In The Name Of Allah

Pathology – Lecture 8
Tuesday 12-10-2010
Dona By: Esra' Soudi

Before we start … I will remind you about many notes:

1) I've put many photos in this tfree3’ because the lecture today was
based on slides & diagrams.

2) The exam will be at Saturday (30/ 10/2010) … we have two times

for two sections ....you can go later and check your e-mail to know your
computer’s number the Dr not allow for any student who don't know
his/her lab..ok…also the Dr said that the material in both sections is
the same…and exam will be the same…

3) The doctor advises you to read your material from your book …

4) The doctor is angry from students who leave the hall before Dr
Finishes

I will start my lecture:

Last lecture we stopped at this slide. Today, we will talk in details

about the process of extravasation of leukocytes to the site of
infection, and the specific steps about adhesive molecules that
activate such extravasation or movement of leukocytes from inside the
blood vessels to outside (the site of infection).

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You remember that we said that initially the leukocytes which margin
at the periphery of blood vessels stick in a transient way to the
endothelium and we called this rolling.

After this, they will have a more firm adhesion and then they will
transmigrate from the intercellular spaces to the outside environment
which is the extracellular matrix or the connective tissue site. The
processes of rolling, adhesion, firm adhesion, and transmigration are
controlled by a group of cellular adhesion molecules and ligands that we
call: (selectins and integrins).

So what are the selectins and intigrins?

Selectins: Receptors that are expressed on the surfaces of
endothelial cells and leukocytes that bind to selected sugars
(sialylated oligosaccharides). They have specific binding sites for
the oligosaccharides and that’s why we call them “selectins”,
because this means they are selective to become apparent or bind
to the ligands which are polysaccharides. They are normally not
expressed on the resting endothelial cells, but are expressed
within 30 minutes of stimulation, by the chemokines or other
cytokines that we will see later on. These chemokines and cytokines
will transform the endothelial cells or leukocytes from low affinity
for binding to high affinity (or fast – off).

 This mean when the cells are in a resting state, they will have
these receptors inside the intracellular space. Then, by the
stimulation they will go through redistribution and transformation
which transforms these receptors to the surfaces. Thus, they
become high affinity receptors (expressed on the surfaces).

 There are many different types of selectins :

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 E-selectin: expressed on Endothelial cells
P-selectin: expressed on Platelets & Endothelial cell
• L-selectin: expressed on different types of Leukocytes

Integrins: obligate heterodimers containing two distinct chains,

called the α (alpha) and β (beta) subunits (selectin has only a single
chain). Integrins only appear on leukocytes (selectin: on leukocytes&
platelets &endothelial cells).

Integrins bind to ligands present in:

 Extracellular matrix
 Complement system
 Surface of other cells

Integrin have cytoplasmic domains and surface receptors which

bind or connect to the cytoskeleton of the cells, which leads to
cytoskeleton transformation, and these transformations will lead to
changes in cell shape allowing it to transmigrate. (Note: the cell
must change its shape in order to transmigrate.)

Integrin-cytoskeletal interactions involve ligand binding to the

integrin’s extracellular domain and the clustering of the integrin
and this is induced by chemokines. This interaction will cause
cytoskeletal changes which will cause locomotion of the cell.
(Remember: the main function of the cytoskeleton is locomotion of
any type of cells from inside opposite to the outside).

Reminder: the difference between migration and transmigration,

migration is the movement of leukocytes from inside to the outside,
but transmigration is the movement of leukocytes within endothelial
cells.

Follow me step by step to know the sequence of extravasation:

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 1) Weak adhesion and rolling: which are mediated by selectin OR
firm adhesion.
2) Firm adhesion: which is mediated by two factors:
• Ig (immunoglobulin), super-family molecules expressed on
endothelial cells such as:
- ICAM-1 (Intra-Cellular Adhesion Molecule )
- VCAM-1 (Vascular Cell Adhesion Molecule)

• Integrins expressed on leukocytes:

a) LFA-1 (the leukocyte function-associated antigen), another name is:
(CD11a/CD18): we used these integrins to test immunodeficiency << not
having a normal level of this integrin indicates immunodeficiency) >>
binds with ICAM-1

b) Mac-1 (CD11b/CD18) ligand (expressed on the endothelial cells)

c) P150,95 (CD11c/CD18)

d) VLA-4 (Very Late Activation antigen 1) >> binds with VCAM-1

NOW … Look at these two diagrams:

Leukocytes have three receptors:

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 1. Sialyl-Lewis x-modified glycoprotein that bind with the selectin
on the endothelial cells....(P-selectin and E-selectin )
2. L-Selectin which binds with L-selectin ligand on the endothelial
cells.
3. Integrins which are found in a low affinity state in two situations
A) Normal flow
B) Rolling or not firm adhesion.

Now we will explain in details the sequence of extravasation of

leukocytes to the infectious area..JUST follow me...

FIRST: in the rolling state and after the macrophages go to the

infection site and secrete cytokines (kemokines), adhesion occurs
between:
A. Sialyl-Lewis x-modified glycoprotein & P-selectin
B. Sialyl-Lewis x-modified glycoprotein & E-selectin
C. L-selectin & L-selectin ligand on the endothelial cell.

IMPORTANT NOTE: in the rolling state integrins still have low

affinity until they arrive to the firm adhesion state.

SECOND: firm adhesion state, when cells (such as macrophages,

fibrin, and leukocytes) produce more chemokines. These chemokines
will be delivered to the endothelial cells by proteoglycans which can be
found in the cell membrane, so proteoglycans expose the chemokines to
the integrin, then conformation occurs (converting integrin from low
affinity to high affinity OR what we called firm adhesion. Integrins
are now ready for adhesion or binding.... look at this diagram...

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 THIRD: migration within endothelial cells (transmigration), this is
activated by chemokines. Chemokines (cytokines) will lead to some
changes in the cytoskeleton of the cell itself, so the cell change its
shape and size and will try to create some pseudopods ( ‫ القدام الكاذبة‬,
which is a method of locomotion in Amoeba.)

The type of chemokine which activate transmigration is (CD31) OR

(PECAM-1) >> Platelet Endothelial Cell Adhesion Molecule - 1

Look at this diagram...

The leukocytes can damage the basement membrane of the endothelial

cells by glycogenosis and other enzymes to create more spaces
between endothelial cells through the transmigration.

FORTH: migration to the site of infection.

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 This diagram explains what we've said before

This is another diagram that shows the function of cytokines …

P-selectin is normally within Weibel-Palade bodies, which are not

expressed on the surface of the endothelial cells, but when these
bodies are activated by cytokines (such as Histamine and Thrombin). P-
selectins are released on the surface of cells. This process is called
upregulation (redistribution) of selectin.

Endothelial and Leukocyte Adhesion Molecule Interactions

 This table is very important:

• ENDOTHELIUM WBC FUNCTION

• P & E-selectins Sialyl-Lewis X Rolling

• GlyCAM-1, CD34 L-selectin Rolling

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 • VCAM-1 VLA-4 Adhesion

• ICAM-1 CD11/CD18 Adhesion,

(LFA1, MAC1)

• CD31 (PECAM-1 CD31 Transmigration

Q: Are the integrins and selectins part of WBC's?

A: They are only receptors, expressed on the endothelial cells &

WBC's…

 General Structure of CAM Families :

Leukocytes

1) Lectin domain which is

polysaccharides, binds with
mucin-like CAMs (ligand)

2) a & b integrins which

usually bind with Ig-
superfamily (ICAM &VCAM).

Endothelial cells
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  Chemotaxis: Migration (movement) of cells along a chemical
gradient (to the site of infection) by the different type of chemo-
attractants. Chemotaxis increases the affinity of Integrin to the
adhesion molecule, and increases the affinity of Integrins to the
adhesion molecule.

 Chemotactic factors:
a) Soluble bacteial products, e.g. N-formyl-methionine
termini
If there is bacteria which has special substance like N-
formyl-methionine termini in the infectious area, this
chemical substance attract leukocytes.

b) Complement system products, e.g. C5a (sequence of

proteins which activate after activation of any part).
c) Lipooxygenase pathway of arachidonic acid metabolism,
e.g. LTB4

d) Cytokines, e.g. IL-8

 Diapedesis: Transmigration of leukocytes between endothelial cells

at the intercellular junctions. This process is activated by PECAM-1
(CD31)/PECAM-1 interaction.

Q: Which type of leukocyte come first to the site of infection?

A: Neutrophils because we have acute inflammation at the beginning of
infection (predominance of neutrophils).

Effects of Chemotactic Factors on Leukocytes:

• Stimulates locomotion
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 • Degranulation of lysosomal enzymes
• Production of AA ( arachidonic acid )metabolites
• Modulation of the numbers and affinity of leukocyte adhesion
molecules

 Look at this diagram please

This is sequence of events following injury:

We take here as example which is myocardial infarction ( ‫)الجلطة القلبية‬
There is ischemic necrosis, and necrosis will initiate an inflammatory
process (transmigration of neutrophils because we have acute
inflammation). Follow me step by step to understand sequence of
events following the injury:
1) We start by edema (accumulation of fluid in the extracellular
matrix)
2) After that (exactly after two days ), we start with delivery of
neutrophils to the site of infection (acute inflammation)
3) Finally macrophages arrive to eliminate from necrotic (dead) cells

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 Q: What are the types of cells responsible for releasing
chemokines?
A: chemokines have different sources; they can be released from the
cells or plasma proteins (this will be explained later on.)

The Dr Focus on this diagram:

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We have here four types of receptors on the plasma membrane:

1) Seven a-helical transmembrane receptors(composed of seven

helixes (rod shaped)). These receptors have intracellular and
extracellular domains. In addition, these receptors bind with many
ligands (such as lipid mediators which can be bacterial product or any
kind of products from dead tissue, chemokines or soluble bacterial
product ( e.g N-formyl-methionyl peptides), they will bind to these
receptors (seven a-helical transmembrane receptor).

2) Toll-like receptor binds with microbe which has LPS

(LipoPolySacchride)
Ligands at specific region called CD14

3) Cytokine receptor: activated by different types of cytokines like

INF (InterFeroN).

4) Phagocytic receptor
I must remind you that all the information on the diagram above is very
important.

Really I did my best

Forgive me if there are any mistakes
Done by: Esraa Mofleh Al-Saudi (PharmD)

Thanks a lot for Basma Deeb & Bayan Abu khalil who gave me this chance
to participate in tfree3'

Also thanks a lot for Rawan Al-Zubaidi & Hanan al-Fayoomi who helped me
in this lecture

* Sama Girls Correction Team *

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