Roles of Phagocytosis

Earlier we studied the basics of phagocytosis using the figure shown to the right, which shows the formation of the phagosome and the subsequent fusion of lysosomes to form the phagolysosome. Now we want to look at roles that phagocytosis plays at various times during an infection. You will find that it is important in both innate (non-specific) immunity and adaptive (specific immunity).

Initial Encounters with Pathogen There are two types of phagocytic cells normally present in interstitial spaces throughout the body, and especially under epithelia. These are the dendritic cells and the resident macrophages, both of which are the first immunological cells to interact with a pathogen. Both are also similar in that they do not need an adaptive immune response in order to phagocytize a pathogen. You can think of these two types of cells as sentinels, always out in the tissues waiting to encounter any microbes that might enter the body. They then alert the immune system so that the various defense mechanisms begin. But the two types of phagocytes have quite different roles. The role of dendritic cells is shown in the figure to the right, which is from the earlier page on MHC molecules. It illustrates that the phagocytosis is not for the purpose of destroying the pathogen, but rather for collectingantigen and carrying it to lymphocytes in lymph nodes, spleen or MALT. In these locations, the dendritic cells serve as antigen presenting cells, which "present" the antigen to helper T cells (lymphocytes). This is an important step in starting an adaptive immune response. Thus, while the phagocytosis and activation of dendritic cells is innate (non-specific), the point of the phagocytosis to help promote an adaptive immune response. But an adaptive immune response requires five days or so in order to have an effect on the pathogens. With this in mind, we are going to leave dendritic cells until we take up adaptive immune responses and concentrate now on macrophages.

Macrophages, on the other hand, start processes that can begin within minutes. At this point, no molecules (antibodies or T cell receptors) are available to specifically identify the pathogen. Thus, these initial processes are part of an innate immune response. In addition, macrophages are important in actually destroying pathogens, especially for infections that are more prolonged and that turn into chronic problems. Recognition of Pathogen A dendritic cell or macrophage must first recognize that it has encountered a microorganism and not a normal cell of the body. Basically, these phagocytes have certain proteins in their membranes that either directly or indirectly recognize various molecules that could not be found on human cells. Reflect that microbes have structures such as cell walls that are never a

part of a human cell. Thus certain molecules are present on microbes that are quite different than those on a human cell. But the recognition molecules on dendritic cells and macrophages are not nearly as precise as the molecules created by an adaptive immune response (antibodies and T cell receptors). The innate recognition molecules only recognize general classes of microbes rather than specific microbes. The resulting phagocytosis is enough to get the immunological processes started, but is restrained, so that it will not get rid of a significant pathogen. For that, an adaptive immune response with antibodies and/or T cell receptors is required. The first and most important of these recognition molecules are the toll-like receptors. There are 11 different molecules in this category, and they recognize a wide variety of molecules found only on microbes. The binding of a ligand to a toll-like receptor sets in motion of sequence of events inside the cell that activates various genes important for orchestrating a innate immune response. Another example of a recognition molecule is the mannose receptor. Some microbes have molecules on their surfaces that contain mannose spaced at specific intervals. (Mannose is a sugar.) Human cells would never have molecules of this type. In addition, there are three or four more types of known recognition molecules on macrophages. But the total number is not large and they are all coded by typical genes. These are normally expressed; the macrophage does not need to be exposed to the microbe to begin making these innate recognition molecules (unlike for antibodies or T cell receptors). A few of the recognition molecules are not on the surface of macrophages, but rather found in the blood plasma. One especially important molecule of this sort is C-reactive protein. This is made by the liver and only binds to microbes. Another protein of this type is called mannose binding lectin. Macrophages in turn have molecules on their surface that bind to C-reactive protein or mannose binding lectin. This binding promotes phagocytosis. A molecule that promotes phagocytosis by binding to a microbe, such as C-reactive protein and mannose binding lectin, is called an opsonin. Another example of an opsonin is C3b, which is discussed on the next webpage. (The best opsonins, however, are antibodies, which are made in a specific immune response.)

What type of cells make mannose binding lectin and C-reactive protein? a. macrophages b. endothelial cells c. TH1 helper T cells d. B cells e. liver cells

Now let us start to follow the sequence of events as microbes first enter a tissue in the body. The very first step is the phagocytosis of a few of the microbes by a few dendritic cells and macrophages. The dendritic cells scurry off to a lymph node and begin the adaptive immune response, which will require about a week to start having an effect on the microbes. For now, we set aside the dendritic cells and start to following the sequence of events that the phagocytosis of a few microbes by resident macrophages sets in motion. All this starts happening within minutes. Release of Cytokines After phagocytizing microbes, macrophages (and dendritic cells) release regulatory moleculess. These serve mainly as paracrines that set in motion many of the further immunological processes during an infection. The term cytokine refers to any small protein that is released to help coordinate an immune response. Most cytokines are released by white blood cells. Two important cytokines released by macrophages when they encounter a pathogen are TNFalphaand interleukin-1 (IL-1). The effects of both are similar, except that when TNF-alpha is released in pathological excess, it can trigger dramatic, life-threatening responses. In addition, macrophages, as well as other nearby cells, release cytokines called chemokines. Roles of Cell Adhesion Molecules The initial phagocytosis of microbes by resident macrophages is not going to end the infection unless there are just a few, stray microbes present. Any significant infection is going to require reinforcements, and recruiting these is perhaps the most important role of the cytokines released by the resident macrophages. The first cells recruited are neutrophils, which are present in substantial quantity in the blood and rapidly enter the infected tissue in order to start phagocytizing microbes. Unlike macrophages, neutrophils are short-lived, lasting only hours to days.

The first step in the migration of neutrophils into the tissues is necessarily the binding of the neutrophils to the endothelium of the blood vessels. Without this step, of course, the neutrophils would simply continue to tumble through and out the vessels serving the region. The binding begins when TNF-alpha and IL-1 from the macrophages cause endothelial cells to begin expressing two types of cell adhesion molecules that promote the binding of

neutrophils to endothelial cells. These molecules allow the binding to occur in two steps. In the first, adhesion molecules called selectinslightly tether the neutrophil to the endothelium, so that it begins rolling along the surface. In a second step, a much tighter binding occurs through another class of newly expressed adhesion molecules on endothelial cells called ICAMs. These cause a tight adhesion of the neutrophils by binding molecules on the neutrophils called integrins. Diapedesis and Chemotaxis Once bound to the endothelium, neutrophils squeeze through gaps between adjacent endothelial cells into the interstitial fluid, a process called diapedesis. Aiding this, an endothelium activated by the cytokines also becomes more permeable than normal. Even blood proteins can now cross. Once outside the blood vessel, a neutrophil is guided towards an infection by various diffusingchemotactic factors. The chemokines released by macrophages and other cells are especially important. Another example is the complement peptide C5a, which is released when the complement system, as discussed on the next page. Certain bacterial molecules are also directly chemotactic. With time, monocytes in the blood also begin adhering to the endothelium and moving into the infected tissue. Once in the tissues these are called macrophages. While the short-lived neutrophils predominate in an acute response to an infection, the long-lived macrophages are important forchronic infections. Another process going on while all of the above are occurring is dilation of the small arteries serving the infected area. This is caused by the cytokines and also various other inflammatory paracrines. This, of course, makes available more substances in the blood to fight the infection and is the reason infected tissue turns red.

The phagocytic process is composed of four interrelated phases: chemotaxis,opsonization, ingestion, and digestion. The function of opsonins is to react with bacteria and make them more susceptible to ingestion by phagocytes. Opsonization of bacteria may occur by three different mechanisms. First, specific antibody alone may act as an opsonin. Specific antibody may also act as an opsonin in concert with complement, by activating C3 via the classic pathway of C1, C4, and C2. Finally, there is a nonspecific mechanism of opsonization present in nonimmune animals called the heat-labile opsonin system. In the heat-labile opsonin system, 7S immunoglobulin from nonimmune animals activates C3 via an alternate pathway distinct from C1, C4, and C2. These different mechanisms may operate concurrently in vivo, but one mechanism may be favored over another depending on the type of organism, the stage of the infection, and the presence or absence of specific antibody. There are a number of different clinical situations in which an increased susceptibility to infection is associated with decreased serum opsonizing activity. These situations serve to illustrate the important role opsonins play in the host's resistance to infection.