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Microbiology: Cytokines pg. 12

Laura Rayne

*The audio began at slide 2

Slide 1: Title slide

Slide 2

This is not something you have to memorize for a test but the point of this slide was to show you that almost every facet of the immune system is regulated by one or more different cytokines. And this slide basically shows adaptive immune responses; it doesn’t really show innate immunity and it doesn’t show development of the immune system as well, which is in part controlled by a number of different cytokines. So you can see that it is an extremely complex system. There is no way we would be able to cover all this stuff, and there is an awful lot we still don’t know about these molecules and how they work, and we’re still discovering molecules that participate in one family or another of cytokine responses in immunity.

Slide 3

These guys are basically the master regulators of the immune system. These are the small molecules that send signals from cells within the immune system, from cells that are infected that aren’t intrinsically part of the immune system themselves to the cells that will respond to infection. They also are molecules that are responsible for development of things like lymph nodes, where you have the collections of T and B cells, dendritic cells throughout the body so you can mount an immune responses to invading pathogens. They also are responsible for tamping down the immune response so there are a number of different cytokines that help shut the immune system down after you have eliminated a pathogen. So they are critical for all facets of the immune system. If you are deficient in certain cytokines, you’re going to be extremely susceptible to infection and have really big problems. You may not even develop certain arms of the immune system as a result of certain deficiencies.

The other reason that these molecules are very important is they have become part of the repertoire of therapy for a variety of different diseases and maybe not so much so for the kinds of clinical settings that you’re going to be involved in, but increasingly so people will find applications, certainly in experimental settings, for a variety of cytokines that resolve inflammation and help in cancer settings. So you’re going to hear and see that as you progress through your career and certainly you’re going to be treating patients who are on cytokine therapy of one sort or another as you go along and that may impact how you treat that particular patient.

Slide 4

Just like everything else in biology the nomenclature for these molecules is a mess. That is in part because as molecules are discovered and functions are assigned to them they get certain names and then as families become established and understood a little bit then the names change again and it goes on and on. So as you look in textbooks or look in literature you going to find a bunch of these different names that are up here.

The first one that was probably used was monokines and these were supposed to cytokines that were produced only by monocytes. There really isn’t a monokine anymore because cytokines are produced by lots of different cell types so this is one of these early names that doesn’t really apply anymore. The same for lymphokines-cytokines that were produced only by activated T cells. Now we know that lots of the cytokines produced by T cells are also produced by a variety of different cell types in and out of the immune system.

Probably the terms that you’re going to see the most are interleukins and interferons. So interleukins are cytokines that were originally described as those that were produced by leukocytes and affected other leukocytes and that’s not really true but the term interleukin still sticks and now we have up to 30-something different interleukins that have been described and more being added to the list almost on a yearly basis. Interferons are some of the earliest cytokines that were described because of their role in regulating viral diseases or infections, and we’ll talk about some interferons and how they control viral infections a little later on.

There are also a number of cytokines that fall under the category of colony-stimulating factors. These are cytokines that are critical for the development of the different subsets of cells in the immune system. So you have lymphocytes, you have monocytes, you have neutrophils, eosinophils, mast cells. These colony-stimulating factors are critical in the development of the early progenitors as the hematopoietic system is developing and helping to push these different cells to their terminally differentiated state and the functions they are going to play in the immune response.

And then there is a large family of molecules called chemokines. I’m just going to touch on those. There are about 60-something different chemokines that have been described now and their function is really diverse throughout the immune response. And there are a number of growth factors that are also critical for stem-cell differentiation and also play a role in a number of other functions in the immune response helping to push these progenitors along and so forth so that you can get all the cell types you need for mounting an effective immune response.

Slide 5

Cytokines as you’ll quickly discover are really a complex system of molecules. Part of the problem in figuring out how these molecules work and what they’re doing is that they’re pleiotropic, and that means that they mediate lots of different functions. So for example a cytokine called interleukin-1 (IL-1) can serve as an important part of the acute-phase response, but it can also help augment adaptive immunity by binding to receptors on T cells and B cells and signaling them to do different things. So all of these different cytokines have multiple functions depending on the type of infection and when they were produced, how much were produced, and the cell types that express the receptors for them. So it can be very complex.

Cytokines are also redundant in their function. We’re going to talk about a number of different cytokines that are important in fighting off bacterial infections – cytokines like Tumor Necrosis Factor-α (TNF-α), but a lot of the same functions are mediated by IL-1 and IL-6. So they have overlapping functions, and that is critical because if they only had single functions and they were deficient then you would really be susceptible to infection and have big problems.

These molecules can also act in a synergistic fashion. That means they can act together, not in an additive fashion, but in a fashion that is much greater than the addition of the two. So if you had for example a biological response when you treated cells with TNF and another response when you treated them with IL-1, it might be that when you put the two of them together you get a much higher response. It really boosts the production of any number proteins or molecules, depending on the context of the infection.

Cytokines are also antagonistic, so that’s really critical for helping to tamp down the immune response. The molecules for example γ-interferon or TNF-α are very potent inflammatory cytokines, helping to promote immune responses. Other cytokines for example IL-4, IL-10, and TGF-β are considered anti-inflammatory cytokines so they help reduce those immune responses and keep them in proportion to the infection you have. So these molecules are very diverse in the functions that they mediate.

Slide 6

By and large cytokines are low molecular weight proteins or glycoproteins, usually 10,000 molecular weight or less, but they can also be multimeric so you can have homodimers or trimers of some cytokines and of course their molecular weights are going to be higher. In some cases, unless these cytokines are multimeric you’re not going to get binding to the receptor or efficient binding to the receptor and a good signal mediated by that cytokine. Cytokines can be synthesized both in an active and in an inactive form so they can be produced and immediately used in some cases and other cytokines are produced in a latent form. For example, TGF-β is a cytokine that can be produced and sit on the surface of a cell. In order for it to be functionally active it has to be cleaved by another protein from the cell surface so that it can be released to bind to its receptor.

Another important feature of cytokines is that their production is generally very brief and self-limiting - on the order of minutes. That’s a very critical factor in limiting what a cytokine can do and how much it can regulate or modulate a given immune response because certain cytokines if they’re produced without any level of control or for extended periods of time can be very damaging to the host, particularly the pro-inflammatory cytokines like TNF.

These molecules are active at very low concentrations so nanomolar or even lower concentrations: 10-8 to 10-9. So they have very high affinity for their receptors and you don’t need very much. A little goes a long way with most cytokines. An important thing to remember is that all signaling mediated by cytokines goes to a receptor. All cytokines use receptors. If a cell doesn’t express a certain cytokine receptor it is not going to mediate anything in terms of changing the function of that given cell type.

Slide 7

As I said every cytokine has a receptor, but sometimes cytokines share receptors. For example there are lots of different α and β interferons but they all use the same receptors to mediate their functions. Some of the chemokines can bind to different receptors. There are lots of different family receptors for cytokines, at least 5 different ones for the main cytokines. There are additional ones for the chemokines. Cytokine receptors can be multimeric, so you can have single-chain receptors, you can have dimers which are very common, and you can even have trimers. Unless you have all the subunits in the case of those multimeric receptors you aren’t going to have proper or fully potent cytokine-mediated signaling for that particular cytokine.

Signaling through cytokines and I’m just going to touch on this because I don’t want you to memorize a lot of signaling stuff but I think you need to at least see how this works because there is an awful lot of biology being looked at for modulating cytokine production through the signaling pathways that they use in order to change activity in the cell. So I think it’s not going to be unusual in the future for people to be treated with drugs that modulate these signaling pathways rather than block the binding of the cytokine to its receptor.

Slide 8

I don’t want you to memorize this. Don’t worry about the structures or the names or anything. I just want you to know that these different receptor families are delineated based on certain structural features and they bind certain different cytokines.

There are some cytokine receptor families that bind for example IL-1 and a number of different related cytokines that have these structural features. This is called an immunoglobulin-binding domain because it has this 60 to 70 amino acid loop that is disulfide bridged. It is the main determining structure in immunoglobulins or antibodies. There are other receptor families that are defined by the presence of certain conserved cysteine residues and other residues. Again you can see that some of these are dimeric. A lot of the chemokines but not all of them bind to what are called 7-transmembrane-spanning receptors and they transmit signals through a family of molecules called G proteins. Interferons bind to dimeric receptors, again defined by these different cysteine residues and some of the receptor families can get extremely complex and large. The TNF receptor family is really large and there are lots of family members and different receptors that mediate signals that can be involved in killing or inactivation, all kinds of different things, depending on the ligand and receptor. So all the families of receptors are determined by certain structural motifs and that’s all I want you to know for that.

Slide 9

There are a couple other things though that you need to know. In the case of cytokine receptors where there are multimeric subunits there is going to be one subunit that is going to be the signaling subunit and others are going to be involved in binding or the initial binding of the cytokine. This particular family is the IL-2 receptor subfamily of cytokines and they all share this common γ chain. It is this common γ chain that is the critical component of that receptor subunit complex that transduces the signal into the cell. The other subunit portions are called the cognate portion of the receptor and these are the parts of the receptor that initially bind the cytokine and then you have the signaling receptor subunit. So if you’re missing this common γ chain that is used by all these different cytokine receptors then you’re in big trouble because these cytokines are critical for development of lymphocytes. So you’re basically not going to develop an adaptive immune response because you won’t have T and B cells or you’ll have very few of them. They may not mature properly under those circumstances. Of course all of these different cytokines mediate different responses so you’re going to have defects in all those different facets of the immune system as well. So defects in certain cytokine receptor subunits are really bad things. They’re also extremely rare.

Slide 10

I just want to take you through a very generalized scheme of how cytokines transduce signals into a cell. This is our cognate receptor. This is the part of the cytokine receptor that binds to the cytokine. Once that binding occurs you’ll have association with what I’m calling the β chain so now you’ll have both subunits for that receptor binding the cytokine. It turns out that inside the cell you continually have association of the cytoplasmic tail of these receptor complexes with a kinase called Janus kinase (JAK), and there are several family members within that group of kinases. When that complex all comes together - both the α and the β chain with the receptor complex, the binding of the cytokine and the association of this kinase - the kinase will become activated, it will phosphorylate itself, it will phosphorylate the cytoplasmic tail of the receptor complex, and that complex then of these phosphorylation sites on this portion of the cytokine receptor forms a docking site for these molecules called the STATs, which is another family of signaling molecules. They’ll bind to that complex and then they get phosphorylated by the kinase too. So cytokine-mediated signaling is regulated in large part by phosphorylation events, at least in certain families. You can turn this process down by dephosphorylating these molecules. So once these STAT molecules are phosphorylated they will homodimerize and they’ll translocate into the nucleus where they’ll serve as DNA-binding proteins to modulate transcription of a variety of different genes.