MicrobiologyTranscriber: Mike Cantrell

08/21/0843:26 – lecture time

Leukocyte/Lmphocyte Trafficking

Quiz

  1. Innate immunity is:
  1. Primarily mediated by IgA
  2. Indispensible
  3. The 1st line of immune defense
  4. None of the above
  5. A and B are both correct
  1. Primary immune organs include:
  1. The Jaw
  2. The Spleen (but only when enlarged)
  3. Activated Macrophages
  4. Lymph Nodes
  5. Thymus and Bone Marrow
  1. Toll-Like Receptors:
  1. The little basket you throw the change in at the Toll Bridge
  2. Recognize both Intra and Extracellular pathogens
  3. Bind to antibodies to mediate innate immune responses
  4. None of the above
  5. Answers B and C are both correct

Slide 1

This afternoon, we are going to talk on two subjects that are inter-related to things we talked a little bit about yesterday. Cytokines come into play and complement comes into play in terms of some of their biological activities and some of the molecules that mediate inflammation and the effects that we are going to talk about today. First, we are going to talk about the trafficking of leukocytes, that is the white blood cells, and we are mostly going to focus on trafficking of neutrophils and monocytes and macrophages as a representative inflammatory cell. A lot of what we are going to talk about today in terms of the families of molecules and the basic steps and mechanisms that we are going to talk about for leukocyte trafficking apply also to lymphocytes but because we only have less than an hour to cover this, I am going to use the inflammatory cells as the kind of prototypical mechanism and that is where a lot of this was really worked out in the 1st place. So, I will just mention briefly a little about trafficking of lymphocytes and then we will go into the steps of all of these different molecules. So, in terms of trafficking, the basic properties that we are talking about involve cell to cell contact and homing for tissue development. So, trafficking is not something that is just exclusive to the immune system, you have cells trafficking all of the time during development and even during repair processes from injury and turnover of tissues, there is always going to be movement of cells and that is all going to be dependent on a number of different cell-surface molecules, receptors, and ligands that mediate that function. In terms of the immune system, trafficking is something that is involved in homing of naïve, effector, and memory cells. So, T-cells and B-cells, for example,all express a variety of different markers as they mature from cells that have never seen antigen to cells that are responding to antigen to cells that can respond to antigen the next time you encounter it and those different molecules that they express on their surface with respect to trafficking determines where they move throughout this entire process. So, a lot of these molecules have been coined addressins because they serve as an address where these cells can be found or will be able to move to during the immune response. Then, what we are going to spend a lot of time on is the recruitment of phagocytic cells. So I have listed up here a good review article, the basic mechanisms haven’t changed even though the article is a little dated.

Slide 2

Lyphocyte Recirculation – This is an absolutely critical aspect of adaptive immunity. You have to have T and B cells moving around all of the time. So, if you get a cut in your foot and you get an infection developing there and the T and B cells that are specific for that particular bacteria are located in a lymph node up under your right arm, somehow you have to get those players together to develop a really good adaptive immune response. Trafficking is, in large part, about getting the cells together that have to respond to a given pathogen with that pathogen so that they can mediate a good immune response. That is why these cells are trafficking around all of the time, moving from different lymph nodes to the spleen and back again so that they have the best opportunity for encountering the pathogen that they are specific for. T and B cells come from the bone marrow and the thymus. Once these cells develop and mature, they move to peripheral lymph nodes, to the Peyer’s patches (lymphoid tissues in the gut), to the spleen, and to specific regions within those different immune organs. That is continually happening because every day you aremaking new T and B cells. They are getting in, moving out, and using some of these adhesion molecules to get into these given tissues and they will continue to circulate like that. Whether theysee an antigen or not, they will continue to circulate and move from one organ to another in an effort to encounter their antigen. If within a couple weeks, they do not encounter their antigen, they will die. If they didn’t turn over and you kept making new cells, you would have a problem – you would have too many cells floating around. So, once you encounter antigen, the antigen is brought to either the germinal center or the T cell zones so that it can encounter specific T and B cells that recognize it and can go through their activation and proliferation steps so that they can direct an immune response to eliminate that pathogen. In the case of B cells, to make antibodies and in the case of T cells, to help those B cells become effector cells and produce antibody and also to direct other cells in the process as well. Those T and B cells, as they mature in response to exposure to antigen, the memory cells will go back to the bone marrow and some of those cells will go to the mucosa in the gut, for example. Some of those cells will also go to sites of infection. T cells in particular will often go to the site of infection and help direct the immune response there. Or, if it is a cytotoxic T cell, it has to go there so that it can kill infected cells whether it be a viral infection or an intracellular pathogen. These kinds of trafficking events are always occurring for T and B cells and they are essential for good adaptive immune responses.

Slide 3

Now, we are going to focus on Neutrophils and Macrophages, I am going to use them interchangeably. This is what I call the BIG PICTURE. Basically, we are talking about events that are occurring at post-capillary venules (small vessels where blood flow is not extremely fast). These are the endothelial cells in that vessel. This is blood flow and here is our representative neutrophil. We are going to talk about 4 basic steps involved in movement of that cell from blood into the surrounding tissue where an infection has occurred:

  1. Rolling
  2. Activation – closely linked to step 3
  3. Firm Adhesion – closely linked to step 2
  4. Diapedesis – transmigration of the cell in between the endothelial cells. There is now evidence that these cells can traffic through the endothelial cell as well to get to the surrounding tissue where the infection may have occurred.

Those are the basic steps and we are going to talk about the different family members of adhesion molecules that mediate these steps and how that all comes together.

Slide 4

1st, I just want to give you an overview of some of these molecules so that when we go into the steps, you will know what I am talking about. 1st, there is a group of molecules called selectins. This is a family of C-type lectins. Lectins bind to carbohydrates. So, we are going to talk about selectins and the carbohydrate ligands that they bind to. These carbohydrate ligands are critical. Next are the integrins. These are heterodimers and there is a large family of integrin molecules (almost 30). We are going to confine our studies to just a couple of the beta 2-integrin family of molecules. There are 4 in this family (CD11a-d) and they pair with another molecule called CD18. Mostly, we are interested in 11a-c. The molecules that the integrins bind to in the context of what we are talking about today are the so-called Immunoglubulin Supergene Family – The CAMs (cell adhesion molecules) such as I-CAM, P-CAM, etc. These molecules are all composed of these Immunoglobulin domains that I showed you yesterday for one of the cytokine receptors. Then, one of the most important parts of this whole process are the signaling molecules. In order for this process to occur, you have to activate all of the cell types involved and a number of different events have to occur in terms of production of various proteins, changes in conformation of these proteins so that they can bind those kinds of things, all mediated by several receptor ligand pairs, chemo-attractant molecules(chemokines). These are goingto be molecules like some of the complement fragments like C5a, some of the chemokines like IL-8, molecules like TNF or IL-1 are all components among many that will stimulate these cells and allow this process to occur.

Slide 5

Blood flow presents a difficulty. These cells are moving along and even if they get the signal, they still have the blood flow pushing them along so that they will not stick. Also, there is the physical barrier of the endothelial cell wall (endothelial cell walls are not usually very leaky). Somehow, you have to make this cell wall permeable so that the cells can get into the surrounding tissue. Also, there must be a mechanism, for the cell that needs to be activated, to detect that there is an infection in the 1st place. So, if there is an infection in your skin, how is it that cells floating in blood vessels nearby can actually recognize that an infection has occurred and that you need to stop that cell and get it to traffic.

Demonstration with super soaker = two people in front (endothelial cells) with one person behind them (nasty bacterial infection). He then sprays water in front of them all to demonstrate blood flow. Cells must get through all of this in order to reach the nasty bacterial infection.

Slide 6

The 1st step is rolling and rolling is mediated by selectins and their carbohydrate ligands. Here, we have our prototypic endothelial cell and our leukocyte (see handout). These molecules here are our 3 different types of selectin molecules: P-selectin (identified on platelets), E-selectin (found on endothelial cells), and L-selectin (found on leukocytes). All 3 are structurally similar, made up of these repeat domains found in a lot of the complement regulatory proteins and they have a carbohydrate binding region. What they bind are a number of different carbohydrate molecules. There are some very heavily glycosylated proteins that will interact with these selectin molecules. This interaction is kind of like weak Velcro- it kind of sticks and comes off = rolling.

Slide 7

This is just an example to demonstrate the complexity of the carbohydrate ligand. So, leukocytes with their carbohydrate ligands will stick to the selectins and then come off. That process is occurring all the time in the post-capillary venuels and in other vessels as well. This is very critical.

Shows Videos:

Video 1: animation of what rolling looks like

Video 2: This is an example of what rolling looks like. You can see the cells just rolling along the surface as opposed to flowing along in the middle. This is in a live animal

Video 3: Same as video 2 but in a chamber set-up. This is rolling of neutorphils. This process is critical for immune responses because if you don’t have these selectin molecules, then the neutrophils and macrophages that express the counter-ligands are just going to float right on by.

Slide 8

If that is what happens, you have a failure of what is called immunosurveillance because that is what these cells do all of the time – they flow through your tissues looking for trouble (infection). The 1st way they can do that is to slowly roll along so it gives them a better chance of encountering the activation signal and responding quickly right at the site where the infection occurred. If they were flowing along at the speed of blood, they wouldn’t have a chance to do that. There are some deficiencies associated with this. If you have incomplete glycosylation of the sLew X molecule, you don’t get any binding to the selectins = Lad II (Leukocyte Adhesion Deficiency type 2). This is not a very common deficiency but if you can’t get those sugars onto those molecules in the proper fashion so that they can bind their receptors, the selectins, then you are not going to have rolling and you have lost the 1st step in the whole process.

Video 4: Without the appropriately glycosylated molecules, these cells just flow right through and do not roll at all.

Slide 9

Step 2 is activation. Now, what we have to do is tell all of the cells in the vicinity of the infection that we have a problem and we do that with a number of these different cytokines, chemo-attractants, complement proteins, chemochymes, etc. Lots of different players can come in and enhance this process. This step is mediated by chemotactic receptors and their ligands. Here is our endothelial cell and our leukocyte (see handout). The receptors that mediate a lot of this, in many cases but not all cases, are these 7-transmembrane-receptor-spanning molecules. These are found on endothelial cells and on the leukocytes and one of the main players that is intimately involved with this activation step is the integrin molecules. This integrin molecule is drawn with a square shape to indicate that it is in an inactive form. It turns out that the integrin molecules are very important in the firm adhesion step that come up next and if they were in an active conformation and could bind their ligands all of the time, then the cells wouldn’t go anywhere on the endothelial cells, they would just stick all of the time. So, they are in an inactive conformation and in order to get firm adhesion, you have to change conformation of these molecules through this activation step. This actually turned out to be a real tour de force in terms of structural biology – they actually have the crystal structure of these molecules both in the inactive and the active conformation now. So, they know that this is a real phenomenon that occurs very quickly when these cells are activated. So, if we have our infection here, we know that bacteria have these different pamps like LPS and a number of other molecules that will activate the host immune response, activate complement so that you can get C5a, and induce the expression of cytokines like IL-8. These and many other inflammatory mediators that are released in response to an infection will bind to these receptors and activate the endothelial cells. These endothelial cells will also produce inflammatory mediators (different cytokines) that will serve to activate the leukocytes. Under the appropriate conditions of inflammation, when the endothelial cell wall becomes leaky, some of the molecules that are liberated here down by the site of infection can also get out here and further activate the leukocytes or activate others that traffic into the area. There are lots of different players that come in and work on both cell types for this activation step. So, we activate both cell types and one of the critical points to make is that this activation causes what is called “inside-out” signaling. This inside-out signaling occurs on the leukocyte. When it receives a signal from some of these inflammatory mediators, that signal goes inside the leukocyte and comes back out in the form of a changed conformation of the integrin molecule. The signal goes in from the inflammatory mediators and comes back out in the form of a changed conformation of the integrin molecules so that it can bind its ligands and adhere firmly in the next step. The ultimate effect of this is that all of these different mediators are involved in a gradient dependent migration of a leukocyte once they manage to get past the endothelial cell barrier. Therefore, these guys are critical in telling the phagocytic cell where to go so that it can find the bacteria and eliminate it.

Slide 10

This is just a blow up of the structure of some of these chemotactic receptors. They span the membrane 7 times, do their signaling through these g-coupled protein complexes. There are lots of players here like the FMLP, IL-8, C5a, etc. Another player that is important here that doesn’t bind to one of these receptors is histamine. Histamine is released by mast cells in the area and mast cells will be triggered to degranulate and release histamine by some of these mediators here because they express receptors for many of these molecules. This will cause them to degranulate and release things like histamine and other vasoactive amines and additional cytokines that will play into this process. So, you have all of these different players that are critical for activating the cell types involved, for increasing vascular permeability, and ultimately these guys chemoattracting the phagocytic cells from blood into the site where the infection occurs.