Download Microbiology Transcriber: Mike Cantrell 08/21/08 43:26 – lecture

Microbiology
08/21/08
Leukocyte/Lmphocyte Trafficking
Transcriber: Mike Cantrell
43:26 – lecture time
Page | 1
Quiz
1. Innate immunity is:
a. Primarily mediated by IgA
b. Indispensible
c. The 1st line of immune defense
d. None of the above
e. A and B are both correct
2. Primary immune organs include:
a. The Jaw
b. The Spleen (but only when enlarged)
c. Activated Macrophages
d. Lymph Nodes
e. Thymus and Bone Marrow
3. Toll-Like Receptors:
a. The little basket you throw the change in at the Toll Bridge
b. Recognize both Intra and Extracellular pathogens
c. Bind to antibodies to mediate innate immune responses
d. None of the above
e. 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
Microbiology
08/21/08
Leukocyte/Lmphocyte Trafficking
Transcriber: Mike Cantrell
43:26 – lecture time
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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 are making 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 they see 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
Microbiology
08/21/08
Leukocyte/Lmphocyte Trafficking
Transcriber: Mike Cantrell
43:26 – lecture time
Page | 3
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
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08/21/08
Leukocyte/Lmphocyte Trafficking
Transcriber: Mike Cantrell
43:26 – lecture time
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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 going to be molecules like
some of the complement fragments like C5a, some of the chemokines like IL-8, molecules like TNF or IL1 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
Microbiology
08/21/08
Leukocyte/Lmphocyte Trafficking
Transcriber: Mike Cantrell
43:26 – lecture time
Page | 5
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
Microbiology
08/21/08
Leukocyte/Lmphocyte Trafficking
Transcriber: Mike Cantrell
43:26 – lecture time
Page | 6
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.
Slide 11
This is the structure of the integrin molecule. It is a heterodimer. There are multiple different
alpha subunits but you only find one alpha subunit per integrin molecule. There is one common Beta
subunit and these molecules all require calcium and magnesium that binds in the “I” domain in order for
them to maintain their appropriate structural conformation. These are expressed on the leukocytes in
an inactive form until they get the right signal to become active and change their conformation.
Slide 12
They bind their ligands and the end of both chains and the ligands that they bind are members
of this immunoglobulin supergene family that are found on the endothelial cells like ICAM 1, ICAM 2,
Microbiology
08/21/08
Leukocyte/Lmphocyte Trafficking
Transcriber: Mike Cantrell
43:26 – lecture time
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etc. Members of the Beta 2 integrin family will bind ICAM 1 quite readily and that is one of the main
reactions that is involved in this firm adhesion step. PECAM is involved in the transmigration step.
Slide 13
Those are all of the players in the next step and the next step is Firm Adhesion. This is our
endothelial cell layer (see handout) and those are our selectin molecules. The first step is rolling - the
leukocyte comes along and sticks to the selectins and their carbohydrate ligands. If there is an infection
present, you have all of these different inflammatory mediators which send their signal into the
endothelial cell which causes the endothelial cell to increase the expression of the selectin molecules.
Now, the rolling process is not as smooth as it was before because you have a lot more selectin
molecules forcing them to stick to the leukocyte. Therefore, instead of rolling along, it drags a little bit
and that increased drag increases the time that it stays at the site where the infection is occurring and
increases the chance that it will get the full activation signal that it needs to firmly adhere and then
move into the surrounding tissue. That is the 1st step.
Slide 14
At the same time, these inflammatory mediators also induce the expression of molecules like
ICAM 1 on the surface of the endothelial cell and the leukocyte will be kind of trapped because it is stuck
with the additional selectin molecules that have come up on the endothelial cell layer. Now, we are
increasing the expression for the ligands of the Beta 2 integrins. These inflammatory mediators
produced by both the endothelial cell and at the site of infection, are going to activate the leukocyte –
producing our inside out signaling. The signal goes in the leukocyte and comes out in the form of a
conformational change in the Beta 2 integrin that binds to the ICAM molecule. Now, we have
production of additional Beta 2 integrin molecules and those that are already on the surface undergo
conformational change; therefore, you have binding of integrins to the ICAM molecules and that is the
firm adhesion step. The cell will stick very tightly and will flatten out very nicely. It changes its shape
pretty dramatically so that it can move into the surrounding tissue.
More Videos:
Video 5: You can see the cells rolling. Then, a chemo-attractant molecule is added. Then,
immediately, the rolling cells stop. The inside out signal is almost instantaneous.
Video 6: Another example of Neutrophil firm adhesion.
Video 7: Example of a Leukocyte Adhesion Deficiency. The Neutrophils just keep rolling along – they
don’t adhere to the surface because the organism doesn’t have the appropriate integrin molecules
Slide 15
If you are missing these integrin molecules, you have what is known as Leukocyte Adhesion
Deficiency type I (LAD I). This is a really bad problem. Individuals with LAD I have either a complete
Microbiology
08/21/08
Leukocyte/Lmphocyte Trafficking
Transcriber: Mike Cantrell
43:26 – lecture time
Page | 8
deficiency of CD18, the common Beta chain that the different Alpha chains can associate with allowing
them to go to the surface of the cell, or they have a variety of point mutations in CD18 which renders it
nonfunctional. So, if you are missing CD18 or have a dysfunctional CD18, you don’t have the expression
of the Beta 2 family of molecules on the surface of the cell which leads to this adhesion deficiency.
These individuals have immediate trouble at birth. One of the 1st signs of this is that the umbilical stump
will not heal which leads to all kinds of infections at the site of the umbilical cord. Then, they have
failure to thrive, they don’t make any puss if they get an infection, and they have all kinds of other
problems. The only chance of survival is if they get a bone marrow transplant. Without treatment, they
will die in a year or two. Therefore, these players that are involved in innate immunity are crucially
important.
Slide 16
The last step in the process is Diapedesis which is the transmigration of the firmly adhered
neutrophil in between the endothial cell layer. You have to undergo a very dramatic shape change for a
molecule that was fat and round floating in the blood to one that sticks to the surface of the endothelial
cell layer, flattens out, and then has to extrude processes between the endothelial cell layer and then
move into the extracellular cell matrix underneath the blood vessel that it has just travelled through.
Both the endothelial cell and the leukocyte express the molecule PECAM on their surfaces. PECAM is
essential in the movement of the neutrophil in between the endothelial cell. So, if you are PECAM
deficient, this process doesn’t occur and you will be prone to infection. There are also a number of
molecules called Junctional Adhesion Molecules (JAMs). JAMs also bind to the Beta 2 integrins and
other integrin family members. Also, they bind to themselves expressed on the endothelial cell or the
neutrophil. Also, integrins bind to ICAM and there are several family members of integrins and CAMs
that can contribute to that. All of this serves to help pull the neutrophil in between the endothelial cell.
The JAM molecules have been shown to act like tank treads to help pull these molecules through.
Video 8: demonstrates neutrophil migration. The cell changes in shape dramatically and looks more like
a worm than a cell.
Slide 17 – Review Slide
The 1st step is rolling and that is a critical step that is occurring all of the time and is very
important in immunosurveillance so that the body can find infection. This process is mediated by
selectins and their carbohydrate ligands. So, if you don’t have appropriate carbohydrate modification
for some of the selectin ligands then you have LAD II. With LAD II, the cells will just float right along in
the blood and won’t stop despite the presence of infection. The 2nd step is activation. This step is
cytokine and chemoattractant mediated. There are several receptor and ligand players involved. These
are found on the endothelial cell layer. Also, the potentially trans-migrating neutrophil has to be
activated as well so that you can have this inside out signaling event occur so that the neutrophil can
firmly adhere to the endothelial cell layer. That is the 3rd step, firm adhesion. This step is mediated by
integrins, CAMs, etc. The 4th step is migration. This step is also mediated by integrins and several other
Microbiology
08/21/08
Leukocyte/Lmphocyte Trafficking
Transcriber: Mike Cantrell
43:26 – lecture time
Page | 9
molecules like the JAMs and the immunoglobulin supergene family molecules like PECAM and those
proteins.