The second line of defense comes into play when a pathogen has penetrated the surface barriers and entered the body. Second line defenses attempt to limit the spread of pathogens through the body by taking a multifaceted approach to pathogen elimination. Although these innate internal defenses are fast-acting, they are nonspecific and provide only a kind of crude protection against any and all pathogens that enter the body. Think of them as being like the guards inside the castle. There are five lines of innate internal defense. One line of innate internal defense consists of the phagocytic cells, primarily the neutrophils and macrophages. A second set of cells, the natural killer (or NK) cells, kill body cells that have turned traitor by becoming either virus-infected or cancerous. Another line of defense does not involve cells at all, but rather antimicrobial proteins such as complement and interferons. The last two lines of defense are neither cells nor proteins, but processes. They are inflammation and fever. Sometimes the innate defenses are no match for invading pathogens, just as a massive invasion can overwhelm the guards on a castle wall. In this case, the armies of the adaptive defenses must be called out. Having already met the enemy, the innate defenses can influence the kind of response the adaptive immune system makes by passing chemical messages to the adaptive immune system, rather like this guard gesturing to his friends. This process is one of the many ways in which the innate and adaptive defenses work together. Pathogens that enter the body are often rapidly ingested by phagocytes, this process begins when a phagocyte recognizes and binds a pathogen. You might wonder how the phagocyte knows that something is a pathogen and not part of the body. Phagocytes use special cell membrane receptors, such as the mannose receptor and the Toll-like receptors (or TLRs), to recognize and bind molecules that are found only on certain pathogens, particularly bacteria, and not on normal body cells. At least 10 different Toll-like receptors have been identified on human phagocytes, each binding to a different pathogen molecule. When phagocytes recognize a pathogen, two events are triggered. The first is the ingestion of the pathogen. The second is the release of chemical alarm signals that mobilize other cells of innate and adaptive immunity. Let’s observe a phagocyte bind pathogens. Pathogens and the immune system are involved in an evolutionary arms race. Many pathogens have evolved strategies to avoid being killed by phagocytes. Let’s watch the macrophage try to destroy each type of bacterium (encapsulated and unencapsulated). Now let’s try the other kind. One strategy certain bacteria have evolved is to enclose themselves in a capsule that makes it more difficult for phagocytes to grab them. In response, the immune system has evolved molecules that can coat these bacteria and provide “hand-holds” that allow the phagocytes to bind and engulf these bacteria. This process of coating bacteria to enhance phagocytosis is called opsonization. Two immune molecules that can act as opsonins are antibodies and complement. We will learn more about both of these later, but here we will focus on antibodies. Observe a bacterium as it is coated with antibody. Phagocytes have receptors that attach to the opsonins. The opsonins form a link binding together the pathogen and the phagocyte, triggering phagocytosis. There are several other strategies that pathogens have evolved to escape destruction by phagocytes. These include secreting molecules that block the fusion of lysosomes with the phagosome, developing resistance to the effects of lysosomal enzymes and reactive oxygen intermediates, and finding ways to escape the phagosome, take up residence, and replicate within the cytoplasm of the phagocyte. The bacterium that causes tuberculosis is known for its ability to hide out and replicate inside macrophages. In response to bacterial strategies such as those already mentioned, the immune system has evolved a counter-strategy. Certain T cells of the more sophisticated adaptive defense mechanisms can enhance the entire killing process within the macrophage. This enhancement only happens when the macrophage presents antigen from such bacteria to the T cell. The interaction between T cells and phagocytes is another example of the interaction between innate and adaptive defense systems. Let’s watch the macrophage try to destroy the tuberculosis bacterium. Now we’ll enhance the macrophage’s killing ability by bringing the T cell to it. Natural Killer (NK) cells are unusual, in that they are a type of lymphocyte, and yet they are involved in innate immunity. They make up 10-15% of the lymphocytes circulating in the blood. Let’s compare these unusual lymphocytes to B and T cells. Like T cells, NK cells kill the body’s own cells under two circumstances— if those cells have been invaded by intracellular pathogens, or if they have become cancerous. NK cells also attack transplanted tissues, playing a role in the rejection of transplanted organs. NK cells are larger than B and T cells, and contain granules in their cytoplasm. Thus, NK cells are sometimes called “large granular lymphocytes”. While B and T cells express specific receptors for antigen, no such receptors are found on NK cells. Yet, NK cells can recognize a variety of cells as abnormal, bind to them and kill them. Now let’s learn how the NK cells recognize abnormal cells. Abnormal cells, such as cancerous cells or those infected by viruses, often reduce the expression of certain membrane proteins. The suppressed proteins are those that tell the immune system that a given cell is “self,” in other words that it belongs to the body. NK cells look for the absence of these “self” proteins. It’s as if NK cells were saying, “If I can’t identify you as one of us, then you are a traitor, and I will kill you.” Let’s watch the NK guard identify the traitor. NK cells and T cells are the two kinds of cells that continually scan our cells for abnormalities, a process called immune surveillance. They act in complementary fashion. T cells look for the presence of abnormal antigens on the cell surface, while NK cells look for the absence of normally occurring self-proteins. How do NK cells kill abnormal cells? The same way that cells called cytotoxic T cells kill— a process we will explore in detail later. For now, let’s just say that killing involves direct contact and induces the target cell to undergo apoptosis, which is programmed cell death or cellular suicide. NK cells are important in the early response to pathogens, acting to contain pathogens before the adaptive immune responses can take over. They continue to play a role after B and T cells are activated. Like macrophages, NK cells become more effective killers following activation by cytokines from certain T cells. Likewise, coating cells with antibody makes them better targets for killing by NK cells, just as coating pathogens with antibodies make them better targets for phagocytes. These processes show of how innate and adaptive host defenses work together to protect us from infections. The first set of antimicrobial proteins we will consider are the interferons. Interferons are members of a larger group of chemicals called cytokines that modulate the immune system. Interferons interfere with viral replication, modulate inflammation, and activate immune cells. The 3 types of interferon— alpha, beta, and gamma—are distinct proteins, but have common as well as unique functions. Gamma interferons act in a variety of ways to signal other immune and non-immune cells, and we will consider later. Here, we will learn about the anti-viral properties of alpha and beta interferons. Let’s begin by examining how viruses replicate within cells. Recall that viruses must enter cells to replicate. This is because a virus is little more than a protein-covered packet of nucleic acids— the genetic instructions for how to create a new virus. When a virus penetrates the target cell's membrane, it releases its nucleic acid and takes over the host cell’s machinery to make more copies of that virus. The presence of a virus replicating inside it causes the cell to produce and secrete interferons. Interferons bind to plasma membrane receptors on nearby cells. They act as warning signals for as-yet-uninfected cells, telling them that there is a virus on the loose. In response, the uninfected cells produce proteins that inhibit viral replication. These proteins act by degrading viral RNA and by preventing the synthesis of viral proteins. Let’s see how this process works. Now let’s see if a virus can reproduce itself when it tries to enter a cell, that has been alerted by interferon. Chewing up viral RNA and blocking protein synthesis are non-specific, in that they work against any virus. In the short term, these mechanisms protect uninfected cells not only against the virus that invaded its neighbor, but also against any other viruses that may be in the area. The next set of antimicrobial proteins we will consider is the complement system. Complement gets its name from the fact that it complements or enhances other components of both innate and adaptive defenses. Like the blood clotting cascade, complement is actually a complex cascade of interdependent plasma proteins. As each protein is activated, it becomes an enzyme that activates the next protein until the final product is formed. When activated, these proteins can mark cells for phagocytosis, promote inflammation, and even kill some bacteria all by themselves. Now let’s watch what happens when complement proteins enter this bacterium and see how it is lysed by the end-products of the complement cascade.