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FUN2: 10:00-11:00
Scribe: Ryan O’Neill
Friday, October 24, 2008
Proof: Caitlin Cox
Dr. Barnum
Leukocyte/Lymphocyte Trafficking
Page 1 of 5
[V] – Video, [SQ] – student question, CAMs – cell adhesion molecules, JAM – junctional adhesion molecules,
sLex - sialyl Lewis X
I. Introduction [S1]:
a. Leukocyte/Lymphocyte Trafficking requires cell to cell contact.
b. Trafficking of neutrophils and macrophages will be discussed later.
c. These interactions of cells are required for tissue development. Different cells need to migrate to new sites for
development and they accomplish this via trafficking
d. Functions in Immune Response:
i. Homing of different types of T cells, B cells and phagocytic cells as naïve cells (never encountered the
antigen before), effector cells (encountered antigen and ready to eliminate it), and memory cells.
1. These three different cell categories all have differential expression of molecules called adhesion
molecules that determine where they do and don’t traffick and are often referred to as “addressins”
because of their ability to determine trafficking location.
e. The recruitment of phagocytic cells to infection sites will be discussed this morning.
II. Lymphocyte Recirculation [S2]
a. One of the hallmarks of the immune system is that it is not a passive system. The immune system actively
seeks out invading pathogens and it does that by trafficking mechanisms.
b. T cells that are generated in the thymus & B cells that are generated in the bone marrow don’t stay in those
tissues or encounter antigen (unless a serious infection is present), but they traffick out to the secondary tissues
(secondary lymphoid tissues) such as: Peripheral lymph nodes, Peyer’s Patch, spleen
1. Each of these different organs have different regions where T and B cells will sit.
2. They go to these tissues, but they don’t just stay there because there are numerous lymph nodes
throughout your body and there are potential sites for infection everywhere.
a. You have to be able to pair up the T cells and the B cells with the invading pathogen.
b. In order to do this, these cells have to constantly move around with tremendous regularity.
3. If these T & B cells do not find what they want, they will traffick out to the next tissue and do the
whole process all over again (hence “lymphocyte recirculation”).
4. If the T cells and B cells do not encounter an antigen, they will die but this is okay considering you
make millions of T cells and B cells all the time.
c. Once these T cells and B cells encounter an antigen, then they will become activated in germinal centers or T
cell zones (depending on the secondary lymphoid tissue) and will migrate out.
i. B Cell Example: B cells will migrate out from the site they were activated to the bone marrow (in this
transition B cells now become plasma cells) where they will “chunk out” antibody.
1. They make this movement through all these different adhesion molecules that allow them to move
out of these tissues and in to the next tissue where they need to go.
ii. T Cell Example: T cells will move to sites of infection where they will produce cytokines that will help direct
the response to that invading pathogen.
III. The Big Picture [S3]
QuickTime™ and a
decompressor
are needed to see this picture.
a. Main topic today: the trafficking of neutrophils and macrophages into sites of infection.
b. This type of trafficking occurs in post-capillary venules, which are small vessels where you don’t have a lot of
room for tremendous numbers of cells to flow through (class videos shows this process)
i. Macrophages (M ) & Neutrophils (PMN)
c. Trafficking into spleen and lymph node occurs in HEV – high endothelial venules (T & B cells).
FUN2: 10:00-11:00
Scribe: Ryan O’Neill
Friday, October 24, 2008
Proof: Caitlin Cox
Dr. Barnum
Leukocyte/Lymphocyte Trafficking
Page 2 of 5
d. “The Big Picture” includes the four different steps and the different families of adhesion molecules that mediate
these different steps.
e. Note the endothelial cells in the post-capillary venules. They are tightly lined up to prevent blood leakage, but
blood flow will occur in these endothelial vessels
f. PMN on slide is a neutrophil.
g. The first step is called “Rolling” and in the presence of an infection in the surrounding tissue, this neutrophil will
become activated (step 2)
h. Once the neutrophil is activated, the shape is changed dramatically and becomes flat and firmly adheres to the
endothelial cell wall (step 3). Then it can migrate in between the endothelial cells into the surrounding tissues
(step 4) where it can attempt to eliminate the bacteria causing the infection.
i. 4 steps: Rolling, Activation, Firm Adhesion, Diapedesis
IV. Adhesion Molecule Families [S4]
a. Different adhering molecules mediate these steps.
b. Selectins – family of C-type lectins
i. Lectins bind carbohydrates.
ii. There are carbohydrate ligands on the surface of the neutrophils that interact with selectins that allow that
interaction to occur.
c. Integrins - we will only be discussing the 2-integrin family (4 different members) with heterodimeric receptors
1. Consists of 2 chains -  &  chains (both are required in order to have efficient ligand binding)
d. Integrin molecules bind to members of the immunoglobulin supergene family that are cell adhesion molecules
(CAMs) that are composed of immunoglobulin domains that are found in antibodies and T cell receptors.
i. This Immunoglobulin Supergene Family are the ligands for the integrins.
e. Signaling molecules are critical and there are several receptor/ligand pairs that are important in these.
i. These are molecules that are chemoattractants to get the neutrophils to move to the site of infection.
ii. Chemokines also play a role here.
iii. Signaling molecules are involved in the activation step (step 2) and ultimately once the neutrophil begins to
migrate through the endothelial cell wall and into the actual tissue itself (step 4).
V. Physical Factors Influencing Lymphocyte/Leukocyte Migration [S5]
a. Neutrophils are being pushed along by the blood flow, and will pass the site of infection if they can’t recognize it.
b. Physical barriers of the blood cells themselves also exist.
c. The infection has to somehow activate the neutrophils that are being pushed along by the blood on the other
side of the endothelial cell barrier.
i. Student Demonstration:
1. Three students: bacterial infection (girl #3) stood behind the other two girls (endothelial cells) and the
blood flow with the neutrophils is going along the side of girls #1 and #2 (without coming in contact of
the bacterial infection). Neutrophils are in the blood and the pressurized blood flow (water gun) will
push them along and the neutrophils have to recognize that there is an infection on the other side of
the endothelial cells and be able to stop there at the exact spot. If they went past the spot, it would
take a tremendous amount of energy to backtrack against the strong blood flow to the infected
tissue. They have to get through the endothelial cells to the infected tissue (was not demonstrated).
d. This is a remarkable process and each step will be demonstrated by a video.
QuickTime™ and a
decompressor
are needed to see thi s picture.
QuickTime™ and a
decompressor
are needed to see this picture.
FUN2: 10:00-11:00
Friday, October 24, 2008
Dr. Barnum
Leukocyte/Lymphocyte Trafficking
Scribe: Ryan O’Neill
Proof: Caitlin Cox
Page 3 of 5
VI. Two model systems to study this process
a. In-vivo/Ex-vivo system where they will take images of cells moving in live animals
b. Chambers with glass plates are used to grow endothelial cells and will inject neutrophils in there to mimic the
pressure and speed that you would find in these post-capillary venules. They can change the experimental
conditions and look and see what happens. This is the process that is done on the class videos.
VII. Step 1: Rolling [S6]
a. This step involves the selectin molecules (the lectins that bind carbohydrates).
b. Several different selectins that are expressed in the endothelial cells in the blood vessels and on leukocytes
such as macrophages and neutrophils.
i. These selectins all have a shared similar structure of short consensus repeats.
c. Note lectin-binding domain on slide.
i. L-selectin = found on leukocytes
ii. E-selectin = found on endothelium
iii. P-selectin = originally found on platelets, but also expressed on endothelial cells
1. All of these bind carbohydrate ligands that are also found on leukocytes and endothelial cells.
2. One of the important molecules they bind to is sLe x but there are also many others
d. Chapter 13 covers a lot of this material. Only what is covered here is important though.
e. This interaction of the selectins and their carbohydrate ligands is a weak Velcro interaction.
i. The neutrophils will his these selectin molecules and will stick transiently and will move on and hit another
selectin molecule (which is expressed all over these endothelial cells)…they just roll along the surface.
ii. This allows the neutrophils to just flow down and not move as fast as the blood.
1. They are allowed to survey what is going on.
iii. These molecules are critical in Immunosurveillance – the ability to detect and infection, seek it out and try
to eliminate it.
f. [S7]: Structure of the complex carbohydrate sLex
i. Don’t worry about memorizing the structure
g. [V1]:
i. Neutrophil interacting with the selectin molecules in a transient interaction, then it moves on. This is
happening all the time in the post-capillary venules in your tissues. It is a way to look for invading
pathogens. Neutrophils slowly roll along because of interaction with selectins.
h. [S8]: Immunosurveillance
i. Previously mentioned, interaction in the rolling stage is critical for the body to find invading pathogens.
1. There are some individuals who have LAD II – Leukocyte Adhesion Deficiency Type 2 – that are
missing an enzyme that adds the sugar moieties onto molecules like sLex, so they have an
incompletely glycosylated ligand for the selectins and as a result, the neutrophils do not bind and it is
difficult for them to detect an infection.
2. Individuals who have this type of deficiency are very predisposed to all kinds of bacterial infections
i. [V2]: LAD II demonstration
i. Neutrophils just pass right along bacterial infection.
j. [SQ1]: Is there not a type of synthetic enzyme you could give to LAD II patients so they could add those sugars
on and overcome the deficiency?
i. It is a rare type of disease. This would require gene therapy and no one has ever done anything using
synthetic enzymes for LAD II. Good idea though.
VIII. Step 2: Activation [S9]
a. This step is very tightly coupled to the firm adhesion step, which is the last step before moving into the tissues.
b. Activation is critical for activation both cells types – endothelial cell and leukocyte.
c. Activation involves the use of all the chemotactic receptors and ligands
i. This also includes proinflammatory cytokines like IL-1 and TNF
ii. Complement fragments like C5a and C3a can contribute to Activation.
iii. There are also some bacterial peptides (tri-peptides) that are extremely potent proinflammatory and
chemoattractant molecule involved in this process in human systems.
d. Activation step is focused on activation of the integrin molecules that are expressed on the leukocyte surface
i. These integrin molecules on an inactivated leukocyte are in an inactive conformation. They don’t bind
tightly to the endothelial cell surface. This is critical because if they were in an active form all the time, they
would just stick to the surface of the blood vessel all the time.
e. Part of this process is designed to activate these integrin molecules to put them in an active conformation so
that they can then bind their ligand molecules (CAMs), which allows them to then tightly interact with the
endothelial cells only when they need to and have received the appropriate signal when there is an infection.
FUN2: 10:00-11:00
Scribe: Ryan O’Neill
Friday, October 24, 2008
Proof: Caitlin Cox
Dr. Barnum
Leukocyte/Lymphocyte Trafficking
Page 4 of 5
f. Note infection on slide 9: When you have an infection you are going to activate a complement such as C5a
g. Note bacterial tripeptide FMLP that also may be generated during an infection
h. Chemokines like IL-8 and cytokines like TNF also may be generated.
i. All of these interact with their receptors on the endothelial cell, which will cause the endothelial cell to become
activated and start releasing cytokines and chemokines in response to the set of signals it receives.
i. Those cytokines and chemokines are going to interact with the leukocyte to complete the activation step.
ii. If you have enough activation where histamine is released because mast cells have become activated,
then you are going to start to have leaky vessels and some of these mediators can leak outside the
endothelial cell barrier and they can also interact with the leukocytes as an additional way of activation.
j. Ultimately, activation will cause inside-out signaling.
i. When the inflammatory mediators hit the leukocyte, they send a signal inside the cell that then comes back
out in the form of activating the integrin molecule into an active conformation (that will then bind tightly to
the endothelium, firm adhere, and then move into the surrounding tissue to eliminate an infection).
k. All of this is dependent on a gradient-dependent migration of leukocytes (due to the chemoattractants).
i. Once the leukocyte tightly adheres, it will migrate in and move in a gradient-dependent fashion to the site of
infection. The concentration of the mediators is critical to this whole process. If you don’t have a really
powerful activation event, you will not have a lot of the activation event occurring.
1. This is another way to regulate the severity of a response to a pathogen.
l. [S10]: Chemotactic Receptors
i. Receptors used for these chemotactic ligands are the seven transmembrane receptors that were discussed
in the Cytokine lecture. There are also standard cytokine receptors that are going to be involved.
ii. These are going to be expressed on endothelium cells and the leukocytes (see slide 9)
iii. Chemotactic receptors pass through the membrane several times and have G-protein complexes that they
use to mediate their signals that go into the cell and come out in the form of activated integrins.
iv. Molecules like FMLP, platelet activating factor (PAF), chemokines, complement fragments all bind to these
type of receptors. They have their own receptor for doing this.
1. This is one of the main mechanisms involved in activation.
v. Also have histamine, which does not interact with this type of receptor. It has it’s own set.
m. [S11]: Integrin Molecules
i. Expressed on the leukocytes.
ii. We will only talk about the 2-integrin family of molecules that have 2 subunits
1.  subunits = 4 different alpha subunits: CD11a-d &  subunits = also called CD18
a. CD18 unit is paired with one of the CD11a-d units
iii. These are also dependent upon the concentration of Ca++ & Mg++ to maintain conformational structure.
iv. These molecules (CAM molecule ligands) bind ligands through the far end of both the  &  chains
v. Two of the 2-integrin molecules CD11b/CD18 and CD11c/CD18 are also complement receptors CR3 and
CR4, respectively, and are bad if you have deficiencies
1. Both these molecules are important during firm adhesion
2. CD11b/CD18 is also frequently called MAC-1
n. [S12]: Immunoglobulin Supergene Family
i. Found on endothelium cells. Made up of immunoglobulin domains like you find on antibodies.
ii. Molecules like ICAM-1 (interact with CD11b and CD11c), ICAM-2, VCAM, PECAM (important in trafficking
into the surrounding tissue by using its ability to bind to the integrin molecules).
QuickTime™ and a
decompressor
are needed to see this picture.
QuickTime™ and a
decompressor
are needed to see this picture.
FUN2: 10:00-11:00
Friday, October 24, 2008
Dr. Barnum
Leukocyte/Lymphocyte Trafficking
Scribe: Ryan O’Neill
Proof: Caitlin Cox
Page 5 of 5
IX. Step 3: Firm Adhesion [S13]
a. If you have an infection and have inflammatory mediators being produced, they interact with their receptors on
the endothelial cell. This will increase the expression of selectin molecules and further slow the neutrophil. This
helps localize the neutrophil at an infection site. Rolling step is slowed down.
b. At the same time of inflammatory mediators being released, you increase the expression of the ICAM molecules
i. Summary: increase expressions of selectins slow down the neutrophil and increase expression of ligands
for the integrin molecules, so you can have a good chance for firm adhesion because you will have several
receptor/ligand interactions to help hold the neutrophil at the site of infection.
c. These inflammatory mediators will also activate the neutrophil which causes the inside-out signaling event and
now you will have increased expression of the integrin molecules and they are all going to be in the
conformational shape they need to be to interact with the ICAM molecules.
d. All of these steps are occurring simultaneously at a very high right.
e. [V3]: Shows neutrophils adding a chemoattractant to slow down their rolling and initiate firm adhesion.
i. Process of activation and firm adhesion happen almost instantaneously.
f. [V4]: Shows neutrophils that have already been stuck.
g. [S14]: shows activated integrins
h. [S15]: LAD I – Leukocyte Adhesion Deficiency Type 1
i. What happens if you don’t have expression of the integrins?
ii. The neutrophil just rolls on by…however the actual rolling step will occur, but no firm adhesion and this is
what occurs in people with LAD I
iii. These individuals are either missing the expression of CD18 ( chain for 2 integrins) or else they have a
mutated CD18 molecules. Under those circumstances you do not have expression of any of the 2
integrins on the surface and molecules like MAC1 and CD11c are no longer on the surface of the cell and
no firm adhesion takes place. These individuals have terrible problems with infections.
iv. Babies are normally diagnosed with this deficiency early because of a massive infection at the umbilicus
v. Bone marrow transplant is the only way to treat this deficiency
X. Step 4: Diapedesis (Transmigration) [S16]
a. This step is mediated by several pairs of integrins and sometimes the ICAM molecules.
b. Can have PECAM:PECAM interactions. PECAM expressed on the endothelial cell and PECAM expressed on
the neutrophil. Those interactions will help drive the process of the neutrophil into the surrounding tissue.
c. Can have Integrin:ICAM interactions that are like in the firm adhesion step will take part in this process
d. Junctional Adhesion Molecules (JAM molecules) contribute to this process by lining up on the membrane and
help pull the cells through.
i. JAM molecules can also interact between themselves because they are expressed on the neutrophil and
endothelial cell.
ii. You have multiple pairings of adhesion molecules expressed on both the endothelial cell and neutrophil
that help these cells move through the tissue
iii. There is a shape change in these molecules, they dramatically reorganize their actin cytoskeleton and push
forward with structures called podosomes. Sometimes they release enzymes to chew through the proteins
that keep cells tightly adhering to each other, so the neutrophil can move into the tissue.
e. [V5]: Neutrophil changes shape begins to work its way into the surrounding tissues to the infection.
f. [S17]: Review: Blood flow pushes the leukocytes (neutrophils) along and the first step in this process is rolling,
which is selectin and carbohydrate mediated. Next step is activation that is mediated by cytokines, chemokines
and other chemoattractants that are liberated in the process of an infection. Complement fragments and
bacterial-derived fragments serve to activate both the endothelial cell and the neutrophil. This causes the insideout signaling that allows the conformational change of the integrins from inactive to active so it can bind its
ligand molecules like ICAM I. When you get the increased expression of all those molecules and everything in
the right conformation, firm adhesion to the endothelial cell layer takes place and then transmigration in between
the endothelial cells to the surrounding, infected tissues. All of this is an integrin, ICAM & VCAM mediated step.
[end 38:51 min]