressed" or damaged in some way. They "select" their target using stimulatory and inhibitory receptors. Inhibitory ligands - particularly MHC I - on healthy cells prevent NK cells from attacking. Thus NK cells do not harm the body they're supposed to be defending - this is self-tolerance. However, on damaged cells MHC I is often under expressed. Damaged cells are also likely to express increased levels of stimulatory ligands (or they may have expressed these when they were healthy as well, but the MHC I levels counterbalanced them). Thus, NK cells "recognise" a lack of MHC I (missing self recognition) and can then be stimulated to attack the cell by the stimulatory ligands. Put more briefly (and more accurately): NK cells attack only if the stimulatory signal is stronger than the inhibitory signal. This is the means by which NK cells select the right target and kill damaged and infected cells, without harming healthy cells. If the NK cell is activated it can release IFNγ and TNFα. The most prominent method of attack, though, is the release of granules. These contain perforin, which forms a pore in the target cell, which allows other substances in the granules to enter the cell and kill it by lysis or apoptosis. NK cells are additionally stimulated by cytokines and can be activated with the help of antibodies.
Monday, 9 June 2014
Natural killer cells
The role of natural killer cells is to attack cells of the body which have become infected by viruses, or which are damaged or dysfunctional in some other way - e.g. cancer cells. The role of natural killer cells in tumour suppression is certainly very interesting, but I will try to focus on their role in defence against pathogens.
To put it very briefly, viruses invade the cells of the body and replicate within them; they hijack the resources and structures of the cell they invade. These are supposed to be used for creating things the body needs and new copies of the cell; instead, the virus repurposes them to create copies of itself. This is how viruses work - very simply. HIV is no different and we will see more of this soon. It is the role, then, of natural killer cells to identify cells which have been infected and to shut them down and kill them. This will destroy the virus and prevent it from replicating; although it will not, of course, undo the creation of any copies of the virus which have already been made and released.
In addition to this, natural killer cells are also able to release cytokines and there is emerging evidence that they are able to adjust to their environment and to "remember" pathogens which have been encountered previously and respond effectively. Thus they seem also to be a part of the adaptive immune system, as well as the innate immune system.
Precisely how natural killer cells achieve all of this is the subject of much current research. We do not yet have all the answers - although that's true of pretty much everything. In this case, though, we don't even have many of them. Nevertheless, for those who are interested, there is a very good paper by David H. Raulet called Missing self recognition and self tolerance of natural killer (NK) cells,1 which I highly recommend and which is able to shed some light on the topic.
For our purposes, though, I think I can be a little cursory, but be sure to take note of the fact that I am simplifying things and eliding a lot of detail. NK cells possess a wide range of receptors - although not all NK cells possess the same receptors. Some of these receptors - such as NKG2D - are stimulatory receptors, while others - such as KIRs (Killer-cell immunoglobulin-like receptors) - are inhibitory receptors. Now, there does seem to be substantial variation in receptors among NK cells, so all NK cells are different and there will, therefore, be some variation in how they behave.
The general principle, though, seems to be that activation of NK cells depends on the balance between stimulatory and inhibitory ligands on a cell surface, or in the environment of a cell. So, if a given cell expresses lots of inhibitory molecules, the NK cell will obviously not be activated. If there is a good balance between stimulatory and inhibitory molecules then, again, the NK cell will not be activated. Again, if there are very few inhibitory molecules (or none), but also few stimulatory molecules (or none), still the NK cell is not likely to be activated. No, one requires the presence of plenty of stimulatory molecules - without there being enough inhibitory molecules to counter-balance them - for the NK cell to be activated. Or, from the NK cell's point of view, the stimulatory signal needs to be stronger than the inhibitory signal.
This, as I have said, is a simplification, however. Some cells do not actually express inhibitory receptors at all. These cells, I gather from Raulet (2006), are hyporesponsive (i.e. less responsive than the norm). I believe these cells can be stimulated to kill, but it is likely that either a strong signal is required, or their activation is dependent on external signalling, perhaps by IL-12 and/or other cytokines.
Now, let's look at this in a bit more detail. The inhibitory receptors generally recognise MHC class I (major histocompatibility complex class I) - something else which will soon be of great importance to us. MHC class I allows the NK cells to recognise "self" cells. So, the presence of MHC class I generally inhibits NK cell activity, which is an effective way of ensuring that NK cells do not attack the body's own cells. When cells become stressed or infected by viruses, MHC class I expression can be affected. In other words, many cells which are infected by viruses (and many cancer cells, too) do not express sufficient MHC class I (if any) to prevent NK cell activation. This is referred to as missing self recognition. NK cells identify damaged and infected cells by the lack of self molecules.
However, this on its own is not enough. NK cells generally do not attack the body's own cells when they are healthy because of the presence of MHC class I. However, absence of MHC class I will not precipitate an immediate attack. Instead, the presence of stimulatory ligands is required. These include heat shock proteins, extracellular matrix fragments, altered membrane phospholipids and other general markers of stressed, infected and cancerous cells. These are expressed on the surface of the cells, but stressed cells can also release stimulatory cytokines and NK cells can be stimulated by macrophages, too.
Seemingly, some molecules present on normal, healthy cells also act as stimulatory ligands. Thus, if MHC class I is poorly expressed on these cells they will be attacked. This is useful, because it means that some unhealthy cells can be killed merely by missing self recognition and expression of additional stimulatory ligands is not necessary, since stimulatory ligands occur naturally on that cell anyway.
So, in conclusion, this balance between inhibitory and stimulatory signalling allows for self-tolerance - the body's NK cells do not attack the body's own healthy cells. Meanwhile, infected cells can be "recognised" by NK cells by virtue of the stimulatory ligands which they produce and the absence of inhibitory ligands, particularly MHC class I (missing self recognition). Notice that it's really a combination of these two factors and, in fact, it seems that the triggering of NK cell activity is dependent on the end result of the interplay of numerous signals. Indeed it may well even be dependent on the particular NK cell in question.
To pick up on that last point, one final thing to add is that, in the case of cells which do not express inhibitory receptors, it appears that hyporesponsiveness is important for self-tolerance. In other words, if these cells were not hyporesponsive, they would regularly attack healthy self cells, as they would not be able to detect the presence of MHC class I and so be prevented from attacking. Thus it is necessary that they be very loath to attack and, in this way, they only attack cells which are strongly requiring of attack.
Now, there are a few ways in which NK cells actually go about killing abnormal cells. The primary method involves the release of granules. The NK cell will release perforin - a protein which creates a small pore in the target cell's membrane. Other molecules from the NK cell granules - generally proteins and proteases - can then enter the cell through the pore. These will then induce apoptosis in the target cell, or kill it via lysis, which we've seen before. Apoptosis, or "programmed cell death," is where a cell shuts itself down in response to certain signalling events. NK cells also release a number of cytokines themselves, most notably IFNγ and TNFα.
Finally, in addition to all the above, NK cells can perform antibody-dependent cell-mediated cytotoxicity (or ADCC). NK cells express FcγRIII (alternatively: CD16) receptors which bind to some antibodies (specifically IgG, I think). When this happens, the NK cells are activated and will induce apoptosis in the cell which has been opsonised with antibodies. This is, of course, in many ways similar to activation by stimulatory molecules and subsequent degranulation. It is important to note, however, that not only do antibodies activate NK cells, but they also seem to be able to expedite the process. Whether or not ADCC can completely bypass the usual mechanism involving other stimulatory receptors and inhibitory receptors is not clear to me. It's worth appreciating, though, another example of just how the adaptive immune system promotes and fine-tunes the innate immune system.
1Raulet, David H. 2006. "Missing self recognition and self tolerance of natural killer (NK) cells." Seminars in Immunology 18 (2006): 145-150
2http://www.nhs.uk/news/2008/05May/Pages/Antidepressantsandimmunity.aspx
To put it very briefly, viruses invade the cells of the body and replicate within them; they hijack the resources and structures of the cell they invade. These are supposed to be used for creating things the body needs and new copies of the cell; instead, the virus repurposes them to create copies of itself. This is how viruses work - very simply. HIV is no different and we will see more of this soon. It is the role, then, of natural killer cells to identify cells which have been infected and to shut them down and kill them. This will destroy the virus and prevent it from replicating; although it will not, of course, undo the creation of any copies of the virus which have already been made and released.
In addition to this, natural killer cells are also able to release cytokines and there is emerging evidence that they are able to adjust to their environment and to "remember" pathogens which have been encountered previously and respond effectively. Thus they seem also to be a part of the adaptive immune system, as well as the innate immune system.
Precisely how natural killer cells achieve all of this is the subject of much current research. We do not yet have all the answers - although that's true of pretty much everything. In this case, though, we don't even have many of them. Nevertheless, for those who are interested, there is a very good paper by David H. Raulet called Missing self recognition and self tolerance of natural killer (NK) cells,1 which I highly recommend and which is able to shed some light on the topic.
For our purposes, though, I think I can be a little cursory, but be sure to take note of the fact that I am simplifying things and eliding a lot of detail. NK cells possess a wide range of receptors - although not all NK cells possess the same receptors. Some of these receptors - such as NKG2D - are stimulatory receptors, while others - such as KIRs (Killer-cell immunoglobulin-like receptors) - are inhibitory receptors. Now, there does seem to be substantial variation in receptors among NK cells, so all NK cells are different and there will, therefore, be some variation in how they behave.
The general principle, though, seems to be that activation of NK cells depends on the balance between stimulatory and inhibitory ligands on a cell surface, or in the environment of a cell. So, if a given cell expresses lots of inhibitory molecules, the NK cell will obviously not be activated. If there is a good balance between stimulatory and inhibitory molecules then, again, the NK cell will not be activated. Again, if there are very few inhibitory molecules (or none), but also few stimulatory molecules (or none), still the NK cell is not likely to be activated. No, one requires the presence of plenty of stimulatory molecules - without there being enough inhibitory molecules to counter-balance them - for the NK cell to be activated. Or, from the NK cell's point of view, the stimulatory signal needs to be stronger than the inhibitory signal.
This, as I have said, is a simplification, however. Some cells do not actually express inhibitory receptors at all. These cells, I gather from Raulet (2006), are hyporesponsive (i.e. less responsive than the norm). I believe these cells can be stimulated to kill, but it is likely that either a strong signal is required, or their activation is dependent on external signalling, perhaps by IL-12 and/or other cytokines.
Now, let's look at this in a bit more detail. The inhibitory receptors generally recognise MHC class I (major histocompatibility complex class I) - something else which will soon be of great importance to us. MHC class I allows the NK cells to recognise "self" cells. So, the presence of MHC class I generally inhibits NK cell activity, which is an effective way of ensuring that NK cells do not attack the body's own cells. When cells become stressed or infected by viruses, MHC class I expression can be affected. In other words, many cells which are infected by viruses (and many cancer cells, too) do not express sufficient MHC class I (if any) to prevent NK cell activation. This is referred to as missing self recognition. NK cells identify damaged and infected cells by the lack of self molecules.
However, this on its own is not enough. NK cells generally do not attack the body's own cells when they are healthy because of the presence of MHC class I. However, absence of MHC class I will not precipitate an immediate attack. Instead, the presence of stimulatory ligands is required. These include heat shock proteins, extracellular matrix fragments, altered membrane phospholipids and other general markers of stressed, infected and cancerous cells. These are expressed on the surface of the cells, but stressed cells can also release stimulatory cytokines and NK cells can be stimulated by macrophages, too.
Seemingly, some molecules present on normal, healthy cells also act as stimulatory ligands. Thus, if MHC class I is poorly expressed on these cells they will be attacked. This is useful, because it means that some unhealthy cells can be killed merely by missing self recognition and expression of additional stimulatory ligands is not necessary, since stimulatory ligands occur naturally on that cell anyway.
So, in conclusion, this balance between inhibitory and stimulatory signalling allows for self-tolerance - the body's NK cells do not attack the body's own healthy cells. Meanwhile, infected cells can be "recognised" by NK cells by virtue of the stimulatory ligands which they produce and the absence of inhibitory ligands, particularly MHC class I (missing self recognition). Notice that it's really a combination of these two factors and, in fact, it seems that the triggering of NK cell activity is dependent on the end result of the interplay of numerous signals. Indeed it may well even be dependent on the particular NK cell in question.
To pick up on that last point, one final thing to add is that, in the case of cells which do not express inhibitory receptors, it appears that hyporesponsiveness is important for self-tolerance. In other words, if these cells were not hyporesponsive, they would regularly attack healthy self cells, as they would not be able to detect the presence of MHC class I and so be prevented from attacking. Thus it is necessary that they be very loath to attack and, in this way, they only attack cells which are strongly requiring of attack.
Now, there are a few ways in which NK cells actually go about killing abnormal cells. The primary method involves the release of granules. The NK cell will release perforin - a protein which creates a small pore in the target cell's membrane. Other molecules from the NK cell granules - generally proteins and proteases - can then enter the cell through the pore. These will then induce apoptosis in the target cell, or kill it via lysis, which we've seen before. Apoptosis, or "programmed cell death," is where a cell shuts itself down in response to certain signalling events. NK cells also release a number of cytokines themselves, most notably IFNγ and TNFα.
Finally, in addition to all the above, NK cells can perform antibody-dependent cell-mediated cytotoxicity (or ADCC). NK cells express FcγRIII (alternatively: CD16) receptors which bind to some antibodies (specifically IgG, I think). When this happens, the NK cells are activated and will induce apoptosis in the cell which has been opsonised with antibodies. This is, of course, in many ways similar to activation by stimulatory molecules and subsequent degranulation. It is important to note, however, that not only do antibodies activate NK cells, but they also seem to be able to expedite the process. Whether or not ADCC can completely bypass the usual mechanism involving other stimulatory receptors and inhibitory receptors is not clear to me. It's worth appreciating, though, another example of just how the adaptive immune system promotes and fine-tunes the innate immune system.
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| (a) An image of two natural killer cells attacking a cancer cell. (Image courtesy the NHS2) |
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| (b) A schematic representation of a natural killer cell. (Image courtesy "A. Rad" (via Wikipedia)) Figure 1.57: The natural killer cell |
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1Raulet, David H. 2006. "Missing self recognition and self tolerance of natural killer (NK) cells." Seminars in Immunology 18 (2006): 145-150
2http://www.nhs.uk/news/2008/05May/Pages/Antidepressantsandimmunity.aspx
Eosinophils
Eosinophils are also produced in the bone marrow, from where they migrate to areas such as the medulla oblongata, the spleen and the lymph nodes. Some of them will remain in the circulation, where they can live for 8-12 hours, while in tissue they can survive for 8-12 days. During an infection, eosinophils are attracted to the site of infection by chemotactic factors such as CCL11, CCL24, CCL5 and leukotriene B4.
At the site of infection, eosinophils must be activated by cytokines released by T cells, e.g. interleukin-3 (IL-3) and interleukin-5 (IL-5). When activated, eosinophils degranulate; eosinophils are very destructive - their activation has a lot less to do with mediating inflammation and a lot more to do with the actual business of killing pathogens. Eosinophil granules contain cationic granule proteins such as ECP (eosinophil cationic protein) and MBP (major basic protein), which are highly cytotoxic. These can be very destructive - including to host tissue. MBP is able to stimulate the degranulation of mast cells and basophils and ECP is able to create pores in the cell membranes of pathogens, allowing cytotoxic molecules to enter the cell. As well as these, eosinophils release harmful oxygen species - such as superoxide and peroxide - eicosanoids, numerous cytokines - such as TNFα and IL-8 - and various enzymes and growth factors. In addition to this, they release RNases - which are able to combat viral infection - are important mediators of inflammation and may be involved in antigen presentation.
At the site of infection, eosinophils must be activated by cytokines released by T cells, e.g. interleukin-3 (IL-3) and interleukin-5 (IL-5). When activated, eosinophils degranulate; eosinophils are very destructive - their activation has a lot less to do with mediating inflammation and a lot more to do with the actual business of killing pathogens. Eosinophil granules contain cationic granule proteins such as ECP (eosinophil cationic protein) and MBP (major basic protein), which are highly cytotoxic. These can be very destructive - including to host tissue. MBP is able to stimulate the degranulation of mast cells and basophils and ECP is able to create pores in the cell membranes of pathogens, allowing cytotoxic molecules to enter the cell. As well as these, eosinophils release harmful oxygen species - such as superoxide and peroxide - eicosanoids, numerous cytokines - such as TNFα and IL-8 - and various enzymes and growth factors. In addition to this, they release RNases - which are able to combat viral infection - are important mediators of inflammation and may be involved in antigen presentation.
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| (a) An image of an eosinophil granulocyte. (Image courtesy "Bobjgalindo" (via Wikipedia)) |
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| (b) A schematic representation of an eosinophil. (Image courtesy "A. Rad" (via Wikipedia)) Figure 1.56: The eosinophil |
Basophils
Basophils are quite few and far between, making up only around 0.01-0.3% of all circulating leukocytes; nevertheless, they pack quite a punch. Now, as I say, basophils are circulating white blood cells, they do not have a home like mast cells - their fellow granulocytes - do. Instead, like neutrophils and immature dendritic cells, they circulate in the blood stream awaiting an invasion by a pathogen. When pathogens are detected by immunocompetent cells, basophils can be recruited from the bloodstream as part of the inflammatory response.
As granulocytes, the primary function of basophils is to degranulate when stimulated to do so. As with mast cells, these granules contain histamine and heparin, as well as cytokines and a number of other things. One of the most notable things released by basophils is interleukin 4 (IL-4), a very important cytokine.
Basophils exhibit a number of receptors, including our friend FcεRI. As with mast cells, basophils bind with IgE antibodies, which are involved in the degranulation process. Again, it is important to appreciate that basophil degranulation will be triggered by specific pathogens - i.e. the ones which bind to IgE antibodies. In this way the right response to a given pathogen is produced.
As granulocytes, the primary function of basophils is to degranulate when stimulated to do so. As with mast cells, these granules contain histamine and heparin, as well as cytokines and a number of other things. One of the most notable things released by basophils is interleukin 4 (IL-4), a very important cytokine.
Basophils exhibit a number of receptors, including our friend FcεRI. As with mast cells, basophils bind with IgE antibodies, which are involved in the degranulation process. Again, it is important to appreciate that basophil degranulation will be triggered by specific pathogens - i.e. the ones which bind to IgE antibodies. In this way the right response to a given pathogen is produced.
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| (a) An image of a basophil granulocyte. (Image courtesy Department of Histology, Jagiellonian University Medical College (via Wikipedia)) |
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| (b) A schematic representation of a basophil. (Image courtesy "A. Rad" (via Wikipedia)) Figure 1.55: The basophil |
Dendritic cells
Dendritic cells also likely originate as monocytes, which then mature into dendritic cells in response to a particular signal, or series of signals. I myself am not sure precisely how dendritic cells arise, but when they have done so, they are very useful. Wikipedia perfectly describes them as "sentinels." Immature dendritic cells use PRRs - e.g. TLRs, which we've seen before - to seek out any pathogens. Some will even phagocytise small amounts of host cells in their search for anything which should not be in the body. This is rather delightfully referred to as "nibbling."
Should an immature dendritic cell successfully phagocytose a pathogen, it will mature and set about its primary function of antigen presentation - which dendritic cells do better than any other cells. Additionally, the chemokine receptor CCR7 - among other things - is upregulated, which helps guide the cell to the spleen or a lymph node. Here the dendritic cell will sensitise cells of the adaptive immune system to that particular antigen (and, therefore, the particular pathogen it has phagocytised) and a powerful response to it is occasioned.
As before, I will now give you a picture, so you can see what they look like:
Should an immature dendritic cell successfully phagocytose a pathogen, it will mature and set about its primary function of antigen presentation - which dendritic cells do better than any other cells. Additionally, the chemokine receptor CCR7 - among other things - is upregulated, which helps guide the cell to the spleen or a lymph node. Here the dendritic cell will sensitise cells of the adaptive immune system to that particular antigen (and, therefore, the particular pathogen it has phagocytised) and a powerful response to it is occasioned.
As before, I will now give you a picture, so you can see what they look like:
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| (a) This is a very detailed image of a dendritic cell, taken from a journal article by Judith Behnsen et al. It is actually a screenshot of a video and the screenshot was uploaded to Wikipedia |
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| (b) A schematic representation of a dendritic cell. (Image courtesy "A. Rad" (via Wikipedia)) Figure 1.54: The dendritic cell |
Neutrophils
The neutrophils are the most plentiful of all white blood cells. They live fairly short lives, lasting for around 5.4 days, and during this time they "roam" the body, searching out pathogens. As I mentioned above, they tend to be the first respondents and will flood a site of infection, navigating by means of chemotaxis.
Once there, being phagocytes, one of the things they do is phagocytise any pathogens that they come across. They are also capable of releasing inflammatory cytokines and recruiting other immunocompetent cells. As I have stated before, neutrophils are granulocytes, as well as phagocytes, and - like mast cells - can be stimulated to release their granules, which are very effective at killing pathogens.
The other extremely interesting thing that they do is form neutrophil extracellular traps (NETs). These are actually reminiscent of nets - they're fibres of DNA. The fibres are composed of chromatin as well as serine and other proteins from neutrophil granules. These proteins then destroy invading pathogens. Brilliantly, it seems that the NETs may actually serve as nets - trapping pathogens. They also prevent the antimicrobial proteins from doing serious damage to the body, as they are kept attached to the neutrophil.
Neutrophils look like this:
Once there, being phagocytes, one of the things they do is phagocytise any pathogens that they come across. They are also capable of releasing inflammatory cytokines and recruiting other immunocompetent cells. As I have stated before, neutrophils are granulocytes, as well as phagocytes, and - like mast cells - can be stimulated to release their granules, which are very effective at killing pathogens.
The other extremely interesting thing that they do is form neutrophil extracellular traps (NETs). These are actually reminiscent of nets - they're fibres of DNA. The fibres are composed of chromatin as well as serine and other proteins from neutrophil granules. These proteins then destroy invading pathogens. Brilliantly, it seems that the NETs may actually serve as nets - trapping pathogens. They also prevent the antimicrobial proteins from doing serious damage to the body, as they are kept attached to the neutrophil.
Neutrophils look like this:
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| (a) Some actual neutrophils (giemsa stained). (Image courtesy Dr Graham Beards (via Wikipedia)) |
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| (b) A schematic representation of a neutrophil. (Image courtesy "A. Rad" (via Wikipedia)) Figure 1.53: The neutrophil |
Macrophages
For the most part, the body's macrophages are to be found in vulnerable locations and in places which are likely sites of infection. Here they can live for many months - and occasionally longer - ready to respond to any pathogen they come across. A small number act as "scavengers," being carried along by the blood, where they may encounter pathogens or damaged cells. Such macrophages have a half-life of only about a day.
Macrophages begin life as monocytes, which will mature, under certain influences, into macrophages or dendritic cells. Monocytes are produced by bone marrow and are released from there into the bloodstream. The majority of them migrate to the spleen - a kind of monocyte base - or vulnerable areas such as the lungs. Once in these areas they generally mature into macrophages, with each location engendering peculiar macrophages. This allows the body to maintain a regular turnover of macrophages in these areas.
There are actually two types of macrophages - M1 and M2. M2 is the phenotype of the majority of resident macrophages which remain in tissue. Here, they can respond to any pathogens which they may encounter - as well as damaged cells - and can initiate the immune response.
In the initial stages of inflammation, however, a lot of the "work" is done by neutrophils. After around 48 hours the initial "wave" of neutrophils will have aged and the neutrophils will have begun to die. The macrophages can then engulf them and clean up the general mess. This work is usually done by the resident macrophages. Macrophages will not tend be drawn to the site of infection in large numbers for some time. Usually, 24-28 hours is required before monocyte levels start to build up.
Macrophages are also heavily involved in tissue repair. M2 "repair" macrophages are capable of producing molecules which aid in tissue repair, but their primary role is to rid the area of any damaged tissue. They also release cytokines which attract other cells which can help to the area and they produce factors which aid in angiogenesis. More generally, M2 macrophages additionally produce anti-inflammatories which regulate the immune response and keep it from harming the body.
Now, when pathogens or damaged cells are detected, the monocytes (and any macrophages which are in the bloodstream) are attracted to the area by inflammatory cytokines. They leave the blood flow and enter the interstitial fluid, where they then move by chemotaxis towards the infected area. Once there, the monocytes are stimulated to become macrophages (or dendritic cells) through numerous processes. In the case of substantial infection, large numbers of monocytes will travel to the site of infection, where they will mature into macrophages.
M1 "killer" macrophages are particularly oriented to hunting down pathogens and cellular debris and phagocytising them. It is possible for T cells - which will become very important to us soon - to cause M1 macrophages to become particularly aggressive. Indeed, macrophages have an incredible appetite and have been known to eat iron filings, for instance.
In general, macrophages are also effective antigen presenting cells and, as we have seen in some detail, are responsible for releasing many inflammatory cytokines and for mediating the immune response as a whole.
For those of you who are interested, the macrophage itself looks like this:
Macrophages begin life as monocytes, which will mature, under certain influences, into macrophages or dendritic cells. Monocytes are produced by bone marrow and are released from there into the bloodstream. The majority of them migrate to the spleen - a kind of monocyte base - or vulnerable areas such as the lungs. Once in these areas they generally mature into macrophages, with each location engendering peculiar macrophages. This allows the body to maintain a regular turnover of macrophages in these areas.
There are actually two types of macrophages - M1 and M2. M2 is the phenotype of the majority of resident macrophages which remain in tissue. Here, they can respond to any pathogens which they may encounter - as well as damaged cells - and can initiate the immune response.
In the initial stages of inflammation, however, a lot of the "work" is done by neutrophils. After around 48 hours the initial "wave" of neutrophils will have aged and the neutrophils will have begun to die. The macrophages can then engulf them and clean up the general mess. This work is usually done by the resident macrophages. Macrophages will not tend be drawn to the site of infection in large numbers for some time. Usually, 24-28 hours is required before monocyte levels start to build up.
Macrophages are also heavily involved in tissue repair. M2 "repair" macrophages are capable of producing molecules which aid in tissue repair, but their primary role is to rid the area of any damaged tissue. They also release cytokines which attract other cells which can help to the area and they produce factors which aid in angiogenesis. More generally, M2 macrophages additionally produce anti-inflammatories which regulate the immune response and keep it from harming the body.
Now, when pathogens or damaged cells are detected, the monocytes (and any macrophages which are in the bloodstream) are attracted to the area by inflammatory cytokines. They leave the blood flow and enter the interstitial fluid, where they then move by chemotaxis towards the infected area. Once there, the monocytes are stimulated to become macrophages (or dendritic cells) through numerous processes. In the case of substantial infection, large numbers of monocytes will travel to the site of infection, where they will mature into macrophages.
M1 "killer" macrophages are particularly oriented to hunting down pathogens and cellular debris and phagocytising them. It is possible for T cells - which will become very important to us soon - to cause M1 macrophages to become particularly aggressive. Indeed, macrophages have an incredible appetite and have been known to eat iron filings, for instance.
In general, macrophages are also effective antigen presenting cells and, as we have seen in some detail, are responsible for releasing many inflammatory cytokines and for mediating the immune response as a whole.
For those of you who are interested, the macrophage itself looks like this:
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| (a) A macrophage of a mouse involved in phagocytising two pathogens. (Image courtesy "magnaram" (via Wikipedia)) |
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| (b) A schematic representation of a macrophage. (Image courtesy "A. Rad" (via Wikipedia)) Figure 1.52: The macrophage |
Phagocytes
The phagocytes are a large group of cells, which actually includes mast cells. Mast cells are a little exceptional, though, so I thought it best to treat them first. Alongside mast cells, we have the three cells I listed in Innate immunity - macrophages, neutrophils and dendritic cells. These are the most notable professional phagocytes - although there are others. Alongside the professional phagocytes, we have a number of non-professional phagocytes. The difference between the two groups is really how well the cells of those groups phagocytose. Professional phagocytes possess receptors which greatly improve their ability to phagocytose, whereas non-professional phagocytes tend to primarily fulfil other roles.
So, I've given you a list of different types of phagocytes and you know from Innate immunity broadly what they do - they ingest pathogens and destroy them. Here, I will go into more detail about what phagocytes are, what they do, and how they do it. Although I will be writing about phagocytes in general, I will have cause to look at specific examples. Then I will treat each of the professional phagocytes I have named individually.
As you know, phagocytes are attracted to sites of infection by chemotactic factors - for example, C5a is a chemotactic factor of neutrophils, among other cells. It is also possible for phagocytes to be drawn to pathogens by the pathogens themselves and it is even possible for phagocytes simply to collide with a pathogen by chance.
Now, upon arriving at the site of an infection, phagocytes phagocytose. Phagocytosis is instigated by the binding of a ligand on the surface of a pathogen to a receptor on the phagocyte. In some cases, this may be the binding of a PAMP to a PRR on the phagocyte. In many cases, opsonins assist in the process, or may be necessary for it to take place at all. Opsonins - such as C3b and various antibodies - work by binding to the receptors on the phagocyte instead of the pathogen's own molecules. In plenty of cases it is only the opsonins which bind to the phagocyte's receptors. The utility of opsonisation is very obvious. Phagocytes are naturally equipped with receptors which bind well to opsonins and this allows the body to respond effectively to all manner of pathogens. The opsonins effectively "draw the attention" of phagocytes, they make the process more efficient, since they tend to coat the surface of pathogens, and - in many cases - they promote phagocytosis which couldn't have happened otherwise. Here we see another example of how antibodies can induce the correct response to specific pathogens. Opsonins also allow phagocytes to overcome the natural repulsive forces they experience from pathogens, which are due to the fact that the cell membranes of both pathogens and phagocytes are negatively charged.
When the receptors of the phagocyte bind to ligands on the pathogen, the phagocyte will begin to project its cell membrane outwards, towards and around the pathogen. This process is mediated by a signalling cascade initiated by the receptors and is achieved in a manner similar to neutrophil chemotaxis (see Chemotaxis). Ultimately, under the direction of the signalling cascade, the actin cytoskeleton is reorganised and in this manner the phagocyte engulfs the pathogen and encapsulates it in a vesicle called a phagosome.
The phagosome is then transported within the cell and fuses with a granule or with a lysosome. A lysosome is simply an organelle (a structure within cells - a bit like an organ of a cell) containing substances which can break down many biomolecules. When the phagosome fuses with a lysosome or a granule, the resulting structure is called a phagolysosome.
The phagolysosome is a place which is extremely hostile to most pathogens and its purpose is their destruction. A few pathogens have evolved mechanisms to survive within the phagolysosome, but it is - broadly speaking - very effective. There are a number of ways in which pathogens may be killed within a phagocyte. Some of these are oxygen-dependent while the rest - as you'd expect - are oxygen-independent.
The first oxygen-dependent method begins with the production of O−
2 - or superoxide. Superoxide is very harmful to bacteria and it can also be converted to hydrogen peroxide - another substance which is damaging to pathogens. The superoxide can then react with the hydrogen peroxide and this produces hydroxyl radicals (•HO), which are highly reactive and, again, destructive to pathogens.
The second of these methods occurs in neutrophils. In this case, when the phagosome fuses with granules, an enzyme called myeloperoxidase produces hypochlorite (ClO−) from hydrogen peroxide and chlorine, which - of course - is used to destroy the pathogen.
These are the most effective mechanisms and the chemicals involved are very damaging - which is why they are contained within lysosomes and released into the phagolysosome (or produced in the phagolysosome itself). If this were not the case, the chemicals involved would be every bit as harmful to the phagocyte as to the pathogen. The oxygen-independent methods are less effective but still quite interesting. One such way uses charged proteins to inflict damage on the cell membranes of bacteria. Another uses lysozymes, which are antibacterial enzymes that I mentioned right at the very beginning. Lysozymes damage the cell wall of bacteria and destroy them that way. A very clever method employed by neutrophils uses lactoferrins. These are antimicrobials which work by essentially trapping iron - which is necessary for microbes to thrive. Finally, proteases and other enzymes may be used to hydrolyse1 peptide bonds between bacterial proteins. In other words, these enzymes digest the proteins of bacteria. I believe this is usually done at the end to "clean up" the remains.
Given below is a very simple - but very useful and informative - diagram showing a broad outline of the process:
In addition to all of this, phagocytes may be stimulated to release nitric oxide, which actually harms and destroys pathogens outside of the phagocyte. However, this does have the disadvantage of causing damage to the body's own cells as well.
Though phagocytosis is really the distinguishing feature of phagocytes, it is far from the only thing they do. As we saw when we discussed cell signalling using the example of macrophages, phagocytes release inflammatory cytokines to draw other phagocytes - and other leukocytes generally - to the site of infection. One cytokine which we have seen before - TNFα - is particularly important as it is also involved in the killing of cancer cells and cells which have been infiltrated by viruses.
Another very important function of phagocytes - particularly macrophages and dendritic cells - is antigen presentation. It really would not be wise to discuss that right now - we will learn all about this when we consider adaptive immunity. Right now, you only need to appreciate that, when the pathogen has been killed, in some cells it is possible for antigens to be recovered from the general debris and these can be "presented" to cells of the adaptive immune system. This serves to sensitise the immune system to that particular pathogen and to instigate a response to it at that time. It also contributes to the process whereby that pathogen can be recognised in future.
1i.e. cleave chemical bonds by hydrolysis
So, I've given you a list of different types of phagocytes and you know from Innate immunity broadly what they do - they ingest pathogens and destroy them. Here, I will go into more detail about what phagocytes are, what they do, and how they do it. Although I will be writing about phagocytes in general, I will have cause to look at specific examples. Then I will treat each of the professional phagocytes I have named individually.
As you know, phagocytes are attracted to sites of infection by chemotactic factors - for example, C5a is a chemotactic factor of neutrophils, among other cells. It is also possible for phagocytes to be drawn to pathogens by the pathogens themselves and it is even possible for phagocytes simply to collide with a pathogen by chance.
Now, upon arriving at the site of an infection, phagocytes phagocytose. Phagocytosis is instigated by the binding of a ligand on the surface of a pathogen to a receptor on the phagocyte. In some cases, this may be the binding of a PAMP to a PRR on the phagocyte. In many cases, opsonins assist in the process, or may be necessary for it to take place at all. Opsonins - such as C3b and various antibodies - work by binding to the receptors on the phagocyte instead of the pathogen's own molecules. In plenty of cases it is only the opsonins which bind to the phagocyte's receptors. The utility of opsonisation is very obvious. Phagocytes are naturally equipped with receptors which bind well to opsonins and this allows the body to respond effectively to all manner of pathogens. The opsonins effectively "draw the attention" of phagocytes, they make the process more efficient, since they tend to coat the surface of pathogens, and - in many cases - they promote phagocytosis which couldn't have happened otherwise. Here we see another example of how antibodies can induce the correct response to specific pathogens. Opsonins also allow phagocytes to overcome the natural repulsive forces they experience from pathogens, which are due to the fact that the cell membranes of both pathogens and phagocytes are negatively charged.
When the receptors of the phagocyte bind to ligands on the pathogen, the phagocyte will begin to project its cell membrane outwards, towards and around the pathogen. This process is mediated by a signalling cascade initiated by the receptors and is achieved in a manner similar to neutrophil chemotaxis (see Chemotaxis). Ultimately, under the direction of the signalling cascade, the actin cytoskeleton is reorganised and in this manner the phagocyte engulfs the pathogen and encapsulates it in a vesicle called a phagosome.
 |
| Figure 1.50: A simplified diagram showing the first three steps of this process. (Image courtesy Graham Colm (via Wikipedia)) |
The phagosome is then transported within the cell and fuses with a granule or with a lysosome. A lysosome is simply an organelle (a structure within cells - a bit like an organ of a cell) containing substances which can break down many biomolecules. When the phagosome fuses with a lysosome or a granule, the resulting structure is called a phagolysosome.
The phagolysosome is a place which is extremely hostile to most pathogens and its purpose is their destruction. A few pathogens have evolved mechanisms to survive within the phagolysosome, but it is - broadly speaking - very effective. There are a number of ways in which pathogens may be killed within a phagocyte. Some of these are oxygen-dependent while the rest - as you'd expect - are oxygen-independent.
The first oxygen-dependent method begins with the production of O−
2 - or superoxide. Superoxide is very harmful to bacteria and it can also be converted to hydrogen peroxide - another substance which is damaging to pathogens. The superoxide can then react with the hydrogen peroxide and this produces hydroxyl radicals (•HO), which are highly reactive and, again, destructive to pathogens.
The second of these methods occurs in neutrophils. In this case, when the phagosome fuses with granules, an enzyme called myeloperoxidase produces hypochlorite (ClO−) from hydrogen peroxide and chlorine, which - of course - is used to destroy the pathogen.
These are the most effective mechanisms and the chemicals involved are very damaging - which is why they are contained within lysosomes and released into the phagolysosome (or produced in the phagolysosome itself). If this were not the case, the chemicals involved would be every bit as harmful to the phagocyte as to the pathogen. The oxygen-independent methods are less effective but still quite interesting. One such way uses charged proteins to inflict damage on the cell membranes of bacteria. Another uses lysozymes, which are antibacterial enzymes that I mentioned right at the very beginning. Lysozymes damage the cell wall of bacteria and destroy them that way. A very clever method employed by neutrophils uses lactoferrins. These are antimicrobials which work by essentially trapping iron - which is necessary for microbes to thrive. Finally, proteases and other enzymes may be used to hydrolyse1 peptide bonds between bacterial proteins. In other words, these enzymes digest the proteins of bacteria. I believe this is usually done at the end to "clean up" the remains.
Given below is a very simple - but very useful and informative - diagram showing a broad outline of the process:
 |
| Figure 1.51: A simplified diagram of phagocytosis. (Image courtesy Graham Colm (via Wikipedia)) |
In addition to all of this, phagocytes may be stimulated to release nitric oxide, which actually harms and destroys pathogens outside of the phagocyte. However, this does have the disadvantage of causing damage to the body's own cells as well.
Though phagocytosis is really the distinguishing feature of phagocytes, it is far from the only thing they do. As we saw when we discussed cell signalling using the example of macrophages, phagocytes release inflammatory cytokines to draw other phagocytes - and other leukocytes generally - to the site of infection. One cytokine which we have seen before - TNFα - is particularly important as it is also involved in the killing of cancer cells and cells which have been infiltrated by viruses.
Another very important function of phagocytes - particularly macrophages and dendritic cells - is antigen presentation. It really would not be wise to discuss that right now - we will learn all about this when we consider adaptive immunity. Right now, you only need to appreciate that, when the pathogen has been killed, in some cells it is possible for antigens to be recovered from the general debris and these can be "presented" to cells of the adaptive immune system. This serves to sensitise the immune system to that particular pathogen and to instigate a response to it at that time. It also contributes to the process whereby that pathogen can be recognised in future.
1i.e. cleave chemical bonds by hydrolysis
Sunday, 8 June 2014
Mast cells
We begin, then, with mast cells. As I said at the beginning, the primary function of mast cells is the release of granules which contain chemicals such as histamine and heparin, which are a useful part of the immune response. Although histamine and heparin predominate somewhat, they are not the only substances released. Upon stimulation mast cells also release numerous other inflammatory mediators, as well as at least one type of cytokine. Together, these help to kick start the inflammatory response.
These substances, sadly, are not unconditionally useful - mast cells are somewhat infamous for their prominent role in allergy and anaphylaxis. Mast cells are a little trigger happy - this activity (degranulation, leading to the activation of the inflammatory response) is very important if there's a pathogen around, but mast cells can be stimulated to degranulate by harmless allergens. This will lead to the initiation of the inflammatory response in the absence of a pathogen and can be very harmful, as the substances I mentioned above can be dangerous to the body as well (see Inflammation).
Mast cells are produced in the bone marrow, from where they migrate in an immature form. Mast cells will either locate themselves in connective tissue, or in mucous membranes and there are slight differences which arise depending on where they are located. They develop in these locations, ready to be stimulated to degranulate as a result of an invading pathogen. These locations are obviously chosen as they are somewhat strategic.
There are three ways in which a mast cell can be stimulated to degranulate - the first is direct injury to the cell itself, the second is stimulation by complement proteins (one way in which the complement system complements other immune activities) and the third is by means of Immunoglobulin E (IgE). IgE is one of the five different isotypes of antibody. In addition, mast cells also express pattern recognition receptors and these may enable the mast cell to recognise pathogens and degranulate unassisted. We will concentrate on stimulation by means of IgE, as this is an informative example. As with all things we've been discussing, this is only one part of a larger picture and there are many other processes which combine to produce an effective response. Recall also that antibodies are part of the adaptive immune system and this is another instance of overlap between the two. Nevertheless, mast cells are part of the innate immune system as they respond generically, do not adapt and can be activated without the assistance of antibodies.
So, expressed on the surface of all mast cells is an important receptor called, helpfully, FcεRI. Now FcεRI has very high affinity for IgE and this tends to cause a build up of IgE on the surface of mast cells. An important subtlety to appreciate at this point is that antibodies will be produced in response to specific pathogens. We will discuss this in more detail later, but specific pathogens instigate the production of antibodies which bind well to an epitope on those particular pathogens. Therefore, when a pathogen - let's say a parasitic worm - instigates the production of IgE in this way, levels of IgE which bind well to that worm will build-up and will bind with the FcεRI receptors on the mast cells, which will then be able to recognise the worm. This can have a dark side if it should be a harmless allergen - like peanuts - which causes this, but it is plainly very useful in the case of harmful pathogens like the worm.
The FcεRI receptor is composed of four subunits - the name given to such a molecule is tetramer (remember that a dimer is a macromolecule formed of two smaller molecules; a tetramer is formed of four). It has one chain called the α chain, a β chain and two γ chains. The two γ chains are linked by a disulfide bridge, meaning that they are joined together by two sulfur atoms (there is one sulfur atom on each γ chain and the two sulfur atoms are bonded to each other, linking the γ chains):
On the α chain there are two domains which form the binding site for IgE. This site binds with a part of the IgE called the Fc region, which we will discuss in more detail later. As I described above, IgE antibodies will bind to the FcεRI receptors and build up on the surface of the mast cells. These antibodies, remember, bind well to epitopes on particular pathogens. When pathogen antigens bind to two (or more) of these IgE antibodies, the mast cell is activated.
The mast cell activation occurs by means of a signalling pathway. The binding of the antigen to the two IgE antibodies (themselves bound to FcεRI receptors), will bring about changes to the receptors, initiating a signaling cascade, leading to the degranulation of the mast cell.
The β and γ chains have as part of the chain an activation motif called ITAM (immunoreceptor tyrosine-based activation motif). When the antigen epitopes bind with the IgE antibodies (which are themselves bound to the α chains of two FcεRI receptors), this causes the receptors to cross-link - that is the chains become linked. The ITAM motif contains a chemical called tyrosine and when the FcεRI receptors become cross-linked, this change allows the tyrosine to become phosphorylated by Lyn tyrosine kinase.
Exactly how this happens is not entirely clear to me, but I believe that Lyn is found in lipid rafts, which are essentially small domains, or regions, of cell membranes. These regions are useful for cell signalling, as they serve to collect the necessary signaling molecules. So, when the receptors cross-link, Lyn is recruited and it associates with the FcεRIβ chain. Lyn becomes activated and phosphorylates the ITAM motifs of FcεRIβ and FcεRIγ. I will confess to not being entirely confident about the finer points of this process, but it seems that the cross-linking causes Lyn to associate with the tail of the FcεRIβ chain and I am given to understand that, since the cross-linking links the chains and brings them close together, this allows Lyn to phosphorylate the ITAM motifs of the FcεRIβ and FcεRIγ chains of all the receptors involved (in our case two, but more than two receptors may be cross-linked).
I mentioned above that my diagram is highly simplified and does not show the cross-linking. Some of you may prefer this more realistic alternative from Sari Sabban:
Now, once this has happened - once Lyn has phosphorylated the ITAM motifs - a tyrosine kinase called Syk is recruited. Syk binds to the phosphorylated ITAM motifs of the γ chains and this causes Syk to become activated and subsequently phosphorylated:
This is a very important stage as activated Syk can go on to activate many other proteins downstream. This, then, is the initiation of the cascade. One of the proteins so activated is LAT (linker for activation of T cells). When LAT is activated, phospholipase C (PLCγ) can bind to it and becomes phosphorylated too. This catalyses the breakdown of another protein called phosphatidylinositol bisphosphate (PIP2), which produces inositol trisphosphate (IP3) and diacyglycerol (DAG):
IP3 serves to increase the level of Ca2+ ions in the cell. Increased levels of both Ca2+ and DAG activates a kinase called protein kinase C (PKC). PKC and the increased levels of Ca2+ combine to direct the reorganising of the cytoskeleton through various intermediaries. The cytoskeleton is like the skeleton of the cell and it also provides the structure along which the secretory granules move to the cell membrane. The secretory granules consist of the chemicals - such as histamine - we mentioned above contained within a vesicle. The vesicle is a bit like a bag containing these proteins and facilitating their movement through the cell and into the outside environment. The vesicle binds with the cell wall and disperses its contents like so:
I have read suggestions1 that PKC may be involved in the process of the secretory granules fusing with the cell membrane. Wikipedia suggests that PKC is involved in severing the links between the surface of the secretory granules and the exoskeleton, allowing it to merge with the cell membrane.
I should also stress that it is difficult to understate how many other processes and sub-processes are involved. It is far beyond me - and this text - to go into all of them, I merely seek to give an overview. However, as I've mentioned before, a number of the the components I've already mentioned go on to do other things and take part in other downstream signaling events and other cascades which help to produce the effect, or produce other effects.
Before I wrap up the discussion of mast cells, I wanted to add a picture:
1http://en.wikipedia.org/wiki/Mast_cell#Degranulation_and_fusion
These substances, sadly, are not unconditionally useful - mast cells are somewhat infamous for their prominent role in allergy and anaphylaxis. Mast cells are a little trigger happy - this activity (degranulation, leading to the activation of the inflammatory response) is very important if there's a pathogen around, but mast cells can be stimulated to degranulate by harmless allergens. This will lead to the initiation of the inflammatory response in the absence of a pathogen and can be very harmful, as the substances I mentioned above can be dangerous to the body as well (see Inflammation).
Mast cells are produced in the bone marrow, from where they migrate in an immature form. Mast cells will either locate themselves in connective tissue, or in mucous membranes and there are slight differences which arise depending on where they are located. They develop in these locations, ready to be stimulated to degranulate as a result of an invading pathogen. These locations are obviously chosen as they are somewhat strategic.
There are three ways in which a mast cell can be stimulated to degranulate - the first is direct injury to the cell itself, the second is stimulation by complement proteins (one way in which the complement system complements other immune activities) and the third is by means of Immunoglobulin E (IgE). IgE is one of the five different isotypes of antibody. In addition, mast cells also express pattern recognition receptors and these may enable the mast cell to recognise pathogens and degranulate unassisted. We will concentrate on stimulation by means of IgE, as this is an informative example. As with all things we've been discussing, this is only one part of a larger picture and there are many other processes which combine to produce an effective response. Recall also that antibodies are part of the adaptive immune system and this is another instance of overlap between the two. Nevertheless, mast cells are part of the innate immune system as they respond generically, do not adapt and can be activated without the assistance of antibodies.
So, expressed on the surface of all mast cells is an important receptor called, helpfully, FcεRI. Now FcεRI has very high affinity for IgE and this tends to cause a build up of IgE on the surface of mast cells. An important subtlety to appreciate at this point is that antibodies will be produced in response to specific pathogens. We will discuss this in more detail later, but specific pathogens instigate the production of antibodies which bind well to an epitope on those particular pathogens. Therefore, when a pathogen - let's say a parasitic worm - instigates the production of IgE in this way, levels of IgE which bind well to that worm will build-up and will bind with the FcεRI receptors on the mast cells, which will then be able to recognise the worm. This can have a dark side if it should be a harmless allergen - like peanuts - which causes this, but it is plainly very useful in the case of harmful pathogens like the worm.
The FcεRI receptor is composed of four subunits - the name given to such a molecule is tetramer (remember that a dimer is a macromolecule formed of two smaller molecules; a tetramer is formed of four). It has one chain called the α chain, a β chain and two γ chains. The two γ chains are linked by a disulfide bridge, meaning that they are joined together by two sulfur atoms (there is one sulfur atom on each γ chain and the two sulfur atoms are bonded to each other, linking the γ chains):
 |
| Figure 1.42: A diagram showing FcεRI and illustrating the four chains which make it up |
On the α chain there are two domains which form the binding site for IgE. This site binds with a part of the IgE called the Fc region, which we will discuss in more detail later. As I described above, IgE antibodies will bind to the FcεRI receptors and build up on the surface of the mast cells. These antibodies, remember, bind well to epitopes on particular pathogens. When pathogen antigens bind to two (or more) of these IgE antibodies, the mast cell is activated.
 |
| Figure 1.43: A diagram showing an antigen binding to the paratopes of two IgE antibodies, which are themselves bound to two FcεRI receptors of a mast cell. This will lead to the activation of the mast cell. This diagram is intended to show a very simplified and approximate view of how things might look at the instant of binding, before any subsequent changes have taken place |
The mast cell activation occurs by means of a signalling pathway. The binding of the antigen to the two IgE antibodies (themselves bound to FcεRI receptors), will bring about changes to the receptors, initiating a signaling cascade, leading to the degranulation of the mast cell.
The β and γ chains have as part of the chain an activation motif called ITAM (immunoreceptor tyrosine-based activation motif). When the antigen epitopes bind with the IgE antibodies (which are themselves bound to the α chains of two FcεRI receptors), this causes the receptors to cross-link - that is the chains become linked. The ITAM motif contains a chemical called tyrosine and when the FcεRI receptors become cross-linked, this change allows the tyrosine to become phosphorylated by Lyn tyrosine kinase.
Exactly how this happens is not entirely clear to me, but I believe that Lyn is found in lipid rafts, which are essentially small domains, or regions, of cell membranes. These regions are useful for cell signalling, as they serve to collect the necessary signaling molecules. So, when the receptors cross-link, Lyn is recruited and it associates with the FcεRIβ chain. Lyn becomes activated and phosphorylates the ITAM motifs of FcεRIβ and FcεRIγ. I will confess to not being entirely confident about the finer points of this process, but it seems that the cross-linking causes Lyn to associate with the tail of the FcεRIβ chain and I am given to understand that, since the cross-linking links the chains and brings them close together, this allows Lyn to phosphorylate the ITAM motifs of the FcεRIβ and FcεRIγ chains of all the receptors involved (in our case two, but more than two receptors may be cross-linked).
 |
| Figure 1.44: At the risk of sounding like a broken record, I would just like to point out once more that the main purpose of my diagrams is to help keep track of the chemicals we're dealing with, since they very often have unhelpful names. I have chosen not to attempt to diagram the cross-linking, instead I have kept everything separate for clarity. The important thing to appreciate, however, is the cross-linking resulting from the antigen binding to the IgE causes a change in the two FcεRI receptors. This change has caused Lyn to phosphorylate the ITAM motifs on the FcεRIβ and FcεRIγ chains. |
I mentioned above that my diagram is highly simplified and does not show the cross-linking. Some of you may prefer this more realistic alternative from Sari Sabban:
 |
| Figure 1.45: A much more realistic, highly detailed diagram showing the cross-linking of two FcεRI receptors and the resulting phosphorylation of the ITAM motifs by Lyn. The original diagram was included by Sari Sabban in their PhD thesis "Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity FcεRI receptor." It was then uploaded to Wikipedia. I have cropped that image so that it shows only what is relevant at this stage and added a key and a few labels. The original image, and therefore this one, is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license |
Now, once this has happened - once Lyn has phosphorylated the ITAM motifs - a tyrosine kinase called Syk is recruited. Syk binds to the phosphorylated ITAM motifs of the γ chains and this causes Syk to become activated and subsequently phosphorylated:
 |
| Figure 1.46 |
This is a very important stage as activated Syk can go on to activate many other proteins downstream. This, then, is the initiation of the cascade. One of the proteins so activated is LAT (linker for activation of T cells). When LAT is activated, phospholipase C (PLCγ) can bind to it and becomes phosphorylated too. This catalyses the breakdown of another protein called phosphatidylinositol bisphosphate (PIP2), which produces inositol trisphosphate (IP3) and diacyglycerol (DAG):
 |
| Figure 1.47: Phosphorylated PLCγ catalyses the breakdown of PIP2 (left) into DAG and IP3 (right) |
IP3 serves to increase the level of Ca2+ ions in the cell. Increased levels of both Ca2+ and DAG activates a kinase called protein kinase C (PKC). PKC and the increased levels of Ca2+ combine to direct the reorganising of the cytoskeleton through various intermediaries. The cytoskeleton is like the skeleton of the cell and it also provides the structure along which the secretory granules move to the cell membrane. The secretory granules consist of the chemicals - such as histamine - we mentioned above contained within a vesicle. The vesicle is a bit like a bag containing these proteins and facilitating their movement through the cell and into the outside environment. The vesicle binds with the cell wall and disperses its contents like so:
 |
| Figure 1.48: On the left we have a vesicle. On the right we see it has moved (this will have been done by moving along the cytoskeleton) to the cell membrane, where is has merged with the membrane itself. You can see that, since the outer surface of the vesicle has merged to become a part of the cell membrane, its contents have been released into the external environment |
I have read suggestions1 that PKC may be involved in the process of the secretory granules fusing with the cell membrane. Wikipedia suggests that PKC is involved in severing the links between the surface of the secretory granules and the exoskeleton, allowing it to merge with the cell membrane.
I should also stress that it is difficult to understate how many other processes and sub-processes are involved. It is far beyond me - and this text - to go into all of them, I merely seek to give an overview. However, as I've mentioned before, a number of the the components I've already mentioned go on to do other things and take part in other downstream signaling events and other cascades which help to produce the effect, or produce other effects.
Before I wrap up the discussion of mast cells, I wanted to add a picture:
 |
| (a) A photograph showing cultured mast cells (100X magnification). The cells have been stained (using Tol Blue) and were activated in the course of an experiment. (Image courtesy "Kauczuk" (via Wikipedia)) |
 | ||
| (b) A schematic representation of a mast cell. (Image courtesy "A. Rad" (via Wikipedia)) Figure 1.49: The mast cell |
1http://en.wikipedia.org/wiki/Mast_cell#Degranulation_and_fusion
Cells of the innate immune response
At the beginning of this section, I gave an extremely cursory overview of each of the cells of the innate immune response and what they do. I couldn't go into detail at that point about how they work, but now I can. In this subsection, therefore, I will "flesh out" the discussion I gave at the beginning. This will, ultimately, be the final component of our discussion of the innate immune response. We have seen both processes and exactly what they do and how they do it at a relatively high level. All that is now left is to delve deeper into the world of the cells which are involved in innate immunity and explain the exact mechanisms by which they effect the responses.
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