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.
Friday, 2 May 2014
Summary
We have now concluded our discussion of the complement system - a biochemical cascade which complements other activities of the immune system. As part of the cascade, proteins are produced which perform these complementary roles and/or go on to react with other proteins to continue the cascade. The complement system produces chemotactic factors, which effectively "summon" immunocompetent cells - which can destroy the initiating pathogens - to the site of the infection. It can also mark pathogens as targets for macrophages and it produces the membrane attack complex, which destroys pathogens through lysis. Finally, it cleans up any neutralised antigen:antibody complexes.
This is all achieved through three different pathways: the classical pathway, the alternative pathway and the lectin pathway. All three of them are different ways of cleaving the important protein C3, which produces C3a and C3b. C3b - which is, additionally, an important opsonin - goes on to form C5-convertase, which cleaves C5 into C5a and C5b. C5b - through a series of intermediate complexes - forms the membrane attack complex, in conjunction with other complement components. This destroys cells through lysis. As a reminder, many of the intermediate products of these pathways, such as C4a, are important anaphylatoxins, which stimulate the release of histamine from mast cells. C5a is, additionally, a chemotactic factor and C3a may be involved in the production of the cytokine ASP.
Due to the dangers that the complement system can pose to the body, it needs to be highly regulated and this need underlies the reality of numerous, complex pathways - for it is important that the complement system only be activated when necessary, but it is equally important for it to be able to be activated quickly.
This is all achieved through three different pathways: the classical pathway, the alternative pathway and the lectin pathway. All three of them are different ways of cleaving the important protein C3, which produces C3a and C3b. C3b - which is, additionally, an important opsonin - goes on to form C5-convertase, which cleaves C5 into C5a and C5b. C5b - through a series of intermediate complexes - forms the membrane attack complex, in conjunction with other complement components. This destroys cells through lysis. As a reminder, many of the intermediate products of these pathways, such as C4a, are important anaphylatoxins, which stimulate the release of histamine from mast cells. C5a is, additionally, a chemotactic factor and C3a may be involved in the production of the cytokine ASP.
Due to the dangers that the complement system can pose to the body, it needs to be highly regulated and this need underlies the reality of numerous, complex pathways - for it is important that the complement system only be activated when necessary, but it is equally important for it to be able to be activated quickly.
Complement system
We come, now, to the complement system - the other prong of the innate immune system (although it has a relationship with the adaptive immune system, too). The complement system complements the activities of immunocompetent cells and of antibodies. The complement system is made up of proteins produced predominantly by the liver which are generally present in the blood plasma, but in inactive states. These proteins take part in a biochemical cascade initiated by means of three possible pathways - the classical complement pathway, the alternative complement pathway, and the lectin pathway - each of which we will look at. As we have already seen to an extent, this cascade serves a number of important functions:
Since the complement system can cause severe damage to the body it's defending, it is of critical importance that it be well regulated. The primary way of doing this is through complement control proteins, which we will talk about very soon, as they're very relevant and have an important role to play in the effective functioning of the system itself, as well.
Before we talk about the complement system, however, we have to discuss antibodies in a lot more detail. We've already made reference to them, but it is time to consider them properly. We will have more to say about them when we look at adaptive immunity; but, for now, we need only understand what they are and what they do.
Antibodies (which have the abbreviation Ab and are also referred to as immunoglobulin (or Ig)) are proteins which play a critical role in the immune system by instigating immunocompetent cells to attack a pathogen, or by directly interfering with the pathogen's ability to invade the body. Importantly, antibodies are capable of engendering particular responses to particular pathogens, which is obviously important for the effective and efficient removal of a threat.
Antibodies function by binding to antigens (antibody generators). An antigen is any substance which binds with an antibody. This is getting to be quite circular and we need to be more specific. An antigen is any substance which is able to be bound by the antigen-binding site of an antibody. Antigen-binding sites are referred to as paratopes and they are highly variable; each antibody will have a slightly different paratope. Each paratope will bind to a particular epitope,1 which is the part of an antigen which actually binds to an antibody. The analogy which is so often used is that of a lock and key. Estimates for the number of different antibodies produced by the human body reach 10 billion, which gives a vast range of paratopes. This, of course, means that there are a vast number of possible epitopes. Antibodies work because of their huge diversity. Billions are produced meaning that vast numbers of different substances can bind to them and can, therefore, be recognised. The definition of an antigen then becomes any substance which the immune system can recognise. In the earlier terminology an antigen was specifically any substance recognised by an antibody; however, we now tend to use the term "antigen" to refer, also, to substances which bind to receptors on cells of the immune system. Antigens tend to be proteins, polysaccharides and, in some cases, lipids. This is because antibodies are generally required to recognise the kind of things which these make up: things like bacterial or viral coats, capsules, cell walls, flagella, fimbrae and toxins. Because these molecules are very large, they may have multiple epitopes. A final, more rounded definition of an antigen, then, is any substance which exhibits one or more epitopes and is, therefore, recognisable by the immune system.
It is worth recognising a distinction, at this point, between antigenicity - the ability to bind to products of the immune response - and immunogenicity - the ability to engender an immune response. Not all antigens are immunogenic, although all immunogenic substances are antigenic, for - if they could not be recognised by the immune system, they could not engender an immune response. This difference is very important. Many of the body's own components have antigens and it is obviously very important that they aren't immunogenic. Unfortunately, in some individuals some of them are, or may become so, and this is what causes autoimmune diseases, such as diabetes - where a self-antigen is recognised and produces an immune response. This is also what is involved in transplant rejection, where a part of another's body is transplanted, recognised as a threat by its antigens, and attacked.
A related problem is allergens which are harmless substances (such as peanuts) which are incorrectly perceived as a threat. They are, then, immunogenic, despite their not being a threat. This occurs as a result of the way paratopes are constructed (more on that later) which is essentially arbitrary. Certainly, the paratope can't tell what it's binding to, it just binds to it. There are mechanisms to subdue the production of low-quality antibodies (which bind poorly to their epitope) and of antibodies which would produce an autoimmune response. However, the construction of paratopes is not guided, the B-cell which produces them is not an intelligence and cannot 'design' an antibody for a particular antigen. It just produces antigens and the immune system relies on the huge variety to recognise as many threats as possible. However, don't get me wrong, as I said above, the impetus of natural selection has furnished us with B-cells which produce antibodies capable of binding to the sort of chemicals which make up (or are produced by) pathogens and there are mechanisms to reduce the risk of an autoimmune response. However, sometimes antibodies will be produced which cause a response to the wrong thing and sometimes things will slip through the net (or, at least, take a long time to be recognised).
There is a lot more to say, and most of it will be said when we turn to the adaptive immune system, to which it is more relevant. Now, however, we need only understand that antibodies bind to antigens. Vast numbers are produced, allowing a vast range of substances to be recognised, allowing a response to be produced to most foreign substances which shouldn't be in the body. There are a number of different responses which can be occasioned, but one of them is the activation of the complement system (which can also be activated by other means). We now return, then, to the complement system.
So, the complement system begins with the activation of C3, which we have already talked about (Inflammation (part 3)). C3 is present in the blood in an inactive state, as we saw earlier, and it needs to be activated as the first stage of the complement system. To reiterate, the complement system is a danger to the body and hence must only be activated in response to a threat. However, it is also a very useful defence. Therefore, the entire basis of the complement system is inactive proteins, which are constantly in circulation, awaiting activation. The genius of the convoluted chain of events I am about to describe is that they ensure that the complement system is only operative when it is required. However, the constant circulation of the components means they can be present in good quantities all over the body, so that the complement system can be readily and quickly activated. The inactive components are harmless and it is their need to be activated that allows them to circulate quite safely until they are needed. The perfect trade-off between a prompt and destructive defence mechanism, and the need to protect the body from its own defences.
C3 is activated by cleavage - the large molecule that is the protein C3 must be split into two smaller proteins - C3a and C3b.
These proteins are useful in themselves (see Inflammation (part 3)) - C3b opsonises pathogens and C3a triggers the degranulation of mast cells, such that they release histamine. C3b is also involved in the rest of the cascade and is necessary for bringing about the other effects of the complement system.
So, the trigger for the complement system is the cleavage of C3. This is what all three of the pathways achieve. The classical pathway achieves the cleavage of C3 by producing a particular C3-convertase, which, in this case, is a complex of two proteins - C4b and C2a (the complex of these two is referred to as C4b2a).
There are actually two different molecules which can cleave C3 and activate the complement pathway, and both of them are C3-convertases (this is because C3-convertase is a description - both of them are convertases which cleave C3 - it is not a name). However, the classical pathway makes use of C4b2a to cleave C3. This, then, is what the classical pathway needs to produce, in the first place. The starting point is another complex of proteins called C1-complex. C1-complex is a complex of 5 molecules: 1 × C1q, 2 × C1r and 2 × C1s. This is denoted C1qr2s2, which we refer to as C1-complex.
The C1q has good affinity for two of the five different types of antibody (specifically IgM and IgG - more on these later). Recall that antibodies can occasion a number of different responses. The "choice" of response is influenced by which of the five isotypes is involved. Only two are involved in the classical complement pathway. This is noteworthy, as this is one of the ways in which specific, appropriate responses can be produced to specific pathogens. So, C1q binds to antigen:antibody complexes - specifically complexes of antigens and IgM or IgG. C1q can also bind to certain molecules on the surface of some pathogens. In either case, this binding causes a conformational change in C1q, which activates the two C1r molecules - forming a catlytically active molecule referred to as C1r*. This then cleaves and activates the two C1s molecules, forming C1s*.
If you're interested in a slightly less stylised and simplified diagram, you may prefer this alternative diagram:
After undergoing this activation, the C1s cleaves another complement component C4. This cleavage produces two smaller molecules - C4a and C4b.
C4a, like C3a, is an anaphylatoxin, meaning it stimulates histamine release from mast cells. Meanwhile, C4b binds to the membrane of the pathogen, before binding to our next complement component C2. This causes the activated C1s to cleave C2 into C2a and C2b.
We now have the components for C3-convertase. The C2a binds with the C4b, forming C4b2a.
C4b2a cleaves C3 into C3a and C3b. We know that C3a is an anaphylatoxin and that C3b opsonises pathogens. C3b also binds with C4b2a, giving C4b2a3b - C5-convertase.
C5 is another complement component and - you will be amazed to hear - is cleaved by C5-convertase into C5a and C5b. In another turn-up for the books, C5a is an anaphylatoxin, while C5b is involved in the membrane attack complex (Inflammation (part 3)).
C5b binds with C6, forming a complex. This complex then binds with C7, forming a complex which binds with C8. This complex catalyses the polymerisation of C9 and these polymers also bind with this same complex, giving C5b-C6-C7-C8-C9{n}. This is the membrane attack complex. When C7 binds to the C5bC6 complex it alters the configuration of the proteins and exposes a hydrophobic site of the C7 (i.e. a site which is repelled by water). The cell membrane of the pathogens affected by the MAC are composed of phospholipid bilayers which have a hydrophobic tail. They also have a hydrophilic head, which has good affinity for water. Since the hydrophobic tails are repelled by water, they aggregate when exposed to it, forming the lipid bilayer cell membrane. This consists of a two-layered sheet with all of the tails in the centre and all of the heads on the outside. This is highly impermeable to water-soluble molecules and allows the cell to regulate the passage of substances into and out of it. The hydrophobic site of the C7 is able to insert into this bilayer.
When the C8 binds to the complex of C5b, C6 and C7, a hydrophobic area is also exposed, which can also insert into the bilayer. The hydrophobic area of C8 (C8 alpha-gamma - C8 is composed of a complex of C8-beta and C8 alpha-gamma) also polymerises C9. Hydrophobic sites of C9 are similarly exposed and associate with the lipid bilayer. Brilliantly, the C9 has this external face which is hydrophobic and a hydrophilic internal face, allowing water to enter the cell. When enough of these pores are opened, the cell is simply flooded with water and, ultimately, falls apart.
This, then, is the complement system. This is what it does and how it does it. Well, it's one way it can do what it does. There are two other pathways and we continue onto the alternative pathway. The alternative pathway is based on the properties of C3. C3 is a little unstable in aqueous solutions and this means that it undergoes occasional hydrolysis. Hydrolysis is a simple process where water cleaves chemical bonds. In this case, it is an internal thioester bond that is cleaved. A thioester has the functional group C-S-CO-C, i.e. a radical bonded to a sulfur atom, bonded to a carbon atom, itself double bonded to an oxygen atom and bonded to a radical. The generic structural formula, then, looks like this:
Anyway, this thioester bond is able to be cleaved by hydrolysis, forming C3(H2O):
This is constantly happening spontaneously, but at a low level. The cleavage brings about a change in the shape of the molecule, which means that it can now bind with a plasma protein called factor B:
After this has happened, another protein called factor D can cleave factor B into Ba and Bb. The Bb remains bound to the C3(H2O), forming C3(H2O)Bb:
This is a fluid-phase C3-convertase, which is capable of cleaving C3 into C3a and C3b, in order to activate the complement system. Because this is constantly taking place, the process needs to be regulated. The C3(H2O) and the C3b that is produced by C3-convertase are inactivated by factor H and factor I (examples of complement control proteins), preventing the spontaneous activation of the entire complement system.
However, naturally, if there are pathogens present, the activation of the complement system is desirable. In this case, it is often true that the C3 will be cleaved by hydrolysis caused by a reaction with a hydroxyl or amino group of a cell surface molecule on a pathogen. This will leave C3b bound to the surface. Alternatively, the C3(H2O)Bb discussed earlier may cleave C3 and produce C3b, which will then bind to the surface of a pathogen. Once the C3b has bound to a pathogen, it is protected from being inactivated by Factor H.
In either case, C3b - now bound to the surface of a pathogen - can bind with factor B, forming C3bB. The factor B can then be cleaved by factor D, leaving us with the C3bBb complex bound to the surface of the pathogen. This is very similar to what happened before:
C3bBb is an unstable molecule; however it can bind with factor P (also called properdin), forming C3bBbP:
This is a stable C3-convertase, which can cleave C3 and produce more C3b, which binds to the same cell surface. Gradually, the amount of C3b will build up, "decorating" the cell and opsonising it. Many pathogens have no way of preventing this; however, the body's own cells possess complement regulatory proteins, such as CD35, CD46, CD55 and CD59. These inhibit this activity, preventing the body's own cells from activating the complement system. Furthermore, factor H can deactivate C3b on self cells, which is achieved by binding to glycosaminoglycans (GAGs). These are present on self cells, but not present on many pathogens. Therefore, the alternative complement pathway is halted when activated C3b binds to self cells, but not to pathogens.
Now, assuming the C3 convertase C3bBbP is bound to a pathogen, which needs to be eliminated, it will now be able to bind to another molecule of C3b which has been produced. This forms C3bBbC3b (sometimes shortened to C3b2Bb) - the C5-convertase of the alternative pathway. This cleaves C5 into C5a and C5b:
C5b can then bind with C6 and, eventually, the MAC will be formed and will be able to destroy pathogens by lysis.
To summarise, then, the alternative pathway is constantly being activated. However, if there are no pathogens around, it is quickly halted by complement control proteins. If, on the other hand, there are pathogens present, the cascade is not halted and the full complement system is initiated and takes effect. Once the pathogens are destroyed, there is nothing to sustain the complement system and it is halted.
Finally, we move on to the lectin pathway, which is very similar to the classical pathway. However, rather than C1-complex, the lectin pathway begins with a protein called mannose-binding lectin (MBL). Alternatively, a group of molecules called ficolins may also initiate this pathway. MBL binds with sugars, such as mannose and glucose, which are present on the surface of many pathogens, including salmonella and listeria. MBL is present in the blood in a complex with MASPs (MBL-associated serine proteases). There are three such MASPs, conveniently named MASP-1, MASP-2 and MASP-3. When MBL, in such a complex, binds with an appropriate sugar (say, for example, mannose), the MASP-1 and MASP-2 are activated and begin to cleave C4 and C2. Thus, we get C4a, C4b, C2a and C2b, whence we get C4b2a, whence we get C3a and C3b, whence we get the rest of the complement system. A similar process occurs with ficolins.
This, then, is the complement system - the second of the two major components of the innate immune system. As I highlighted above, the complement system does "interface" with the adaptive immune system and can be initiated by that system. It is, however, a much older and more primitive defence. Its response is also non-specific and the alternative pathway can be activated "automatically" and is not reliant upon antibodies. The key point to stress, though, is that it does not adapt over the course of the lifetime of an individual
1Some paratopes can bind to more than one epitope
- Some of the proteins serve as chemotactic factors for immunocompetent cells involved in the inflammatory response
- Some proteins opsonise pathogens, "marking" them as targets for phagocytosis
- As we saw above, the membrane attack complex destroys pathogens through the process called lysis
- Finally, "clearing up" antigen-antibody complexes (more on these shortly) is the last role that the complement system plays
Since the complement system can cause severe damage to the body it's defending, it is of critical importance that it be well regulated. The primary way of doing this is through complement control proteins, which we will talk about very soon, as they're very relevant and have an important role to play in the effective functioning of the system itself, as well.
Before we talk about the complement system, however, we have to discuss antibodies in a lot more detail. We've already made reference to them, but it is time to consider them properly. We will have more to say about them when we look at adaptive immunity; but, for now, we need only understand what they are and what they do.
Antibodies (which have the abbreviation Ab and are also referred to as immunoglobulin (or Ig)) are proteins which play a critical role in the immune system by instigating immunocompetent cells to attack a pathogen, or by directly interfering with the pathogen's ability to invade the body. Importantly, antibodies are capable of engendering particular responses to particular pathogens, which is obviously important for the effective and efficient removal of a threat.
Antibodies function by binding to antigens (antibody generators). An antigen is any substance which binds with an antibody. This is getting to be quite circular and we need to be more specific. An antigen is any substance which is able to be bound by the antigen-binding site of an antibody. Antigen-binding sites are referred to as paratopes and they are highly variable; each antibody will have a slightly different paratope. Each paratope will bind to a particular epitope,1 which is the part of an antigen which actually binds to an antibody. The analogy which is so often used is that of a lock and key. Estimates for the number of different antibodies produced by the human body reach 10 billion, which gives a vast range of paratopes. This, of course, means that there are a vast number of possible epitopes. Antibodies work because of their huge diversity. Billions are produced meaning that vast numbers of different substances can bind to them and can, therefore, be recognised. The definition of an antigen then becomes any substance which the immune system can recognise. In the earlier terminology an antigen was specifically any substance recognised by an antibody; however, we now tend to use the term "antigen" to refer, also, to substances which bind to receptors on cells of the immune system. Antigens tend to be proteins, polysaccharides and, in some cases, lipids. This is because antibodies are generally required to recognise the kind of things which these make up: things like bacterial or viral coats, capsules, cell walls, flagella, fimbrae and toxins. Because these molecules are very large, they may have multiple epitopes. A final, more rounded definition of an antigen, then, is any substance which exhibits one or more epitopes and is, therefore, recognisable by the immune system.
It is worth recognising a distinction, at this point, between antigenicity - the ability to bind to products of the immune response - and immunogenicity - the ability to engender an immune response. Not all antigens are immunogenic, although all immunogenic substances are antigenic, for - if they could not be recognised by the immune system, they could not engender an immune response. This difference is very important. Many of the body's own components have antigens and it is obviously very important that they aren't immunogenic. Unfortunately, in some individuals some of them are, or may become so, and this is what causes autoimmune diseases, such as diabetes - where a self-antigen is recognised and produces an immune response. This is also what is involved in transplant rejection, where a part of another's body is transplanted, recognised as a threat by its antigens, and attacked.
A related problem is allergens which are harmless substances (such as peanuts) which are incorrectly perceived as a threat. They are, then, immunogenic, despite their not being a threat. This occurs as a result of the way paratopes are constructed (more on that later) which is essentially arbitrary. Certainly, the paratope can't tell what it's binding to, it just binds to it. There are mechanisms to subdue the production of low-quality antibodies (which bind poorly to their epitope) and of antibodies which would produce an autoimmune response. However, the construction of paratopes is not guided, the B-cell which produces them is not an intelligence and cannot 'design' an antibody for a particular antigen. It just produces antigens and the immune system relies on the huge variety to recognise as many threats as possible. However, don't get me wrong, as I said above, the impetus of natural selection has furnished us with B-cells which produce antibodies capable of binding to the sort of chemicals which make up (or are produced by) pathogens and there are mechanisms to reduce the risk of an autoimmune response. However, sometimes antibodies will be produced which cause a response to the wrong thing and sometimes things will slip through the net (or, at least, take a long time to be recognised).
There is a lot more to say, and most of it will be said when we turn to the adaptive immune system, to which it is more relevant. Now, however, we need only understand that antibodies bind to antigens. Vast numbers are produced, allowing a vast range of substances to be recognised, allowing a response to be produced to most foreign substances which shouldn't be in the body. There are a number of different responses which can be occasioned, but one of them is the activation of the complement system (which can also be activated by other means). We now return, then, to the complement system.
So, the complement system begins with the activation of C3, which we have already talked about (Inflammation (part 3)). C3 is present in the blood in an inactive state, as we saw earlier, and it needs to be activated as the first stage of the complement system. To reiterate, the complement system is a danger to the body and hence must only be activated in response to a threat. However, it is also a very useful defence. Therefore, the entire basis of the complement system is inactive proteins, which are constantly in circulation, awaiting activation. The genius of the convoluted chain of events I am about to describe is that they ensure that the complement system is only operative when it is required. However, the constant circulation of the components means they can be present in good quantities all over the body, so that the complement system can be readily and quickly activated. The inactive components are harmless and it is their need to be activated that allows them to circulate quite safely until they are needed. The perfect trade-off between a prompt and destructive defence mechanism, and the need to protect the body from its own defences.
C3 is activated by cleavage - the large molecule that is the protein C3 must be split into two smaller proteins - C3a and C3b.
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| Figure 1.24: A highly simplified graphical representation of C3. This is C3 in its normal, inactivate state. It is a large protein composed of two smaller proteins C3a and C3b. The separation of these from one another is what is achieved by cleavage |
These proteins are useful in themselves (see Inflammation (part 3)) - C3b opsonises pathogens and C3a triggers the degranulation of mast cells, such that they release histamine. C3b is also involved in the rest of the cascade and is necessary for bringing about the other effects of the complement system.
So, the trigger for the complement system is the cleavage of C3. This is what all three of the pathways achieve. The classical pathway achieves the cleavage of C3 by producing a particular C3-convertase, which, in this case, is a complex of two proteins - C4b and C2a (the complex of these two is referred to as C4b2a).
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| Figure 1.25: The C3-convertase C4b2a |
There are actually two different molecules which can cleave C3 and activate the complement pathway, and both of them are C3-convertases (this is because C3-convertase is a description - both of them are convertases which cleave C3 - it is not a name). However, the classical pathway makes use of C4b2a to cleave C3. This, then, is what the classical pathway needs to produce, in the first place. The starting point is another complex of proteins called C1-complex. C1-complex is a complex of 5 molecules: 1 × C1q, 2 × C1r and 2 × C1s. This is denoted C1qr2s2, which we refer to as C1-complex.
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| Figure 1.26: C1-complex: C1qr2s2 |
The C1q has good affinity for two of the five different types of antibody (specifically IgM and IgG - more on these later). Recall that antibodies can occasion a number of different responses. The "choice" of response is influenced by which of the five isotypes is involved. Only two are involved in the classical complement pathway. This is noteworthy, as this is one of the ways in which specific, appropriate responses can be produced to specific pathogens. So, C1q binds to antigen:antibody complexes - specifically complexes of antigens and IgM or IgG. C1q can also bind to certain molecules on the surface of some pathogens. In either case, this binding causes a conformational change in C1q, which activates the two C1r molecules - forming a catlytically active molecule referred to as C1r*. This then cleaves and activates the two C1s molecules, forming C1s*.
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| Figure 1.27: As we've been going along, I've been making self-deprecating remarks about my own diagrams. This isn't just for the purposes of humour or anything like that, I want to stress that they're for illustration only. They're a bit like the tube map - they don't really show what the things they represent actually look like, they use straight lines and bright colours in the hope of providing a useful guide. This time, though, I really need to emphasise this again. I have chosen to leave my diagrams the way they are because I think it is clearer and because I'm bad at drawing. However, C1-complex looks nothing like this. Still, I hope the diagram above gives a flavour for what is going on. The change in the colour of C1q indicates the conformational change in C1q as a result of binding with the antigen:antibody complex. I have chosen to take this as an example and I have chosen to use an IgG antibody purely as an example. As I explained above, there are other possible alternatives |
If you're interested in a slightly less stylised and simplified diagram, you may prefer this alternative diagram:
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| Figure 1.28: An alternative diagram of the same process, showing a more accurate rendering of C1-complex. This is taken from a Wikipedia image and was authored by "Tossh_eng." I have only included the relevant part of a much larger image available here. We will come to the processes shown in the rest of that image in due course |
After undergoing this activation, the C1s cleaves another complement component C4. This cleavage produces two smaller molecules - C4a and C4b.
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| Figure 1.29: Here, C4 is shown in its normal, inactive state, composed of two proteins - C4a and C4b. I have tried to show it moments before it is cleaved by activated C1s. This cleavage, of course, will separate C4a from C4b |
C4a, like C3a, is an anaphylatoxin, meaning it stimulates histamine release from mast cells. Meanwhile, C4b binds to the membrane of the pathogen, before binding to our next complement component C2. This causes the activated C1s to cleave C2 into C2a and C2b.
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| Figure 1.30: Again, I am attempting to show the process moments before the cleavage takes place. You can see that C2 is shown in its inactive state, that it has bound with C4b - which has now bound to the membrane - and that it is about to be cleaved by activated C1s. Note, also, that C4a has now moved away from where this is all taking place |
We now have the components for C3-convertase. The C2a binds with the C4b, forming C4b2a.
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| Figure 1.31: You may recognise the C3-convertase C4b2a from before |
C4b2a cleaves C3 into C3a and C3b. We know that C3a is an anaphylatoxin and that C3b opsonises pathogens. C3b also binds with C4b2a, giving C4b2a3b - C5-convertase.
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| Figure 1.32: This time I have chosen to show unactivated C3 moments before it is cleaved on the left. On the right is the result of this cleavage - C3a goes off to fulfil its role as an anaphylatoxin, while C3b binds with the C3-convertase (C4b2a) to form C5-convertase (C4b2a3b) |
C5 is another complement component and - you will be amazed to hear - is cleaved by C5-convertase into C5a and C5b. In another turn-up for the books, C5a is an anaphylatoxin, while C5b is involved in the membrane attack complex (Inflammation (part 3)).
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| Figure 1.33 |
C5b binds with C6, forming a complex. This complex then binds with C7, forming a complex which binds with C8. This complex catalyses the polymerisation of C9 and these polymers also bind with this same complex, giving C5b-C6-C7-C8-C9{n}. This is the membrane attack complex. When C7 binds to the C5bC6 complex it alters the configuration of the proteins and exposes a hydrophobic site of the C7 (i.e. a site which is repelled by water). The cell membrane of the pathogens affected by the MAC are composed of phospholipid bilayers which have a hydrophobic tail. They also have a hydrophilic head, which has good affinity for water. Since the hydrophobic tails are repelled by water, they aggregate when exposed to it, forming the lipid bilayer cell membrane. This consists of a two-layered sheet with all of the tails in the centre and all of the heads on the outside. This is highly impermeable to water-soluble molecules and allows the cell to regulate the passage of substances into and out of it. The hydrophobic site of the C7 is able to insert into this bilayer.
When the C8 binds to the complex of C5b, C6 and C7, a hydrophobic area is also exposed, which can also insert into the bilayer. The hydrophobic area of C8 (C8 alpha-gamma - C8 is composed of a complex of C8-beta and C8 alpha-gamma) also polymerises C9. Hydrophobic sites of C9 are similarly exposed and associate with the lipid bilayer. Brilliantly, the C9 has this external face which is hydrophobic and a hydrophilic internal face, allowing water to enter the cell. When enough of these pores are opened, the cell is simply flooded with water and, ultimately, falls apart.
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| Figure 1.34: A very highly stylised diagram of the MAC, giving a broad idea of how it works and the proteins involved. I would imagine there would be more units of C9 and it should not be presumed that the proteins would join together in this way. This is merely the principle |
This, then, is the complement system. This is what it does and how it does it. Well, it's one way it can do what it does. There are two other pathways and we continue onto the alternative pathway. The alternative pathway is based on the properties of C3. C3 is a little unstable in aqueous solutions and this means that it undergoes occasional hydrolysis. Hydrolysis is a simple process where water cleaves chemical bonds. In this case, it is an internal thioester bond that is cleaved. A thioester has the functional group C-S-CO-C, i.e. a radical bonded to a sulfur atom, bonded to a carbon atom, itself double bonded to an oxygen atom and bonded to a radical. The generic structural formula, then, looks like this:
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| Figure 1.35: The structural formula of a thioester. (Image courtesy of Ben Mills and "Vladsinger," via Wikipedia) |
Anyway, this thioester bond is able to be cleaved by hydrolysis, forming C3(H2O):
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| Figure 1.36: A very unrealistic representation of C3(H2O), purely for diagrammatic purposes |
This is constantly happening spontaneously, but at a low level. The cleavage brings about a change in the shape of the molecule, which means that it can now bind with a plasma protein called factor B:
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| Figure 1.37 |
After this has happened, another protein called factor D can cleave factor B into Ba and Bb. The Bb remains bound to the C3(H2O), forming C3(H2O)Bb:
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| Figure 1.38: The Factor B is cleaved by Factor D |
This is a fluid-phase C3-convertase, which is capable of cleaving C3 into C3a and C3b, in order to activate the complement system. Because this is constantly taking place, the process needs to be regulated. The C3(H2O) and the C3b that is produced by C3-convertase are inactivated by factor H and factor I (examples of complement control proteins), preventing the spontaneous activation of the entire complement system.
However, naturally, if there are pathogens present, the activation of the complement system is desirable. In this case, it is often true that the C3 will be cleaved by hydrolysis caused by a reaction with a hydroxyl or amino group of a cell surface molecule on a pathogen. This will leave C3b bound to the surface. Alternatively, the C3(H2O)Bb discussed earlier may cleave C3 and produce C3b, which will then bind to the surface of a pathogen. Once the C3b has bound to a pathogen, it is protected from being inactivated by Factor H.
In either case, C3b - now bound to the surface of a pathogen - can bind with factor B, forming C3bB. The factor B can then be cleaved by factor D, leaving us with the C3bBb complex bound to the surface of the pathogen. This is very similar to what happened before:
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| Figure 1.39 |
C3bBb is an unstable molecule; however it can bind with factor P (also called properdin), forming C3bBbP:
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| Figure 1.40 |
This is a stable C3-convertase, which can cleave C3 and produce more C3b, which binds to the same cell surface. Gradually, the amount of C3b will build up, "decorating" the cell and opsonising it. Many pathogens have no way of preventing this; however, the body's own cells possess complement regulatory proteins, such as CD35, CD46, CD55 and CD59. These inhibit this activity, preventing the body's own cells from activating the complement system. Furthermore, factor H can deactivate C3b on self cells, which is achieved by binding to glycosaminoglycans (GAGs). These are present on self cells, but not present on many pathogens. Therefore, the alternative complement pathway is halted when activated C3b binds to self cells, but not to pathogens.
Now, assuming the C3 convertase C3bBbP is bound to a pathogen, which needs to be eliminated, it will now be able to bind to another molecule of C3b which has been produced. This forms C3bBbC3b (sometimes shortened to C3b2Bb) - the C5-convertase of the alternative pathway. This cleaves C5 into C5a and C5b:
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| Figure 1.41: Again I show unactivated C5 moments before it is cleaved on theleft. On the right is the result of this cleavage |
C5b can then bind with C6 and, eventually, the MAC will be formed and will be able to destroy pathogens by lysis.
To summarise, then, the alternative pathway is constantly being activated. However, if there are no pathogens around, it is quickly halted by complement control proteins. If, on the other hand, there are pathogens present, the cascade is not halted and the full complement system is initiated and takes effect. Once the pathogens are destroyed, there is nothing to sustain the complement system and it is halted.
Finally, we move on to the lectin pathway, which is very similar to the classical pathway. However, rather than C1-complex, the lectin pathway begins with a protein called mannose-binding lectin (MBL). Alternatively, a group of molecules called ficolins may also initiate this pathway. MBL binds with sugars, such as mannose and glucose, which are present on the surface of many pathogens, including salmonella and listeria. MBL is present in the blood in a complex with MASPs (MBL-associated serine proteases). There are three such MASPs, conveniently named MASP-1, MASP-2 and MASP-3. When MBL, in such a complex, binds with an appropriate sugar (say, for example, mannose), the MASP-1 and MASP-2 are activated and begin to cleave C4 and C2. Thus, we get C4a, C4b, C2a and C2b, whence we get C4b2a, whence we get C3a and C3b, whence we get the rest of the complement system. A similar process occurs with ficolins.
This, then, is the complement system - the second of the two major components of the innate immune system. As I highlighted above, the complement system does "interface" with the adaptive immune system and can be initiated by that system. It is, however, a much older and more primitive defence. Its response is also non-specific and the alternative pathway can be activated "automatically" and is not reliant upon antibodies. The key point to stress, though, is that it does not adapt over the course of the lifetime of an individual
1Some paratopes can bind to more than one epitope
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