03/04: innate immunity

Transcribed by Mandy Weil July 2, 2014

General Pathology - The Innate Immune Response by Dr. McCutcheon [1] – [The Inate Immune response] [McCutcheon] – Welcome to General Pathology. This is the immunology portion of General Pathology. For those of you who don’t know me, I’m Dr. McCutcheon and I’m going to be taking you through the journey of the rest of the summer in this class. I want to start by talking a little about this topic. You are all here, so that means that you were very successful last year. Most of last year was a lot of memorization and memorization got you where you needed to go. Unfortunately, Dentistry is an essay profession. Okay? Your patient is going to walk in your room and they’re going to sit in your chair and its up to you to figure out what you’re going to figure out in order to treat them. Okay? They don’t come with a bib that tells you what to look for. It’s up to you to figure this out. It’s up to you to look at whatever complaint they have an diagnose the symptoms and that is not something that goes along with memorization. So starting this year, you’re going to need to learn to do something different. You’re going to need to learn to integrate information. You should have done it last year in Organ Systems, but most of you probably didn’t and you got away with it. You’re not gonna get away with it if you continue to do that. You’re not going to learn to diagnose. So, immunology is the first topic where there’s memorization and there’s a lot of it and it’s not enough. Okay? You need to go beyond memorization and you need to put pictures of the puzzle together. And the way I teach this course is I tell a story and you know that you can understand the story when you can explain the story to someone else. So it’s a good way to study this in groups cause you can talk yourself into anything no matter how illogical, but your friends will say “Oh no, that doesn’t make sense.” I’m helping you out posted along with every lecture is a study guide. Some of the questions you’re going to be able to look at the slides and know the answers. Some of the study guide questions, you’re going to have to figure out the story to answer. When we do our conferences, our conferences will be splitting into groups and then you the students will answer all of the questions in the study guide. I do not give you the answers. I have given you all the information that you need in lecture. So it’s up to you to put the answers together and answer the questions in the study guide. Do this is in groups. It’s really good to study this stuff in groups. Okay? You learn a lot better from studying with your friends. So, one of the things that I’m after are concepts and to help me do this, Dr. Phelan has actually given me an extra hour, so that when we’re working with a difficult concept—and this lecture series is actually going to be one of the, if not the most difficult things you learn in dental school—If you learn immunology, all of pathology drops into place. Rather than trying to memorize a set of symptoms that don’t make any sense, you’ll know immunology and you can say “Oh! that’s how this works!” And then you understand the disease rather than try and memorize the disease. And you’re going to talk about B and T cells, you’re going to talk about cytokines in every single class that you have and you’re going to deal with these drugs, you’re going to deal with these cytokines in every single solitary patient that you treat. So it’s really worth doing the work now. And part

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of the reason, we move immunology in the summer is because you’re somewhat less busy and you’ve had a whole month to rest so you’re not as tired. Plus that way, the test is just about immunology, cause traditionally, they tend to not study the other subjects on the test. So, we did this to help you guys. So, I have extra time, that frequently means that I’m not going to end a lecture at the last slide. I’ll end when we run out of time. I’ll pick that topic up at the next lecture. I’ll finish that topic. I’ll start the next topic after that. So you know, be prepared, we’re going to take the time that we need to go through the concepts. There are a lot of details your book has—although we picked a book thats deliberately not detail heavy. Parum. There still are details in there. When I want you to know a detail, I will tell you specifically “you have to know that.” Okay? If there are details on the slides and I’m not talking about them, you need to know the CONCEPT, you don’t need to know the detail. And you are used to knowing details and a lot of people try to do that. It’s certainly isn’t going to hurt you. But it’s not going to get you where you need to go. Spend the time learning the concept. Putting this piece and that piece and that piece together. A lot of you have never really practiced doing that, so As we going through the study guides—you’ll see that there are more ‘how?’ and ‘why?’ questions as we get further on into the lecture series. How and why are not things you can memorize. How and why are things you have to figure out. Alright? One of the other things that’s really important to do and if you talk to the upperclassmen who did well in this class—they will tell you: “stay caught up.” This is a building lecture series so everything we talk about now, you’re expected to understand that for the next lecture. So if you think you’re going to study for this two days before the test, I’ll tell you now, you’re going to flunk. Alright? You can’t do it. So stay caught up! and we’ve got this spread out so you have time to do that. Alright so… we’re going to pick up where we left off in May. We learned about the lymphoid organs and now we’re going to talk about the immune system itself. The order that I’m telling this story is the order that a lymph, an infection, a reaction to an infection occurs. So we’re going to start at the beginning and we’re going to go through to the end. Now there are a couple times where I have to drop out of that sequence and explain something before we can move forward. But they’re very complicated B and T cell development and I didn’t want to start there because what a great way to lose your entire audience, to start off with the most difficult thing you can talk about. So we’re gonna do that in order…One of the annoying things about this is that there are always things that have happened that we haven’t talked about. I’ve got those down to the minimum number of places, but I can’t get rid of all of them and I apologize in advance. It’s the nature of the beast. By the way—It’s lovely to see you all here at 8 o’clock in the morning.

[2] – [“Immunology is the study…”] [McCutcheon] – So, Immunology is the study of the physiologic mechanisms that humans and other animals use to defend their bodies from invasion by other organisms. And they keyword in that—or keywords—are physiologic and defend. One of this course’s pathology. And pathology as a rule is what happens when

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physiologic immunity doesn’t work. Ok? And then you go from an acute inflammatory process to a chronic inflammatory process and all of the things that are going wrong in a necrotic inflammatory process. I’m going talk about normal. I’m going to talk about a physiologic immune response. And everybody who follows be is going to talk about the things that happen when an immune response goes wrong. and to understand this and to defend—this is your defense mechanism. The Army, the Navy, the Marines and the Air Force put together And, in order to know when we’re defending against, we need to define something. And we’re going to define an antigen. An antigen is a part on a pathogen—bacteria, virus, fungi, parasite, some debris or ragweed pollen—an antigen is the part of the pathogen that’s recognized by the specific receptors of a B cell, the antibodies, or a T Cell. So the antigen--and I’ll use the terms ‘antigen’ and ‘pathogen’ somewhat interchangeably--but in fact, antigens are the spot on the pathogen that the immune system recognizes. Antigens are the spot on the pathogen that the immune system recognizes. Because we don’t want our immune system to react against us, so the antigen is something that is not found on a host cell. Cause otherwise, you’d have immunity against you and those are called autoimmune disease and they’re all bad. So the antigen is a spot on the pathogen that is different from anything on host and because it’s different, it can be recognized by the immune system. [3] – [Unique Features] [McCutcheon] – We talked about this in May. There are several unique features of the immune system that don’t happen for other cells. Lymphocytes move. Their job is to be in the circulation, to move around in the circulation, to crawl out of the circulation and into the tissues. They move. Lymphocyte cells change their function over time. They’re secreted as inactive cells. They become activated, they do their thing. Some of them stop being activated. Others have very short half lives. Only 2-3 days and they die. Okay? Some of them have long half lives. They do their thing, they can stop doing their thing. They’re release as inactive cells for both kinds of immunity, innate and adaptive. They have to be turned on and we’ll spend a fair bit of time talking about that this summer. And their functions are specific and in May, I told you I would explain that and we’re gonna start explaining that today and we’re gonna pick that up Friday the 11th. [4] – [Picture (Intrinsic epithelial barriers to infection] [McCutcheon] – Now before an immune response occurs, You have to get the pathogen inside the body. Okay? So there are barriers that, while not officially apart of the immune response, keep the immune system from needing to be active. And the first barrier is fairly obvious—it’s the physical barrier of your skin and the endothelial cells line all of the cavities of the body that come into contact with the outer world. Cause as long as you’ve got the pathogens out here, you don’t need an immune response. So before you can get an immune response going, you have to break the physical barrier. There are some other barriers that help out. So the next one is chemical. Your skin, saliva, tears, they secrete chemicals that help

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degrade viruses and keep bacteria from growing. So low pH enzymes like pepsin, there are small molecules called defensins and all of those help keep skin, bacteria and any viruses from being able to attach and replicate. So there’s a chemical barrier. And then, surprisingly, there’s a microbiological barrier. So any of you who’ve always thought all pathogens, all bacteria are bad, you’re wrong. and you can tell that now because they have all these commercials for probiotic this and probiotic that. You know what they’re giving you bacteria. Some bacteria keep you healthy. One of the things to think about is, all these antibacterial soaps and antibacterial this-es and antibacterial thats wiping off all of the bacteria on your skin is not necessarily a good thing. You have bacteria on your skin that have colonized and they filled the niches and they get all the resources in. If you wipe them out, you can get something else that can colonize and now it can get to food and resources and it can grow and may be bad for you. So part of our defense is normal flora. For anybody who has taken a broad spectrum antibiotic like tetracycline and then puked for the next week, you broke the microbiological barrier and you allowed something else, you wiped out the normal flora in your gut and you allowed something else to grow there that caused you to throw up or gave diarrhea. So the three barriers are mechanical, chemical and microbiological. And you have to break at least one of those before you’re going to need an immune response. Right? So you’re not making an immune response against your normal flora because you don’t need to. [5] – [Picture (Adherence to epithelium…)] [McCutcheon] – So this is one way to break the barrier and this is breaking the physical barrier and this is the perfect slide for you as dentists. That’s your finger—the epithelium they’re showing you there—and you’ve just had an explorer in somebodies mouth and your scraped a bunch of plaque—and what’s in plaque? Bacteria. And now you’ve stabbed that plaque-laden explorer into your finger. You will do this at least once in your career. Ok? And since it’s somebody else’s bacteria, you may or may not have an immune response already against them. If you don’t, you now have to start an immune response. So the first thing that happens in you break the physical barrier. You have to start an immune response… [6] – [Picture (The colon is colonized…] [McCutcheon] – If you take antibiotics and you wipe out the normal flora in the guy, you can allow something else, like Clostridium difficile to colonize. That is an opportunistic pathogen. It causes nausea, vomiting and diarrhea. Because you’ve wiped out the flora that kept all of the resources to themselves, this is now allowed to grow up. You get sick. So two different ways to break a barrier. You have to break the barrier before the immune response starts. [7] – [The Players] [McCutcheon] – So we talked about this in May. The Players. We have cells. The first set of them, neutrophils, macrophages, dendritic cells, Natural Killer cells,

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mast cells, eosinophils and basophils. These are all players in the innate immune response. Mast cells, eosinophils and basophils are things we’re not really going to talk about because they don’t really have a huge role in a physiologic immune response. They have a very large role in a lot of pathologic immune responses, so other people are going to talk about them a lot more than I will. The last two on the list are B Cells and T Cells—those are part of the adaptive immune response and they are antigen specific. And we will explain that starting today and continuing on. But it’s not just cells that make up the immune response. Soluble molecules. There are a series of proteins that live in the serum, you always have them. They are called Complement. You have antibodies once you’ve had a B cell reaction. Your macrophages, dendritic cells, neutrophils and T cells all secrete cytokines and we’re going to start talking about those today. Chemokines are a form of cytokine that have a specific ligand site and specific function. And then you have various vasoactive mediators. You’re going to learn a lot more about a lot of those from other people. But these are reactive oxygen species. The prostaglandin pathway, things that are associated with causing pain and inflammation. So this is our component. These are what we’re going to talk about in the physiologic immune response. [8] – [Dichotomies] [McCutcheon] – There are some traditional dichotomies of how people divide up the immune system. The two common, traditional dichotomies are the innate immune response versus the adaptive immune response and the humoral immune response versus the cell mediated immune response. So the innate immune response is really talking about those macrophages, dendritic cells and neutrophils and complement. That’s innate immunity. And then humoral, that’s immunity mediated by soluble factors, so complement and antibodies versus immunity mediated by cells, mostly T Cells and macrophages. And I’m gonna put to you that these are not really good, they’re real dichotomies but they don’t explain how an immune response works. So there’s a better way to think about how an immune response works and we’re going to talk about that today. [9] – [Picture (Recognition mechanisms…] [McCutcheon] – So what is innate immunity? Well, the real advantage of innate immunity is that it’s fast. Within four hours of stabbing yourself in the finger with that explorer, neutrophils are on site. Complement is there immediately. Neutrophils show up within four hours. Macrophages and monocytes are not far behind. Alright? So it’s very fast. In contrast, it takes at least a week to get your T cells going. Five days at the fastest and it takes another couple days to get your B cells going. So the first, beginning of that first week of an immune response, you don’t have B or T cells yet. Everything doing the work are innate immune cells, mostly. They have a limited repertoire of things they can do. They can do the things that they can do and that’s the only thing that they can do. In contrast, B and T cells have a number of different mechanism of dealing with pathogens and unlike innate immunity—where they do what they’re going to do, and if they do

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it on a bacterium, it kills a bacterium, but if they do it on a host cell, it kills the host cell—the variable part of the adaptive immune response is it pretty much only kills the pathogen. It leaves the host cells in tact. I have to tell you, I have allergies and so I start coughing while I talk; so I’m going to keeping sipping water. Um, innate immunity only recognizes a limited number of things and those are all on bacteria. Alright? Do you guys know where viruses are made? Have you had that microbiology yet? Okay? Viruses are made in host cells. So all of the proteins made by a human host cell are going to be what kind of proteins? …Take a guess… Human proteins! Yea! Do you want the immune system to be able to recognize human proteins? No. So the innate immunity really doesn’t recognize anything on a virus because it’s made in a human cell. Innate immunity can recognize some unique features of bacteria. In contrast, B and T cells, you’ll have one or two that will recognize each and every single solitary bacteria and virus and that’s all you need to get enough adaptive immunity. So they’re very specific and they don’t recognize host unless something’s gone wrong. And then innate immunity, it does what it’s gonna do and it can’t change. And some of what it does is against host cells and that’s not good for you. Adaptive immunity gets better over time. So the longer the infection goes on, the more efficient the immunity becomes. And, in the end, (this is one of the reasons it’s not a good idea to split the immune system up into innate and adaptive) is you have to have both things to get rid of a pathogen. You have to have all of the innate immunity working and you have to have all of the adaptive immunity working if you want to get rid of a pathogen. If they don’t all work right, the pathogen doesn’t go away and you end up with a chronic inflammatory disease. Does any dentist in this room want to tell me a chronic inflammatory disease? I want a dentist…the key was dentist. Periodontal disease. Okay? Periodontal disease. The patients in your office who have periodontal disease have it because something isn’t working right. Patients in your office who don’t have periodontal disease—it’s because something in their immune system is working right. We’re beginning to figure out the things that go wrong with people who have periodontal disease—we don’t know them all yet. But it’s not the bacteria. Okay? People with periodontal disease have a different bacterial profile than people that don’t have it, but its because their immune system couldn’t get rid of them. People who don’t have periodontal disease can still have seen those bacteria but they didn’t cause disease. So, it’s the host response that causes periodontal disease. [10] – [Picture (Healthy skin is not…)] [McCutcheon] – So what happened: you have your healthy skin, you get an owie and you have some blood and dirt or bacteria, or something gets under your skin so you’ve opened the physical barrier. And the beginning of the innate immune response, it causes a change in the vasculature. You get fluid into the area. You get cells into the area. The area becomes red, swollen, hot and painful. So ruber, tumor, calor, dolor. The four symptoms of an infection. Somebody else is gonna tell you this: so in Latin, rubor is red, tumor is swelling, calor is heat, dolor is pain. So classic signs of inflammation: rubor, tumor, calor, dolor—it’s because the immune

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system has opened up the blood vessels, you have fluid, you have cells, it swells things so that the redness is because the blood has leaked out. The swelling is from the extra tissue. The heat is because you’re starting to cause a fever and the pain is because you’re swelling the tissue and you’re pushing on the nerves. [11] – [Location, Location, Location (I)] [McCutcheon] – Now, in the end, so we have the innate versus the adaptive; we have the humoral versus the cell mediated immunity. Here is the real dichotomy in the immune system: it’s where the pathogen lives. Pathogens can live outside the cell in the extracellular space; pathogens can live inside a cell. There’s nowhere else. Outside the cell, inside the cell, there are no other options. And the way the immune system works depends on whether or not you’re trying to get rid of something that’s living in the extracellular space or something that’s living inside a cell. And that’s really where the dichotomy comes in. And so for extracellular, you have additional proteins that play a role against extracellular pathogens that don’t work against things that live inside host cells. For intracellular pathogens: you have an additional cell that comes into play that does not play a role in extracellular pathogens. And except for the couple of extra proteins, and extra cell, everything else is the same. Okay? Everything else is the same. So the way to think about this is: where does the pathogen live? Because that tells you what you need to get rid of it. [12] – [Location, Location, Location (II)] [McCutcheon] – So, because we have these two different places for pathogens to live, we have two different places for an immune response to start. So for extracellular, the immune system starts with complement, those germ proteins complement, those extra proteins that are part of an extracellular response. And toll-like receptors on macrophages, dendritic cells and neutrophils. So one of the principles is: in order for an immune cell to recognize something, there has to be a receptor. No receptor— no recognition—no activity. There has to be a receptor. For the intracellular pathogens, they’re inside a cell. So are proteins floating around in the serum or extracellular spaces going to be able to get to something inside a cell? No. So complement plays very little role in an intracellular pathogen. To get rid of it, you need a different kind of cell. So intracellular response starts with a dendritic cell. That’s DC stands for dendritic cell. [13] – [Extracellular First Responders] [McCutcheon] – So for extracellular pathogens: the first thing that comes into play is complement and if we have them, and we probably have them, it’s antibodies. And then its these three cells: neutrophils, macrophages and dendritic cells are the cells that are resident in the tissue. They’re call Langerhans cells. Dr. Wishe probably called them tissue macrophages. They’re really dendritic cells. Now, I said we have antibodies. So how is it that if we have a bacterium that we’ve never seen before we have an antibody? In May, I talked about a subset of B Cells called CD5 B cells or B1 B cells. These B cells live in the tissues.

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[14] – [Complement] [McCutcheon] – Alright.. I’ll pick that up in a minute….So complement. Complement is a series of proteins that are found in the serum. They exist in inactive forms. The inactive forms get converted to active forms usually be previous complement pieces. Does anyone remember the song The Hole in the Bottom of the Sea? So…(Singing in italics) There’s a hole in the bottom of the sea, There’s a log on the hole in the bottom of the sea… and then you end up eventually with: There’s a flea on the tail of the frog on the log in the hole in the bottom of the sea. That’s how complement works. We’re going to start off with a piece and then we’re going to put a piece on that and then we’re going to put a piece on the piece on that and eventually we end up with something that will get rid of bacteria. [15] – [Picture (Classical pathway)] [McCutcheon] – There are three ways to start complement. The classical pathway begins with antibody. We’re gonna talk about where that comes from in a minute. You always have antibodies. After you’re a week old you have antibodies. The alternative pathway does not start with antibody and it leaves off the first several proteins of the classic pathway. The alternative pathway can always be activated. It actually is always being activated and then you have ways to shut it down. These two pathways are not exclusive. They can run simultaneously. Once the classical pathway gets to the convergence point, it can drive the alternative pathway to become more effective. The lectin pathway is similar to the alternative pathway and we’re not gonna talk about that. [16] – [Picture (B-1 cell binds bacterial . . .)] [McCutcheon] – So B1 B cells are CD5 B cells. They live in the tissues. They can make antibodies without any T cell help and they only make a very limited specificity of antibodies. They recognize bacterial carbohydrate residues. So every species of streptococcus has a similar carbohydrate residue. Once you make an antibody against a carbohydrate residue of one kind of streptococcus, it will be against all other streptococcus. Once you make an antibody against one kind of staphylococcus carbohydrate it will work against all staphylococcus carbohydrates. Now the staphylococcus antibody won’t bind to the streptococcus and the strep won’t bind to the staph, but the staph will bind any staph; the strep will bind any strep. Early on, you know just within weeks of when you’re born, you get these antibodies and these are IgM antibodies. They’re HUGE. They have really low binding efficiencies and it doesn’t matter because one IgM antibody can be a landing pad for the first component of the classical component pathway. So you only need one and it doesn’t have to stick on very long. [17] – [Picture (C1r, C1q, C1s)] [McCutcheon] – So this is the first piece of complement. It’s called C1. It has components—we don’t care. Okay? Details you don’t have to memorize. You do

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have to know about C1. C1 binds to an IgM molecule, the IgM is this big thing. It’s a landing pad. C1 sits down on the landing pad. Once the C1 is bound, it activates itself. [18] – [Picture (Pentameric IgM molecule. . .)] [McCutcheon] – C1 can also bind to IgG, a different isotope of antibody. Except, it takes about 1,000 IgG’s to scatter themselves around on a bacterium in order for five of them to be in the right position for the C1 to bind. So one IgM; 1,000 IgG. C1 binds, it activates itself. [19] – [C1 “Converts” C2 and C4] [McCutcheon] – It starts recruiting in other factors of complement. So the next piece of complement that binds is not C2, but, C4. The complement pieces were numbered in the order they were found, which then turned out not to be the order that they acted. So C1 binds C4 and it cleaves C4 into two pieces. The big piece binds, the little piece goes away. Okay? C1 also cleaves C2. The big piece binds, the little piece goes away. Alright? C1 binds to IgM, it cleaves itself and becomes active. It starts converting. We call it a convertase because it converts C2 and C4. It binds C4, the little piece goes away. It converts C4, the little piece goes away, the big piece binds. It converts C2, the little piece goes away, the big piece binds. The C4/C2 pieces now become a convertase. They convert C3. The little piece goes away, the big piece binds. Okay? So the two bound pieces of complement, C4 and C2, become a C3 convertase. They convert C3, the little piece goes away, the big piece binds. [20] – [Activation of C3] [McCutcheon] – So, here we have our C4/C2 piece. They convert C3. The little piece goes away, the big piece binds. Now C3 is the convergence point between the classical and the alternative pathway because not only can C3 be cleaved by C4/C2, it can also spontaneously cleave. And so you can end up with C3 binding—The C3b piece—the bound piece—binding to an alternative protein called Factor B, B as in boy. B gets cleaved into two pieces. The little piece goes away, the big piece binds. Now, in the alternative pathway, C3b, big B, little B, that also is a C3 convertase. Okay, so you can have the classical pathway C4/C2 cleaving C3; you can have C3 cleaving itself spontaneously; you can have C3 plus factor B cleaving…C3 plus factor B that gets cleaved forming C3 convertases. So at this point in time, you have three things that all cleave C3. So now you get lots and lots and lots and lots of C3 cleaved. The C3b binding—and C3b is the piece you have to remember, okay? You need to remember C1 and you need to remember C3—C3b is the only complement piece that plays a role in viral infections. And remember, it can cleave spontaneously, so that’s how it gets there. C3b binds and it’s going to do two things… [21] – [Complement Activation]

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[McCutcheon] – So, before we talk about the two things C3b does, we’re going to look at a movie. The sound worked perfectly when I did this yesterday. And it’s not working at all….I just rearranged my slides so sorry about that. So C3 turns into C3a and C3b. C3a is a chemotaxin. It starts recruiting cells—Neutrophils—to the site of infection. C3b binding causes opsonization. Opsonization is Greek. It means to coat with butter. So we’ve coated the bacterium with butter so that would make it very easy for what? …What are the cells that are showing up at this point? …Macrophages—and what does macrophage mean? …Big eater. And so what’s going to be eating? —Something that’s coated with butter. So what this means is that when C3b is bound, theres a receptor on macrophages and neutrophils thats a high affinity receptor. So now it’s very easy for the macrophage and neutrophil to eat, phagocytose, the bacterium. So C3b, it can cleave itself, so you get this massive auto-loop of ramping up complement. And that can work in both the alternative and the classical pathways. It’s an opsonin so it makes it easy for cells with receptors to eat whatever the C3b is stuck to. And then, eventually, once you get C3b, you’re going to end up with end stage of complement, which is the Membrane Attack Complex—or the Big MAC Attack. [22] – [Picture (Fixation of complement)] [McCutcheon] – So C3 cleaves. It coats the bacterium. Just so you know, this is ridiculous not in scale. If this were in scale, the C3b would be this tiny little blip that you couldn’t see. So it’s not the same size as a bacterium. [23] – [Picture (Fixation and action of the C3 . . . )] [McCutcheon] – So we’ve got C3b all over the pathogen and it doesn’t matter if its C3b that got there because it was cleaved by the C4/C2 complex or if it was cleaved by the the C3bB complex. As long as it’s there, it’s an opsonin. So now macrophages have receptors That can bind to the C3b. They’re high affinity receptors. The pathogen gets engulfed. [24] – [Activation of C5] [McCutcheon] – You ready? The C4b2a3b becomes a convertase. It cleaves C3 and C5. Okay? So the 423 piece cleaves C3 and C5. In the alternative pathway, you get the BC3b—two of them, C3b, C3b plus B piece, becomes a convertase—cleaves C3 and C5. I’ll say it again. So, we had our convertase for C3 which was 4 and 2. They cleave C3. The C3b falls in with the 4 and the 2. So now we have a 423 piece that cleaves both C3 and C5. Classic Pathway: 423 cleaves C3 and C5. Alternative Pathway: C3b plus C3b plus the B piece, so two C3b’s and a B, cleaves C3 and C5. So we’ve got both the C42 cleaving C3; We’ve got the C3b-B piece cleaving C3; Now we have the 423 piece cleaving C3 AND the 33B piece cleaving C3—and C5. So we have a massive amount of C3b being cleaved. And of complement of the only piece if you have deficiencies in C3b. That’s the only thing we really see disease associated with. And you’re never gonna see those people because they don’t live long enough to have teeth. It’s a very rare mutation but it’s quite lethal.

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Okay, C5a is an anaphylatoxin so it goes off and it starts recruiting inflammatory mediators and it’s about 1,000 times more potent than C3a. So 3a and 5a go off and do other things and they’re good at it. So this is how you help start recruiting the neutrophils and the macrophages to the site of infection. Remember, complement is in the serum. The minute you start breaking capillary walls, complement is where the infection is. Okay? cause the serum comes out of the capillary, the complement is there—off it goes. Within, you know, a minute, this starts working. So, we have the C5 convertase of 423 cleaving C5 (it also can cleave C3). We have the alternative pathway convertase BC3bC3b cleaving C5, also C3. Once C3 gets into play, the MAC attack is going to occur. So C5 gets cleaved, C5, the big piece binds the little piece goes away. [25] – [Picture (pathogen)] [McCutcheon] – When the big piece binds and the little piece goes away, it recruits the next three pieces: C6, C7 and C8. They don’t get cleaved, they bind together and they insert themselves onto the membrane and they insert themselves onto the membrane of the bacterium. So 5 recruits 6, 7 and 8. Doesn’t matter how we got the 5—if it came from alternative or classical pathway. 5 is 5 is 5. It makes the 6, 7 and 8 bind together. And then once they insert into the membrane, that causes the polymerization of C9. You get 10-15 C9 pieces that polymerize and they form a pore. The pore pokes all the way through the bacterial membrane and if there’s a big hole in the bacterial membrane, what happens? …All the inside stuff rushes outside and the bacterium is dead. Alright? So that’s the MAC. Now as cool as the MAC attack is, in reality, if you don’t have it, the only bacteria ((NOTE: ?--she names something, I THINK this is what she is referring to)) leishmaniasis listerium monocytogeni… So every other bacterium in the planet, you can get rid of without a MAC attack. [26] – [Complement] [McCutcheon] – Now, try the movie… So when I did this last night, the sound was fine and when we tried to do this today, there was no sound so I may have to narrate this for you. If she does talk, wait….go back…Okay thats not gonna work….Okay, she’s not gonna talk. If she does talk, she mispronounces lots of words. Okay…Well, it’s running on my computer. Um…Or we’re not gonna watch movies unless I can figure out how this works… [27] – [Functions of C3] [speaker] – Sorry about that. I’ll get the guys back in here at break and we’ll get the movies running. So C1 is important, and I’ll talk about why in a minute. C3b if you don’t have C3b, you’ll be dead. C3b is important because it’s an opsonin and macrophages and monocytes have receptors. Dendritic Cells have receptors. Neutrophils have receptors and most importantly, B lymphocytes have receptors. Without C3b, you don’t get an antibody response. And so the only piece of complement that plays a role in an intracellular infection is C3b. And the reason

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why isn’t for the macrophages, monocytes and neutrophils, it’s to get the B cells going. If you don’t have C3b bound to that virus, you don’t get an antibody response. Alright? In truth, the pore of complement is bigger than a virus. So if you form that MAC attack complex, the virus would just go through it. IgM—bigger than a virus. So you’re never going to be able to get C1 to bind, because the virus would just sit inside the IgM and nothing would happen. Okay? So complement has NO role against viral infections, except for C3b. Intracellular pathogens, you have to have C3b. Nothing else. Extracellular pathogens: you want all of it. And then you need C3 as the C3/C5 convertase. But you get this massive expansion of the immune response beau se every C3 convertase, be it the C42 or the C3Bb piece, can cleave 1,000 C3 molecules. And every one of those C3b’s that binds can then form a convertase that can cleave another 1,000 C3 molecules. So you get a massive expansion of the complement system. [28] – [Picture (Chart: CR1, CR2…)] [McCutcheon] – Okay so briefly, there always has to be a receptor. There are actually several receptors that recognize several forms of C3. So we have C3…this ‘i’ is the inactivated C3. C3d is a breakdown product and that’s part of the B cell receptor. You don’t need to memorize that. You need to know there are receptors but you don’t need to memorize which one is where. [29] – [Picture (The terminal complement…)] [McCutcheon] – Functions of the other factors, we talked about them: C5 forms the membrane attack complex. 6,7 and 8 bind together. 9 polymerizes and pokes a hole. [30] – [Complement must be controlled] [McCutcheon] – Now, complement is very powerful. Anyone with glomerular nephritis, that was caused by complement binding to things bound on the kidney endothelial walls and it destroys the endothelium and it get replaced with scar tissue and the kidney doesn’t work. Alright? Because complement is powerful, it is very tightly regulated. So for every factor that can do something, we have several inactivating factors. You need to know about these. [31] – [Picture (Activated C1r and C1s…)] [McCutcheon] – There are two forms of inactivating factors. Soluble inactivating factors will inactivate complement no matter what it’s found on. Host cells have membrane inactivating factors that will prevent complement from activating on host cells, but it will not prevent complement from acting on bacterial cells. So the first factor is C1 inhibitory factor. C1 inhibitory factor inhibits C1—it stops C1 from being a convertase. People who have defects in C1 inhibitory factor have hereditary angioedema, a disease you will deal with as dentists. What happens is their lips and tongue swell and they can’t breathe. That’s caused by a mutation in the C1 inhibitory factor.

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[32] – [Picture (Formation and action…) ] [McCutcheon] – The rest of the factors are pretty much directed at C3—not surprisingly. So, you have soluble factor, um, so we have C3b binding…okay…and then we can have factor D inhibit this, so we get an inactive C3b. [33] – [Picture (Inactivation of the classical…) ] [McCutcheon] – We have C4-binding protein that prevents C4 from cleaving C3. C4-binding protein inactivates the C3 convertase. We have Factor H that does the same thing that does the same thing with the C3bBb convertase. So Factor H binds to the C3b and makes it inactive. Okay so C4-binding protein inactivates the C42 C3 convertase. Factor H inactivates the C3bBb convertase. [34] – [Picture (DAF dissociates C3 convertases. . .)] [McCutcheon] – So those are our soluble factors. Membrane, we have DAF, which inactivates the C3b by … binds to the C3b and displaces the Bb, so it’s no longer C3 convertase. We have MCP, which cleaves off a portion of the C3, so it’s no longer active. This inactive form of C3b… [33] – [*back to previous slide*] [McCutcheon] – Even though it’s inactive, there are still receptors that bind that…so it can still act as an opsonin. It just can’t cleave anymore. So the inactivating factors prevent the C3b from being a cleaving agent. They do not prevent it from being an opsonin. Nicely designed system. [35] – [Picture (On microbial cells…) ] [McCutcheon] – And then finally, host cells have a protein called CD59 that prevents the polymerization of C9. So you prevent the C3, you prevent the pore from forming on host cells. It does nothing for bacterial cells because bacterial cells don’t have human host cell proteins on them. So the pores can still form on bacterium. They cannot form on host cells. [36] – [Complement starts inflammation] [McCutcheon] – So, C3 and C5 are anaphylatoxins. They alter the vascular endothelium and we’re going to show pictures of that in the next hour. They recruit inflammatory cells. [37] – [Picture (Anaphylatoxins act on blood vessels…) ] [McCutcheon] – So, C3 and C5, what they cause is they cause the capillaries to become leaky. That allows more complement to go out of the capillary into the inflamed, infected area. It also allows anything else in the plasma. So any antibodies that are around can get out, into the infected area. And then it allows cells to diapedese in between the endothelial cell walls so that the neutrophils and the monocytes can get out of the capillary and into the tissue. Any reason why

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these cells are going in and out of capillaries? … Why not arteries? Why can’t they go in and out of an artery? … Somebody mumble really loudly… They’re too thick! What are they thick with? Collagen..what’s the thing that arteries have that veins don’t? Starts with an M….Muscle! Yea. You’re not gonna have something be able to crawl through thick collagen and several layers of muscle cells. So the inflammatory cells get out of the circulation into the tissues through capillaries and really, really, really small venues and it’s not 8:50—I’ll give you a ten minute break and see if I can’t get AV to come help. [38] – [The Initial Response to Extracellular Bacteria] [McCutcheon] – Alright…having argued with my computer… So initial response to the extracellular bacteria—It starts with complement. Second after you’ve disrupted the capillary beds, the inactive proteins start becoming active by cleaving themselves or cleaving other things. C3b is an opsonin for any cells that have receptors. Cells that don’t have receptors, it doesn’t do anything. Always have to have a receptor. C3a and C5a are inflammatory mediators. So seconds after you’ve poked yourself with that explorer, you now have signals that start changing the endothelium and telling the neutrophils and monocytes to show up. In the blood, monocytes and monocytes. When they migrate out into the tissues, they differentiate into macrophages. Okay? [39] – [Pathogens Reside in Different Compartments] [McCutcheon] – So, that’s an extracellular response. Now, for purposes of this class, we’re going to say…when I say bacterium, I’m talking about something that lives outside a cell. When I’m talking about a virus, I’m talking about something that lives inside a cell. There are obligate intracellular pathogens that are bacteria. Okay? So i’m using virus as the model. it does not mean there cannot be bacteria that reside inside the cell. If there is bacteria that reside inside the cell, it is dealt with the same way a virus would be dealt with. If the something lives outside the cell, it’s dealt with as an extracellular pathogen. If it lives inside the cell, it’s dealt with as an extracellular pathogen. I’ll say bacterium as extracellular and virus meaning intracellular—but that’s a shorthand, that’s not inclusive. Okay? [40] – [Picture (Site of infection. . .) ] [McCutcheon] – Typical extracellular pathogens, viruses can also be outside the cell and you know this, although you may not know that you know this. Because when you came here, they drew blood and they did what? They titer-ed you. What does that mean? They looked at antibodies. And what disease did they look at antibodies for? … Pardon? … Yep. Measles, mumps, chicken pox. Okay, what type of pathogens are measles, mumps, chicken pox? They’re all viruses. So they’re looking to see if you have antibodies against a virus. And knowing the state of your immunity is by knowing how good your antibodies are against a virus. So even though viruses live primarily in the cell, there are periods when they’re out and about in the extracellular space and that’s when the antibodies bind them. If you don’t have antibodies, you are not going to live. I’m telling you this now,

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we’re going to talk about it over and over and over. You have to have antibodies against a virus. When things are in extracellular spaces: it’s complement, antibodies and phagocytosis. When things are inside, so viruses, things like kvidia ((???)), things like microbacterium, microbacterium only live inside vesicles inside cells—then you have to have T lymphocytes and macrophages. So extracellular, you have extra molecules, complement… Intracellular, you have extra cells. CD8 T cells. And that’s the difference. Everything else is both extracellular and intracellular. You need both innate and adaptive to get rid of extracellular and intracellular pathogens. You need both humoral and CMI immunity to get rid of extracellular and intracellular pathogens. The difference: you have some extra proteins in the beginning of an extracellular response. You don’t have those proteins, you have an extra cell at the end of an intracell response. Everything else, same. So the real dichotomy is where something lives. [41] – [Extracellular Pathogens] [McCutcheon] – So how do we recognize bacteria? I’ve told you, it has to be a receptor. It turns out that bacteria make some proteins and carbohydrates and fatty acids that are different than anything a mammalian cell can make. They’re different than anything a mammalian cell can make. We call these things “Pathogen Associated Molecular Patterns” or PAMPs. And if a bacterium can make something that is not host, then the host can make a receptor that will specifically bind to the bacterium but won’t bind to host. If the bacteria has something that’s the same as the host, you can’t make a receptor against it, because then your immune cells would act against you. You’d have nasty, awful autoimmune disease. So the bacteria are only recognized by things unique to them. So what’s the first amino acid in all proteins? Methionine. And bacteria, they have the first three amino acids identical: methionine, leucine, phenylalanine. Where all of our proteins start with a Met, theirs start with Met, Leu, Phe. Not only to they start off with methionine—they can formulate it. We can’t formulate methionine. So there’s this unique structure: formulates methionine, leucine, phenylalanine that start every bacterial protein. Since that’s not something found in host, you can make a receptor that will recognize that. So f-Met-Leu-Phe glycopolysaccharide is found on every gram negative bacteria. It’s not something mammalian cells can make. If the bacterium can make something that humans can’t make, we can make a receptor against it. So, there’s a receptor against glycopolysaccharide. Bacterium can do different things with carbohydrates. They can make glycan structures and mannose structures. Human cells cannot do those things. Because human cells or mammalian cells will never have glycan or mannose, you can make receptors against them. We call these receptors “toll-like receptors.” These receptors reside primarily on neutrophils…that funny nucleated thing…and macrophages. They’re not very high affinity, but they still work. Anything that has an f-Met-Leu-Phe, an LPS, glycan or mannose, a neutrophil and a macrophage will have a receptor against one or more of those things that allows the neutrophil or macrophage monocyte to bind to the pathogen and then it can eat it. Phagocytosis.

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[42] [No title —Video] [McCutcheon] – Cross your fingers Ach! I keep setting it on duplicate, and then it goes back…I’ll play later… Okay… duplicate…Ahh! So this is a neutrophil. This is in the bloodstream. These are RBCs. That are staphylococcus that have been introduced into the bloodstream. The neutrophil has receptors that recognize the staphylococcus. The staphylococcus are being bounced around by Brownian motion… Eventually the neutrophil catches up—phagocytosis. So, that’s toll-like receptors. [43] – [Toll Receptors] [McCutcheon] – So, toll-like receptors. There are ten, they’re a family of ten receptors. If you can have extracellular pathogens, where will you have toll-like receptors? Says so on the slide…on the cell membrane. If you can have intracellular pathogens, where do you have to have toll-like receptors? Says so on the slide… You have to have receptors inside the cell. So somewhat unusually, you have toll receptors both on the cell membrane and in the endosomes. [44] – [Cartoon of TLR Receptors] [McCutcheon] – So this is a cartoon of toll-like receptors. You need to know there are toll receptors. You need to know there are things they bind against. You do not need to know the names of the receptors that bind the things. So you have some on the cell membrane; you have some that are in vesicles. Notice that that in the vesicle part, the membrane end is in the vesicle and the cytoplasmic domain is in the cytoplasm. [45 – [Picture (Structure of Toll-like…)] [McCutcheon] – So this is the real structure of toll-like receptors. This is the pathogen recognition domain out in the extracellular space. And this is the cytoplasmic domain and that stands for Toll Isle-1 Receptor Domain. [46] – [Picture (Sensing microbial products…) ] [McCutcheon] – We can see our toll-like receptor recognizing something on bacterium. A different one recognizing something different on bacterium. We can see toll-like receptors that are against single stranded and double stranded RNA. DNA and RNA—which is what viruses are made up of. [47] – [Picture (A complex of TLR4)] [McCutcheon] – I have been telling classes for years that they did have to know anything about 2nd messengers until we had drugs against 2nd messengers. And you guys drew the short straw. There’s now a drug against them, so we’re going to learn about them. All of the immune receptors work through a 2nd messenger system. So something is going to bind the cytoplasmic domain. You go through a

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cascade kinase. You don’t need to know the specifics of that. You end up turning on a transcription factor that translocates into the nucleus and that transcription factor turns on cassettes of immune related genes that give you cytokines. All receptors, all immunology receptors, work through a 2nd messenger system. The receptor binds to what it binds to, that phosphorylates a 2nd messenger in the cytoplasm, you go through a cascade kinase chain and you end up turning on a transcription factor. THat transcription factor translocates into the nucleus, it binds to DNA and it causes the transcription of groups. Cassettes of related genes. So different transcription factors turn on different sets of cytokine genes. [48] [Toll Receptors] [McCutcheon] – Toll Receptors are critical in an immune response because they dictate the end result. SO the Toll receptors that you turn on and the proportion of cytokines that get turned on help drive you to have primarily what’s called a humoral response driven by B cells or a cell-mediated response driven by TH1 T cells. And that all starts at the beginning depending on the toll receptors that bind. So, you activate the different toll like receptors, you go through a 2nd messenger system, that turns on transcription factors. The transcription factors then drive the differentiation of the various T cells depending on what cytokines are secreted. [49] – [Macrophage Activation (TLR)] [McCutcheon] –So when macrophages, the neutrophils, get to the site of infection, they are end-stage cells. They eat bacteria. That’s all they do. They do that for 2-3 days. They die. They’re replaced. Okay? Without neutrophils, you cannot keep the pathogen at bay long enough to get anything else in there to do the deed. So anybody who has gone through chemotherapy, they’re in isolation until their neutrophils come up enough that they can defend against common bacteria. So neutrophils are the first thing thats there. The function of the innate immune response is to keep the pathogen whittled down enough so that the adaptive immune response can show up. The function of antibiotics is to help the innate immune system keep the pathogen down enough so that the adaptive immune response can show up. Antibiotics will never cure you of anything if your adaptive immune response cannot show up, you’re going to die. Doesn’t matter what antibiotics they put you on. So innate immunity is there simply to keep bacteria from overwhelming you. A lot of bacteria have a 40 minute replication cycle. If you started off with one and divided it in 40 minutes to two, and 40 minutes later it’s divided to four. At the end of 24 hours, you have a million-something. Okay? So the function of the neutrophils and eventually the macrophages, is to keep those numbers down. It’s the adaptive immune response that actually gets rid of it. When a macrophage is activated through toll-receptors, it does two things. It’s very phagocytic, so it can eat things and it starts secreting cytokines. [50] – [Picture (On sensing…)]

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[McCutcheon] – Before we talk about the cytokines the macrophage secretes—and these are it-- [51] – [General Principles of Cytokine Biology] [McCutcheon] – We’re gonna talk about cytokine biology in general. So cytokines—how many people know what one is? Okay…two. Cytokines are small soluble molecules, they have a myriad of functions. Originally they were discovered being secreted by white blood cells, leukocytes. So a lot of the early ones were named IL for leukocyte, interleukin because they were secreted by white blood cells and they thought they acted on white blood cells. So interleukin: between white blood cells. We now know that almost any cell in the body can secrete cytokines given the right stimulus. Any cell in the body can be acted on cytokines provided that they have a receptor. So, all cytokines are pleiotropic. They have many, many, many functions. Some of them have more functions than others, but they all have more than one. All cytokines, all of the functions of cytokines (except one, and we’ll talk about that) are redundant. So cytokine X can do this, cytokine Y can do this, cytokine Z can do this. What that tells you is that that function is so critical that you have to have more than one thing do it. So all cytokines, their functions, except for one, are redundant. More than one cytokine causes that function to happen. They have really broad activities. And the more we know about immunology, the more broad the activities are. One of the things that we’re discovering is that pretty much all diseases have something to do with the immune response. Even disease that you wouldn’t have thought have anything to do with the immune response. And a lot of the effects don’t particularly have anything to do with the immune system. A lot of them do, but some of them don’t. And then they come in families. What that means is that there are groups of structurally related proteins that generally have similar redundant functions. So they have many, many, many functions. All but one of those functions are done by more than one cytokine. They have very broad activity, including non-immune effects. And they come in families. All of these act through 2nd messengers. They now have their first drug against a 2nd messenger and we’ll talk about that. [52] – [Examples] [McCutcheon] – So, the two major families of cytokines are pro-inflammatory cytokines—things that ramp up inflammation. And, anti-inflammatory cytokines. Things that calm down inflammation. And then, the third family of cytokines are the chemokines. Chemokines all have a cysteine binding motif. So the part of the ligand, the chemokine, that binds a receptor has some kind of a cysteine and they’re named after their cysteine motif. So pro-inflammatory IL1, TNF-alpha, Interferon-gamma and IL6 are the major players in the pro-inflammatory cytokines. There’s also IL17 and related proteins that are also pro-inflammatory cytokines. The major players of anti-inflammatory cytokines are IL4, IL10 and TGF-beta and then the more recently discovered IL22 function. Now, one of the things that has happened over the past several years is there’s been kind of a sea change in the way we understand cytokine biology. The old way

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was, you knocked it in or you knocked it out of a mouse. And then, you said it does this, that, this, that, this and that. Well, because some of these cytokines are so structurally related and the functions are redundant, a lot of times, when you knock something out, the functions still happen because the other cytokines that have the redundant properties were still there. And the other thing is that, and this is gonna come as a shock to you guys, mice are not people. Okay? And especially with cytokines, the functions in mouse are not the same thing as the functions in human. So you knock it out in a mouse and you say “Oh! It does this.” And then you knock it out in human cells and nothing happens, or something completely different happens. So trying to use the mouse knock-in, knock-out model to explain what cytokines do is really kinda inadequate. So we’ve gone to broader categories. And experiments nowadays, if you knock something out of a mouse, you have to go prove that it does the same thing in humans before you can go anywhere with it. So the science has really changed in the past 5-10 years. [53] – [Receptors] [McCutcheon] – All cytokines work through a receptor. Receptors like the cytokines themselves, come in families. So pro-inflammatory cytokines generally have very similar looking receptors. The receptors have widely variable structure. Okay? And then, what in part explains the redundancy are the shared chains. So, these three chains can be shared between main cytokine receptors and because they’re =shared that means you’re activating the same 2nd messenger, which means you turns on the same transcription factor, which means you turn on the same cassette of genes. And that’s why the properties are redundant. [54] – [Picture (A,B,C)] [McCutcheon] – Looking at these, we can see that IL2, which is pro-inflammatory, IL4, which is anti-inflammatory, IL15, which is pro-inflammatory and IL21, which is pro-inflammatory all share this gamma-C chain. So the difference is that IL2 has a different IL2 alpha and IL2 beta chains compared to IL4, which has an IL4 alpha chain. And then IL7 has a different chain, so it’s an IL7. Of these, you need to know that they share a chain. I want you to remember this structure because we’re going to about it when we talk about T cell activation. The rest of these: you should know that there’s receptors. And so each receptor is specific for a specific cytokine but with shared chains that means that you can have the same 2nd messenger systems activated. So, cytokines have a combination of unique cytokines, unique chains and a shared chain. You need to know the structure of the IL2 receptor. The rest of them you just need to know that they have specific chains. Each cytokine has a specific chain. And a shared chain. [55] – [Macrophage Cytokines] [McCutcheon] – So, macrophage cytokines. So IL1, also, although it’s not listed in your textbook, IL18 is a part of the IL1 family. And it does the same things that IL1 does. And if you knock IL1 out IL18 does it anyway. So, it activates soluble inflammatory mediators. It does a couple of things. It goes off, and it starts

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activating locally on the prostaglandin pathways. So you start getting these small molecules that can cause pain and inflammation. It activates the vascular endothelium; ’ll show you a picture of that in a minute. That does several things. First of all, activated vascular endothelium changes shape. IT changes the receptors that are expressed on the activated vascular endothelium. It puts up the receptor for the chemokine CXCL8. And it’s a concentration gradient. So the closer you get to the site of infections, the more receptor for CXCL8. And so eventually that stops the incoming cells from rolling. They stop long enough that they can crawl in between the tissues. So IL1 does those things. IL1 causes the production of gamma interferon. When we get cells that can secrete gamma interferon. IL1 also has a host of systemic effects. We’ll talk about those in a minute. And IL1 can activate the resident dendritic cells and cause them to go from an immature tissue phenotype to a mature phenotype. TNF-alpha. Redundancy. It activates the same soluble inflammatory mediators that IL1 can activate. TNF-alpha…also activates the vascular endothelium. It causes the same shape changes in the endothelium. It causes the same change in the receptors on the endothelium. And it puts the CLCX8 on the endothelium. Unique function of TNF-alpha: TNF-alpha, in addition to activating the vascular endothelium, TNF-alpha causes the venues to occlude. TNF-alpha, and only TNF-alpha, causes the venules to occlude. Okay? We’ve opened up the capillary beds, we’ve clamped off the venules. Where’s all that fluid going to go? …Capillaries are open. …Say it loud…It’s gonna go into the lymph. All of that fluid ends up into the lymphatics. And where do the lymphatics drain into? They drain into the lymph node—good for you. Okay, so we’ve got the infection. We’ve got all this bacteria and good replicating. And we’ve closed off the venous return. Okay? So now can the bacteria spread throughout the body? —No. They all get forced up into the lymph node. And what’s one of the functions of the lymph node? —It’s a meeting place. What’s another function?…It’s meeting place—pardon?— It traps things. So all of the bacteria that are at the site of infection…They can be at the site of infection or they can end up in the lymph node. They don’t get out into the body, most of the time. Okay? So TNF-alpha and IL1, they open up the capillary beds. TNF-alpha and only TNF-alpha occlude the venules. So the blood does not go back out of the site of infection carrying pathogen. It all gets forced into the lymph. TNF-alpha, it causes IL1 production. Okay? TNF-alpha has the same systemic effects as IL1, TNF-alpha can cause dendritic cells to go from immature to mature phenotypes. TNF-alpha, directly, this stands for tumor necrosis factor. You learned what necrosis is. Tumor necrosis factor can cause cell death. IL1 doesn’t do that. So if you look, you can see that these have similar overlapping, redundant functions, but that they’re not identical. So, it’s important to realize that. Just because you have redundant functions doesn’t mean that TNF-alpha and IL1 do the same things. They do some of the same things, they do some unique things. [56] – [histological slide I] [Mccutcheon] – Does anybody wanna grab the pointer and point at, um, an endothelial cell in this normal capillary? …Be brave. What’s the worst thing that’s

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gonna happen? …Capillaries…where are the endothelial cells? ((throws clicker)) Oops…It’s under your seat. …Oh, without any batteries. This was not my best idea…Okay, top button is the laser. Yes, exactly, that’s the vascular endothelium. Now…push the right arrow… [57] – [histological slide II] [McCutcheon] – Now where’s the vascular endothelium? …Exactly.. Those flat cells in the previous slide…push the previous slide ..((back to #56).. So the flat cells here, flat connected, flat connected, next slide…((back to #57))…Big puffy cell. Big puffy cell. Big puffy cell. Space. That’s activated vascular endothelium. Okay, so the cells round up, and they lose their hemidesmosomes and desmosomes. So there are big spaces now in between the capillary cells, the cells that line the capillary bed. And if there are big spaces, what’s gonna happen to all the fluid in all the capillaries? …Let’s give him a hand. ((clapping)) Thank you very much. So if there are big spaces in between the capillary cells, what’s gonna happen to all the stuff in the capillaries? It’s gonna end up in the tissue. What’s gonna happen to cells that come into the area? They’re gonna have an easy time of getting from the capillary into the tissue. So that’s activated endothelium. So the endothelium changes shape. Plus the endothelium has changed their receptors. They’ve changed their receptors. They’ve put up the receptor for CXCL8. When you have enough CXCL8 receptor, the cells following that concentration gradient come to a dead stop. That allows them the time to migrate into the tissue. [58] – [Chart (Molecular basis *mutations)] [McCutcheon] – So, I said that immunology is now the cause of many diseases. These are diseases that have something to do with IL1. Sometimes we know what they have to do, sometimes we don’t. But if you treat these people with a drug that treats IL1, you make the disease go away. So some of these make sense. Arthritis is an autoimmune disease. We’ve known that for a long time. How about diabetes? How many people have you thought that type II, not type I, type II diabetes has a autoimmune component. There was a clinical trial in Europe. They treated people with one round of an anti-IL1 drug and they caused remission of type II diabetes. The last I heard, it had been going on for over a year. So, um, you know, vitiligo. Who thought that vitiligo would have an autoimmune component? How about high blood pressure? Cancer? We knew cancer. So many, many, many of the disease that you’re going to see—you’re not going to treat these people. You’re dentists, not physicians.—But they’re going to come in and they’re gonna be on biological drugs against cytokines because many, many diseases have a cytokine component. All sorts of things that we never thought the immune system had anything to do with. We were wrong. [59] – [Picture (Local infection with…)] [McCutcheon] – So, TNF-alpha, and only TNF-alpha, can occlude blood vessels. And what does that mean? So you stabbed your finger with an explorer, right? So you’ve got localized inflammation in your finger. And you know that may spread a

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little bit to the surrounding hand. So the TNF-alpha and IL1 have opened up the capillary beds. And so you’ve got lots of fluid, and lots of complement and antibodies and cells all moving into the area of the infection. And then TNF-alpha has occluded the venules so all of the blood, everything that went off into the extracellular space, now has to go into the lymphatics, and they end up in the axial lymph nodes. Okay? What would happen if there were bacteria all over the body? So you have a systemic infection…okay? IL1 and TNF-alpha open up the capillary beds throughout the body. What happens to you when your capillary beds open up throughout the body? …There’s a massive drop in blood pressure….That’s called? Anaphylactic shock. Okay, so first you go into anaphylactic shock. And what do you think happens to all your major organs if you’ve occluded all the blood vessels in them? …Yeah…they die. So you die. Alright. That’s why TNF-alpha and only TNF-alpha does this. This one function is so potent that if it happens widespread, it’s death. It’s massive organ failure—it’s quick, painful and you can’t stop it. So TNF-alpha in a localized area is fantastic. TNF-alpha spread throughout the body is death. Only TNF-alpha occludes blood vessels. [60] – [Picture (Selectin-mediated adhesion…)] [McCutcheon] – Red blood cells get pushed through the blood system by the beat of the heart and the forces from the heart beat. White blood cells roll along the walls. And they always roll along the walls. So there are receptors on the capillary walls, on the endothelium, and then there are ligands on the white blood cells bind to the receptors. This is a very low affinity interaction. The push from the blood flow causes the white blood cells to roll along the walls. This is how all white blood cells move throughout the body all the time. When you have an infection, you change these receptors and that allows only certain white blood cells to migrate to the site of infections. Neutrophils are one of them, macrophages are the other. And then the adaptive cells, the B cells and the T cells that are specific to the infection can go to the site of infection. The rest of them don’t. When you get enough CXCL8 receptor, that stops the rolling interaction long enough to allow the cell to diapedese through the endothelial walls, which should be puffier than they are in this cartoon. And then, that causes the neutrophils and macrophages to go to the site of infection. So one of the causes of juvenile periodontitis— and I know they changed the name, and I don’t know what they changed it to. What did they change the name to?—One of the causes of juvenile periodontitis is the mutation in CXCL8. So that it saturates out too soon. So you have the bacteria here, and you have neutrophils that get to here and instead of going all the way out to here to diapedese where the infection is, they diapedese here and they go to work here. And since what they’re going to work here on, A, isn’t getting rid of the bacteria over here, and B, the only thing that’s there are host cells, what they do is, they kill a lot of host cells. So, juvenile periodontitis is an autoimmune disease. There are other causes, but that’s one of them. [61] – [Video slide]

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[McCutcheon] - So, let’s cross our fingers…If this doesn’t work, I’ll skip it and try it later….Ach…Oh! Okay so…this is a zebrafish. They poked a hole in the fin of the zebrafish. This is a vessel, and this is a capillary bed. And the reason it looks like that is because the red blood cells are moving so quickly that you can’t visualize them. So here is a neutrophil thats crawled out of the extracellular space and it’s moving up towards the puncture. So these are fibroblasts that are in the tissue. But all of the neutrophils, they don’t just move anywhere. They only move to the site of infection. And they’re following that CXCL8 concentration gradient, so they only go to where the infection is…see them crawling out? Alright…let’s try this one… So we’ll do the cartoon…So the white blood cells (those look like monocytes) are moving through the blood vessels. As you start to change the receptors on the endothelium, that causes the white blood cells now to start sticking. ((Video zooms in to depict sticking mechanism)) Um, you don’t need to know that. So the white blood cells are rolling along. You change the receptors…Now…see this has hit enough CXCL8 receptor that it stuck and they diapedese though the tissues and they migrate towards the infection… So looking at this in real time. This is a vein. Okay, this is an artery and again…nothing’s going to come out of an artery because of that muscular wall. So we can see the cells rolling along the in vein…and the reason this is moving up and down is because the mouse is breathing. So we can see the cells rolling along…that is not gonna diapedese… (mumbling indistinctly)… So these have diapedesed out. They’re moving toward the site of infection. This one is stuck. Now they’ve clamped the blood vessels a bit so that they’ve slowed down the circulation so you can see…and you can see how this is pulsing. That’s the artery. The vein…the pressure isn’t as strong, so it’s more of a slow flow. But the millions of red blood cells in here—you can see how crowded they are. And they’re just being pushed along. The white blood cells are crawling along the endothelial cell walls. They always crawl. They get moved forward, but they crawl… [62] – [Macrophage cytokines] [McCutcheon] – So in addition to IL1 and TNF-alpha, macrophages secrete IL6. IL6 primarily has systemic effects. IL6 can help with B cell activation in the right places. And then, IL6 helps B cells make more antibodies once the B cells are activation. CXCL8, it used to be called IL8, so if you’re reading older literature and they say IL8, they’re talking about CXCL8. So this means that cysteine, any amino acid, cysteine-leucine is the binding motif for the receptor. Okay? If I ask you the question on the test and you say IL8, it’s wrong. The new name is CXCL8. It is chemotactic. So, the chemokines are all chemotactic. It calls the cells, primarily neutrophils and macrophages, to come to the site of infection, where the macrophage is secreting…um, CXCL8. And then, along with TNF-alpha, it helps activate the neutrophils that get there. The neutrophils get there. So, the neutrophils get there; they have CXCL8 receptors—we knew that. And they have

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receptors for TNF-alpha. And those two things cause the neutrophil to become highly phagocytic. They start eating everything in sight. And then IL12 activates a member of the innate immune cells called the natural killer cell. And then IL12 also will drive activated T cells to become TH1 T cells, and we’ll talk about that several lectures from now. [63] – [Selected IL1 and TNF-alpha Non-Immune Effects] [McCutcheon] – Non-immune effects. Femtomole. Does anyone know how many zeroes are in a femtomole? How about millimole? Close. How about milli-mol? 6? Nanomole? 9. Picomole? 12. Femtomole? 10 to the minus 15. IL1 activates osteoclasts in the Femtomole range. So it’s something like four molecule of IL1 will make an osteoclast turn on. TNF-alpha also activates osteoclasts, but it’s in the nano mol range, so you need a million times more. But what’s one of the functions of TNF-alpha? That’s one of them…what’s the function related to this? What does TNF-alpha cause the production of? —IL1. So even thought TNF-alpha isn’t very good at activating osteoclasts comparatively, it can make lots of IL1, which is really good. So, one of the disease pathways for rheumatoid arthritis, inflammatory bowel diseases is IL1. So, there are drugs now, either against IL1 agonists, and for the IL1 receptor or against TNF-alpha. And one set of those will cause good remission of people with rheumatoid arthritis and inflammatory bowel disease. And you hear the commercials for these all the time. Humira. Remicaide. Infliximab. These are all drugs that block IL1 or TNF-alpha. And in some people, blocking TNF-alpha, even though that’s not the thing that activates osteoclasts—it prevents the production of IL1 and that works really well… So as a dentist, you are now looking at the cause of all the bone loss that you’re going to deal with. [64] – [Picture (IL-1/IL-6/TNF-alpha)] [McCutcheon] – So the systemic effects. IL1, TNF-alpha and IL6 go off to the liver. They cause production of acute phase proteins. C-reactive protein. And then the lectins. And that helps activate the alternative. And then the lectin binding pathways of the complement system. So, the liver makes proteins that help activate complement—Just in case it wasn’t going well enough on its own. They go to the bone marrow endothelium, and that gets the neutrophils up out of the bone marrow so that they go off to the site of infection, and there they’re phagocytic. They go to the hypothalamus. And they cause fever. So when you’re sick, the fever is not caused by the pathogen. The fever is caused by IL1, IL6 and TNF-alpha. The higher your body temperature, the less well bacteria and viruses can replicate. They cause fat and muscle to metabolize, and that causes you to shiver, and that keeps the bacteria from replicated—and viruses. So those are the systemic effects of IL1, IL6 and TNF-alpha. IL6, the other systemic effects of IL6, are lymphocyte activation and antibody production. [65] – [Summary]

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[McCutcheon] – So in summary, our extracellular pathogens, an immune response to extracellular pathogens begins with complement. There’s always IgM from the B1 B cells. So you have both the classical and the alternative complement pathways in play. That causes inflammation that gives you the vascular changes that allows other cells to get into the area and prevents the bacterium from getting out of the area, except for the efferent lymphatic, which is going to drain into the lymph node where the bacteria will be trapped and either eaten by macrophages or acted on by other cells. Neutrophils eliminate the bacteria and then the macrophages and dendritic cells secrete cytokines. Macrophages are phagocytic. They’re not fully functional yet. It takes a TH1 T cell to make them fully functional. The dendritic cells are going to pull up stakes. They’re going to follow the pathogens off to the lymph node and that’s where we’ll begin the adaptive immune response, starting with the dendritic cells. [66] – [Intracellular Pathogens] [McCutcheon] – Intracellular Pathogens. Intracellular Pathogens start by infecting dendritic cells. Now, one of the unique things about a dendritic cell is that it’s capable of being infected by all types of viruses. So viruses, generally speaking are tissue specific. The HIV virus can infect T cells, CD4 T cells and some kinds of macrophages. Rhinovirus can infect nasal epithelium. Okay? Uniquely, dendritic cells can be infected by any type of virus. It’s a unique property. What happens when the dendritic cell is infected by the virus..so it starts off with dendritic cells being infected by the virus, and then the C3b piece of complement, you have to have that on the virus when it’s in the extracellular space because you have to have that to get B cells activated. So this where the immune response starts for intracellular pathogens. They infect the dendritic cells. And then you have the C3b pieces which are then for the B cells. [67] – [Tissue Macrophages=Dendritic Cells] [McCutcheon] – So, these are dendritic cells in epithelium. The red stuff, that’s the body of the cell. You can see some of the dendritic cell arms. So there are lots of dendritic cells in your epithelium because epithelium is one of the places where you break the barrier and you get pathogens. [68] – [A Dendritic Cell] [McCutcheon] – A scanning micrograph of a dendritic cell, so again you can see how many arms it has. [69] – [Intracellular Pathogens] [McCutcheon] – And…we will pick up…I’ll start the intracellular pathway again on Friday the 11th, so we’ll finish the last few slides of this, and then we’ll go into our next lecture. So, have a fantastic 4th of July. Study—which is not what you want to hear…and I’ll see you a week from Friday.

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03/04: innate immunity

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