The slides will give you an overall idea about the extravasation of leukocytes through the endothelial cells.

1. LEUKOCYTE EXTRAVASATION
By
Devi Priya Sugathan
Dept of Biochemistry &
Molecular Biology

3. LEUKOCYTES
• The cellular components of blood include erythrocytes,
leukocytes and platelets.
• Leukocytes are a heterogeneous group of nucleated cells.
• Normal human blood contains 4000-10000 leukocytes/µl.
• Leukocytes are classified onto five:
Neutrophils (40-75%)
Eosinophils (1-6%)
Granulocytes
Basophils (less than
1%)
Monocytes (2-10%)
Agranulocytes

4. TISSUE INJURY

5. TISSUE INJURY

6. Due to tissue injury, bacteria and other pathogens takes their entry on to
the injured site:
 The host cells will then begin to secrete endogenous molecules called
DAMP (Danger associated molecular patterns) which is recognized by
PRR (Pattern Recognition Receptors) present on the immune cells and
initiates innate immune responses.
 The pathogen provide exogenous signals called PAMP (Pattern
associated molecular patterns) recognized by PRR that would alert the
immune system to the presence of pathogen.

7.  The APC present near the injured site will
also phagocytose these bacteria and
present their peptide fragments on their
membrane to activate the T lymphocytes.
 Non phagocytic granulocytes near the
injured tissue will release histamine which
increases the blood vessel permeability and
smooth muscle activity.
 Cytokines are also secreted by neutrophils,
eosinophils, macrophage, activated T
cells….
 Cytokines secreted will direct the movement
of leukocytes to the injured area.

8. LEUKOCYTE EXTRAVASATION
• Also called Diapedesis.
• It is the movement of leukocytes out of the circulatory system and
towards the site of tissue damage or infection.
• Chemokines or chemoattractant cytokines are a family of pro
inflammatory mediators produced at the inflammatory site which induces
the mechanism of extravasation.

9. Leukocyte extravasation is orchestrated by the
combined action of cellular adhesion receptors and
chemotactic factors, and involves radical
morphological changes in both leukocytes and
endothelial cells.

10. Recruitment of Leukocytes to Inflammation Site
Multistep Model
1. Tethering (Capture)
2. Rolling
3. Activation
4. Firm Adhesion
5. Transendothelial
migration

11. TETHERING AND ROLLING
 The generated inflammatory chemicals, bind to seven
helix receptors on endothelial cells and stimulate the
fusion of cytoplasmic vesicles called Weibel-Palade
bodies with the plasma membrane.
 This exposes, P-Selectin formerly stored in the
vesicle membranes , on the cell surface facing the
blood.
 Selectins bind mucins that are constitutively exposed
on the surface of neutrophils, tethering them to the
surface.
 The bond forms and break rapidly, allowing the
neutrophils to roll along the surface of the
endothelium at rates greater then 10µm/s as the
blood wall pushes them along.

12. Interaction between Selectin and carbohydrate ligand present on the leucocytes.
• WBC and Platelets use selectins to interact with
vascular endothelial cells.
• Selectins are a group of transmembrane molecules,
expressed on the surface of leukocytes and activated
endothelial cells.
• Selectins contain an N-terminal extracellular
domain with structured homology to Ca-dependent
lectins, followed by a domain homologous to
epidermal growth factor and 2-9 consensus repeats
similar to sequences found in complementary
regulatory proteins.

13. • All selectin ligands are transmembrane glycoproteins
which present oligosaccharide structures to the
selectin.
• All three selectins recognize glycoproteins or
glycolipids containing the tetrasaccharide Sialyl-
Lewis.
• This tetrasaccharide is found on all circulating
myeloid cells and is composed of sialic acid, galactose,
fucose and N-acetyl galactosamine.
The low affinity nature of selectins is
what allows the characterestic rolling
action of leukocytes.

16. Activation & Firm Adhesion
• During the process of leukocyte rolling the contact of
leukocytes with the luminal endothelial surface allows
leukocytes to effectively ‘sense’ the endothelial
surface-bound chemokines.
• These surface-deposited chemokines are presented to
rolling leukocytes.
• By interaction with the G-protein coupled chemokine
receptors on leukocytes chemokines induce
intracellular signals leading to inside-out integrin
activation and firm leukocyte adhesion.

17.  Binding of chemokines to their receptors on leukocytes
results in the inside-out signalling activation of leukocyte β1-
integrins and the β2-integrins LFA-1 and Mac-l, that
mediate firm arrest of leukocytes.
 Neutrophils use both LFA-1 and Mac-l for adhesion.
 The endothelial counter-ligands for leukocyte integrins are
members of the immunoglobulin superfamily and include
(ICAM)-1–5, (VCAM)-1 and mucosal addressin cell adhesion
molecule-1 (MAdCAM-1).
 LFA-1 and Mac-1 bind to endothelial ICAMs such as ICAM-
1 and ICAM-2.
 ICAM-1 and ICAM-2 are constitutively expressed, and
ICAM-1 expression is further increased after endothelial
activation.
 In contrast, endothelial VCAM-1 is recognized by β-1
integrin receptors predominantly found on lymphocytes and
monocytes .

19. Transmigration
 Transmigration of leukocytes through the vascular endothelium can
take place in a paracellular or transcellular manner.
 Actin rearrangement ocuurs in leukocytes, pseudopods are formed and
then they begin to squeeze through the endothelial cells moving
towards higher cocentration of chemokines.

20. Paracellular
• Leukocytes encounter multiple endothelial cell junctional
molecules and molecular complexes during paracellular
transmigration.
• CD99 and CD31 (platelet endothelial cell adhesion molecule-1
(PECAM-1)) are expressed on endothelium and are enriched at
cell-cell lateral junctions.
• These molecules also are expressed on most leukocyte types.
• Both molecules interact through homophilic interactions, that is,
CD99 on one endothelial cell binds to the same molecule on
adjacent endothelial cells.
• Transmigrating leukocytes must cross endothelial tight junctions
(TJ) and adherens junctions (AJ), which contain numerous
proteins involved in selective permeability, growth control, and
cell-cell adhesion.
• The tight junction and its subjacent adherens junction (AJ)
constitute the apical junctional complex (AJC).

21. • Adherens junction include VE-Cadherin and tight junctions that incorporate members of the junctional adhesion
molecule(JAM) family, endothelial cell selective adhesion molecule(ESAM) and claudins.
• A number of other adhesion molecules are also enriched at borders of adjacent endothelial cells such as PECAM 1,
CD99,ICAM 2.
• These multiple junctional endothelial cell adhesion molecules are thought to recycle in a variety of intracellular
compartments or vesicles including the membranous lateral border recycling compartment (LBRC).
• LBRC supports leukocyte TEM through efficient recruitment of key molecules to sites of leukocyte diapedesis.
• These molecules will undergo various mechanisms and causes the loosening of the junctions between endothelial
cells to enable leukocyte damage.

22. Transcellular
 Integrin-mediated leukocyte adhesion can trigger clustering of endothelial
ICAM-1, and recruitment of VCAM-1 into membranous structures.
 Ligation of ICAM-1 and VCAM-1, along with CD9, CD151 or CD47 can
also elicit multiple signaling events in endothelial cells postulated to reduce
endothelial barrier properties.
 This includes increased intracellular Ca2+, reactive oxygen species (ROS)
generation, and activation of p38 mitogen-activated protein kinase (MAPK).
 In addition, ICAM-1 ligation can result in tyrosine phosphorylation of key
endothelial cell junctional molecules through activation of endothelial
proline-rich tyrosine kinase 2 (Pyk2) and Src kinase.
 These responses can couple to the triggering of RhoA (a small GTPase
involved in regulation of actin cytoskeletal networks) and its downstream
Rho-associated protein kinase (ROCK), as well as to endothelial myosin light-
chain kinase.
 These responses finally result in the formation of intracellular channels
through which the leukocytes enter.

23. An overview