1. Leukocytes traffic from the bloodstream to sites of infection or injury by adhering to and transmigrating through endothelial cells lining blood vessels. 2. This process, called the leukocyte extravasation cascade, involves selectins, chemokines, and integrins that mediate tethering, rolling, activation, firm adhesion, and transmigration of leukocytes. 3. Leukocytes must open endothelial cell-cell junctions in order to transmigrate through the vessel wall in a multi-step process that is tightly regulated to recruit immune cells only when and where needed.

1. Leukocyte Adhesion and Trafficking

2. Leukocytes
Granulocyte Monocyte Lymphocyte Erythrocyte

3. Leukocytes
Granulocyte Monocyte Lymphocyte Erythrocyte
Innate Immunity
- immediate reaction
- not antigen-specific
Adaptive Immunity
- delayed reaction
- antigen-specific

5. Circulation
(blood)
Lymph and
Lymphatic
organs
Leukocyte Trafficking

6. Inflammatory leukocyte recruitment starts with a wound or infection

7. When a wound gets infected, the immune system needs to react

8. Blood contains immune cells that need to cross the vessel wall

9. Recruited immune cells „fight“ against invading pathogens

10. How do immune cells get across the vessel wall?

11. EM images of immune cells adhering to the vessel wall

12. EM images of a neutrophil migrating on endothelium

13. EM image of a neutrophil probing transendothelial cell contacts

14. Immune cells need to open endothelial cell contacts to transmigrate
90% of transmigration events are paracellular

15. Inflammation facilitates opening of endothelial cell contacts
Endothelial control layer Endothelial layer with TNF-α
VE-Cadherin actin
10 μm

16. Immune cells need to open endothelial cell contacts to transmigrate
90% of transmigration events are paracellular

17. Leukocyte trafficking to sites of inflammation

18. Leukocyte trafficking to sites of inflammation

19. Leukocyte trafficking to sites of inflammation

20. Leukocyte trafficking to sites of inflammation
activation
is local!

21. Leukocyte trafficking to sites of inflammation

22. Leukocyte trafficking to sites of inflammation

23. 1. Tethering and Rolling
3. Firm Adhesion
4. Transmigration
2. Activation
Bloodflow
Leukocyte – Endothelial Cell Contacts:
The “Adhesion Cascade“

24. 1. Tethering and Rolling
3. Firm Adhesion
4. Transmigration
2. Activation
Bloodflow
1. Selectins
Leukocyte – Endothelial Cell Contacts:
The “Adhesion Cascade“

25. 1. Tethering and Rolling
3. Firm Adhesion
4. Transmigration
2. Activation
Bloodflow
1. Selectins
2. Chemokines
Leukocyte – Endothelial Cell Contacts:
The “Adhesion Cascade“

26. 1. Tethering and Rolling
3. Firm Adhesion
4. Transmigration
2. Activation
Bloodflow
1. Selectins
2. Chemokines
3. Integrins
Leukocyte – Endothelial Cell Contacts:
The “Adhesion Cascade“

27. 1. Tethering and Rolling
3. Firm Adhesion
4. Transmigration
2. Activation
Bloodflow
1. Selectins
2. Chemokines
3. Integrins
4. Ig-SF molecules
(Immunoglobulin superfamily)
Leukocyte – Endothelial Cell Contacts:
The “Adhesion Cascade“

28. 1. Tethering and Rolling
3. Firm Adhesion
4. Transmigration
2. Activation
Bloodflow
1. Selectins
2. Chemokines
3. Integrins
4. Ig-SF molecules
(Immunoglobulin superfamily)
Leukocyte – Endothelial Cell Contacts:
The “Adhesion Cascade“

29. Selectins
L-selectin E-selectin P-selectin
C-type
lectin domain
binds carbohydrates
binds Ca 2+

30. Selectins

31. Regulation of E- and P-selectin expression
Lipopolysaccharide (LPS)
Cytokines: IL-1, TNF-a, INF-g
activation
Weibel-Palade bodies
surface expression:
within minutes
surface expression:
within hours
E- and P-selectin expression in endothelial cells is induced
If E- and P-selectin were constitutively expressed on endothelial cells,
neutrophils would constantly adhere because L-selectin on leukocytes
is constitutively expressed

32. Regulation of L-selectin expression: shedding
metalloproteinase
(sheddase)
naive
lymphocytes
activated
lymphocytes

33. Transcriptional regulation of L-selectin expression
naive
lymphocytes
memory
lymphocytes
DNA
mRNA
DNA
L-selectin

34. Function and Importance of E-, L-, and P-selectin
L-selectin E-selectin P-selectin
lymphocyte homing
to lymph nodes
inflammation inflammation inflammation
defective normal defective
E-/P- double KO mouse:
very strong defect
Function
Immune
system in
knock-out
mouse

35. PSGL-1:
P-selectin glycoprotein ligand-1
(leukocytes)
ESL-1:
E-selectin ligand-1
(granulocytes)
PNAd:
Peripheral node addressin
(PNAd is a collective name for
several L-selectin binding
proteins on HEV)
PNAd:
Selectin ligands

36. 1. Tethering and Rolling
3. Firm Adhesion
4. Transmigration
2. Activation
Bloodflow
1. Selectins
2. Chemokines
3. Integrins
4. Ig-SF molecules
(Immunoglobulin superfamily)
Leukocyte – Endothelial Cell Contacts:
The “Adhesion Cascade“

37. Chemokines
Chemokine =
chemotactic cytokine
Small secreted
proteins ( 8 – 12 kDa)
Characterised by
certain patterns of
cystein residues
Nomenclature:
CCL1 - CCL28
CXCL1 - CXCL17
CL1 - CL2
CX3CL
Synonyms:
e.g. RANTES, SDF-1, MCP-1
Source:
Many different cell types
e.g. leukocytes and
endothelial cells

38. Chemokine receptors:
proteins with
7 transmembrane domains
Mostly GPCR
Leukocyte surface
Chemokines

39. Chemokine
receptors
expressed on
leukocytes

40. Chemokine secretion

41. Chemokines

42. Proteoglycan
Heparan Sulfate:

43. 1. Tethering and Rolling
3. Firm Adhesion
4. Transmigration
2. Activation
Bloodflow
1. Selectins
2. Chemokines
3. Integrins
4. Ig-SF molecules
(Immunoglobulin superfamily)
Leukocyte – Endothelial Cell Contacts:
The “Adhesion Cascade“

44. a b a b a b
Integrins
Integrins

45. Integrins

46. Integrin activation

47. Integrin activation

48. Integrin affinity vs avidity

49. 1. Tethering and Rolling
3. Firm Adhesion
4. Transmigration
2. Activation
Bloodflow
1. Selectins
2. Chemokines
3. Integrins
4. Ig-SF molecules
(Immunoglobulin superfamily)
Leukocyte – Endothelial Cell Contacts:
The “Adhesion Cascade“
flattening of firmly
bound leukocytes

50. 1. Tethering and Rolling
3. Firm Adhesion
4. Transmigration
2. Activation
Bloodflow
1. Selectins
2. Chemokines
3. Integrins
4. Ig-SF molecules
(Immunoglobulin superfamily)
Leukocyte – Endothelial Cell Contacts:
The “Adhesion Cascade“

51. Molecules involved in Transmigration
Junctional Adhesion Molecule
Platelet Endothelial Cell Adhesion Molecule
Molecular class:
Integrin
Ig-SF
Mucin
(highly glycosylated)

52. N-CAM
Member of
Ig-Superfamily
Many others:
ICAM-1
VCAM-1
PECAM-1
JAM
...

53. Ig-SuperfamilyMembers during diapedesis
ICAM-1

54. Transendothelial Migration

55. Transmigration cup
Green: ICAM-1
Red: LFA-1
Carman & Springer, JCB, 2003

56. Transmigration:
Deformation of transmigrating cells

57. Transmigration:
Deformation of transmigrating cells

58. Transmigration:
Deformation of transmigrating cells II

59. Migration through the inflamed tissue:
Chemokines cause directional movement
2nd task:
chemotactic
gradient

60. The Leukocyte Extravasation Cascade: Summary
Schnoor et al., J. Immunol., 2015

61. Literature
Textbooks
• Janeway‘s Immunobiology, 8th Edition, 2012, Garland Science
• Lodish et al., Molecular Biology of the Cell, 6th Edition, 2007, Freeman
Reviews
• Ley et al., Nat. Rev. Immunol., 2007
• Voisin & Nourshargh, J. Innate Immun., 2013
• Kolaczkowska & Kubes, Nat. Rev. Immunol., 2013