Immunology – Innate Immunity Page 1 Dr. Wei-Chiang Shen, Ph.D. August 24th, 2009 Immunology Notes by Tony Dao Innate Immunity The first subject we are going to talk about is innate immunity. Today and Wednesday we will have a 1 hour lecture of each. Before we talk about innate immunity, we need to have some objectives. Why do we need to learn innate immunity? The first objective is we need to explain to you what the immune system is. Then we will go over the two different branches: innate and acquired. Then we will go over how innate immunity works, and what diseases are due to the deficiency of innate immunity, and what treatments are there for these diseases. General Terms What is immunology? It’s the study of the physiological mechanisms that humans and other animals use to defend their bodies from invasion by microbes or foreign substances. Immunity would be defined as the ability to respond to the invasion of microbes and foreign substances. Immunity does not consider small molecules though, only large molecules. This is because the small molecules can be taken down by enzymes and such, not so much by phagocytosis or the similar means. The collective system of the body that can defend and provide immunity to itself via generating molecules, cells, tissues, and organs is the termed the immune system. Note that in other body systems, we can actually identify the organs and tissues associated in those systems. For example, we can identify the heart and the vessels in the circulatory system, and likewise the intestines and stomach in the GI system. However, the immune system is different. In invasion, the motility of the body is very important. The body has to be able to move the immune system to the spot to defend itself. Different types of cells are therefore the most important parts of the immune system. We will go over the cells in future lectures. Why Pharmacists Need to Study Immunology Why do we need to learn immunology? First, most of the diseases are rooted in the immune system. AIDS, cancer, infection, diabetes, arthritis, asthma, multiple schlerosis , etc are examples of these diseases. The more we learn about disease, the more we see how they are rooted in immunology. Secondly, we also have to know how to use immunological agents as drugs – antibodies, cytokines, vaccines, immunomodulators, etc. We can use these to treat disease. Thirdly, pharmacists would need to perform immunotherapy and immunodiagnosis, so we need to understand immunology. Lastly, we need to know immunology because of how it is important that we need to know drug allergies, drug myelotoxicity (via cancer drugs, immunosuppression) , new drug developments, and etc. How does immunity work? When we look at “how” immunity works, the answer would depend on how we look at immunity. It can be seen through different mechanisms, different locations, or different responses. Different Mechanisms: Cellular Immunity vs. Humoral Immunity Let’s start with a little bit of history on this. Two scientists of the late 19th century each found evidence of different mechanisms of immunity. Elie Metchnikoff researched and discovered through his findings that immunity is due to the response of cells, hence he termed cellular immunity. However, Paul Ehrlich’s research led him to discover that cells were depending on factors in the blood, termed humoral immunity. These two scientists basically were at odds with their respective theories until 1908, in which they were both awarded the Nobel prizes for their theories. Immunology – Innate Immunity Page 2 Now let’s go into what cellular immunity is. Cellular immunity is an immune response in which there are no antibodies involved, but rather involves the activation of macrophages, NK cells, antigen specific cytotoxic T lymphocytes, and the release of various cytokines in response to an antigen. Basically, this response is due to the cell response. On the contrary, humoral immunity does not involve cellular response, but rather its protective function can be found in secreted proteins or other factors inside the humor, or serum. Different locations: Mucosal Immunity vs. Serosal Immunity Immunity can be classified through the location of which it occurs. The mucosal immunity defends the body against pathogens before they even get inside. Mucosal immunity occurs on surfaces with a mucous layer, such as epithelium of which can prevent invasion of pathogens into the body. Serosal immunity is immunity in the blood circulation and the lymphatic circulation. Once invasion gets into the body, you rely on the serosal immunity to fight the pathogens. Different responses: Innate Immunity vs. Acquired Immunity You can also classify by response. Innate immunity is a type of immunity that is received from birth, and the acquired immunity is when the body learns through infection and generation of antibodies. Most of the lecture we will learn is about acquired immunity, and today’s lecture is innate immunity. Innate Immunity vs. Acquired Immunity Innate immunity is the protection against infections that relies on mechanisms that existed before the infection. Acquired immunity is protection against infections that was induced and stimulated by exposure to infectious agents. How are they different? The following table summarizes the answer to this question. Property Innate Immunity Acquired Immunity Recognizing Cells Non-clonal, all cells of an identical All cells of a distinct class class Receptors Fixed in genome, no rearrangement Encoded in gene segments, (Pattern recognition receptors, PRR) rearrangement necessary (T cell receptors and immunoglobulins) Recognition Conserved molecular patterns Details of molecular structure (pathogen-associated molecular (antigens) patterns, PAMPS) Self-Nonself discrimination Perfect: selected over evolutionary Imperfect: selected in individual time somatic cells The innate immunity and acquired immunity recognize cells differently. Innate immunity cells belong to one single class and/or they have the same way to identify foreign cells. Acquired immunity have cells that can be of the same class but they can recognize different particles. This is why innate immunity is very limited compared to acquired immunity. But why is innate immunity so limited? The innate immunity’s receptors are fixed in the genome. There is no rearrangement. These PRR (pattern recognition receptors) are always there and always the same. However, the acquired immunity receptors are encoded in gene segments, and they can be rearranged if necessary. Some examples of these receptors are the T-cell receptors and the immunoglobulins. You can have a wide range of combinations of receptors from these gene segments so therefore you have more specific bindings and a wider range of immunity possibilities with the acquired immunity. Immunology – Innate Immunity Page 3 The recognition of the receptors of the innate immunity recognizes conserved molecular patterns (PAMPS) because of the nature of their fixed receptor genome. The acquired immunity can recognize very unique bindings because of the rearrangement possibilities. These structures that they recognize are called antigens. Also, because the innate immunity is fixed genome, there is perfect discrimination between self and nonself. It was selected over evolutionary time what these genes are, whereas for acquired immunity isn’t. It is actually imperfect because the rearrangement possibilities lowers the self-nonself discrimination. Pattern recognition receptors Pattern recognition receptors are receptors in the innate immune system in which they can identify pathogen- associated molecular patterns, or PAMPs, that are associated with microbial pathogens or cellular stress. There are two types of PRRs. One is soluble PRRs and the other is cell-associated PRRs. Soluble PRRs These PRRs do not remain associated with the cell that produces them. These circulate and bind to the pathogen via the PAMPs. One major example is the MBL (mannose-binding lectin) which binds to a wide variety of viruses, bacteria, fungi, and protozoa. Binding of MBL would initiate the lectin pathway that leads to a complement cascade, eventually leading to formation of a membrane-attack complex that would ultimately rid the invading pathogen. C-reactive protein, or CRP, is another example of a soluble PRR that may be important in the innate immune system. Cell-associated PRRs These PRRs can undergo two types of mechanisms. The first is phagocytosis. An example of this is that macrophages have a mannose receptor on their surfaces that would bind to the carbohydrates on the surfaces of infectious cells. Binding would stimulate phagocytosis to occur. The second is signaling activation. An example of this would be toll-like receptors (TLRs) that induce the expression of inflammatory factors. Toll-like receptors These are single membrane spanning non-catalytic receptors that can recognize structurally conserved PAMPs. These receptors were first recognized because they were very similar to the Toll gene in drosophila. The Toll gene is important in embryo formation in drosophila. Since 1994, there were many different TLRs that have been identified. Examples include: TLR2 recognizes PAMPs from gram-positive bacteria, TLR1/2 and TLR2/6 recognize glycolipids and zymosan; TLR3 recognizes dsRNA; TLR5 recognizes flagellin; and TLR9 recognizes CpG and DNA. For TLR9, CG combination is low in humans but high in microorganisms, so TLR9 recognizes the number of CG combinations to discriminate the microorganisms from humans. Also note that inflammatory factors induced by TLR binding can also regulate the acquired immunity responses. Immune response Type of immunity Specificity Time required Memory Innate immunity Nonspecific Short – minutes to hours No Acquired immunity Highly specific Long – days Yes Innate immunity responds really fast because the body already has all these factors. Acquired immunity takes days because the body needs to wait for rearrangement, then binding, then factor release, etc. However, innate immunity Immunology – Innate Immunity Page 4 has no memory so you will always get the same way of defense. The acquired immunity can remember previous infections so then the response is faster with subsequent same infections. This is an important basis in making vaccines. In this diagram here, it shows that the first thing that fights an infection is the innate response. However, it’s not a very efficient method of fighting. As time goes on, you see here the acquired immunity kicks in. If you look at a recurrent infection on the right, the acquired infection response became faster and stronger, but the innate immunity is still the same as the primary infection. Note that in the primary infection, the infection is allowed to increase again until the acquired immune response kicks in, while in the recurrent infection it immediately gets knocked out. This implies the necessity of both innate and acquired responses to fight off an infection, and it also shows the memory trait that the acquired immune response features. If you have no innate immunity, the person can be killed because of the rapid infection. If they lack acquired immunity, the innate can still function and fight the infection initially, but the lack of acquired immunity will allow proliferation of infection and kill the patient. Only in normal humans can you have the patient fully recover because the acquired immunity in combination with the innate response prevents the infection from proliferating in the long run. Components of Innate Immunity Components of innate immunity include physical barriers, cellular components, and soluble factors. Physical barriers The skin provides a barrier against pathogens to invade the body. You see infection when you get cuts in the skin, so the skin is a very important barrier. Your mucus is a viscous liquid that can prevent the penetration of the pathogen, and can also get rid of the pathogen because it can stick to it. The cilia are fur-like cells that continuously move up so you can cough up the pathogens. The stomach acid can kill many microorganisms, so the acid is a good factor in terms of digestion and immunity. You also have a lot of flora of nonpathogenic microorganisms. These are important to compete with pathogen microorganisms. Cellular components There are two types of cells: phagocytic and cytotoxic cells. Phagocytes that can eat other cells. These cells engulf pathogens. We have granulocytes in the blood, and one important one that functions as a phagocyte is the neutrophil. These constantly generate bone marrow and are in circulation with a short half life (12 hours in circulation). This is why cancer patients have a low cell count and high turnover rate. The other phagocytes are in tissues and are called monocytes. These migrate to tissues and differentiate to macrophages and they can eat more than neutrophils. Immunology – Innate Immunity Page 5 The second type is cytotoxic cells. The granulocytes in the blood that function as cytotoxic cells are called basophils and eosinophils. There are also NK cells that can recognize virus-infected cells and possibly tumor cells. Cellular Actions in Innate Immunity Cellular actions include phagocytosis, cytotoxicity, and chemotaxis. Phagocytosis This illustration displays the process of phagocytosis. The phagocyte usually forms finger-like projections called pseudopods that would surround the offending microorganism. After surrounding it, the microorganism is engulfed into what is called a phagosome. The phagosome is then pulled back into the lysosomes, and what follows is the formation of a phagolysosome. Factors that increase phagocytic activity can be classified as either endogenous or exogenous. For the endogenous factors, we have interferon gamma and CD40 ligand, both from T lymphocytes. For the exogenous, we have Lipopolysaccharides, or LPS. This gets released an binds to receptors on phagocytes to activate them and increase phagocytic activity. We can also see increase in phagocytic activity by opsonins. Markers on the surface would bind first. The 1st binding causes 2nd to bind faster like a zipper. These markers on the pathogens by which can be recognized are called opsonins. An example of an opsonin is mannose. The process by which a pathogen is covered with opsonins is opsoninization. Cytotoxic actions Extracellular degranulation is when cell attaches to the target and releases toxic stuff around the cell and kill the cell. Eosinophils usually secrete peroxidase or lysophophatases. Example is eosinophils attacking schistosome larva. A lot of eosinophils will surround the SL and release toxic substances and eventually the SL will be killed. Immunology – Innate Immunity Page 6 Intracellular degranulation is when cell release, for example, of perforin and granzyme by NK cells INTO the target. In this diagram, it shows a tumor cell killed by the NK cell. The NK cell recognizes the tumor marker and attaches to it, then it forms a channel and releases toxic substances directly into the tumor cell and kills it. Chemotaxis Chemotaxis is defined as the movement of cells through a concentration gradient. How do the white blood cells know where to go if they are in the blood and there’s a site of inflammation somewhere? The infection would release chemotaxic factors. The cells will recognize the chemotaxic factors and go UP the gradient to go TOWARDS the infection. The reasoning here is that as the infection releases the factors, a gradient of factors is formed; high concentration near the inflammation site and lower further away. In chemotaxis, there are endogenous and exogenous factors. Endogenous factors are released by inflammation and can be either chemokines or tufsin. Chemokines are a group of small proteins released from the sites to attract and activate phagocytes, such as CXCL8 (IL8). Tufsins are tetrapeptides consisting of Thr-Lys-Pro-Arg. Exogenous factors are unique to microbial pathogens, such as N- formylmethioinyl peptides. These can also attract phagocytes. In blood circulation, you have neutrophils and on the surface you have the s-lex: sialyl lewis x antigen. At the site of infection, you have inflammatory factors released and chemokines released. These factors will make the endothelium express more of ICAM-1 (intercellular adhesion molecule 1). These will help attract phagocytes (neutrophils) to it. You also get vasodilation effect so the junction will be loose in the cell junctions, so the phagocytes will be easier to cross the junction. Once the phagocytes move across, they will follow the chemokines to find the infection and fight it.