Leukocyte extravasation

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

2. 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 Lymphocytes (20-45%) They are made in the bone marrow and are found in lymph and blood. They play major role in phagocytosis and immunity and therefore in defense against infection.

3. TISSUE INJURY

4. TISSUE INJURY Production and release of inflammatory mediators Chemokines Adherence Migration Phagocytoses Vasodilation Increased capillary permeability Vasoactive mediators Chemotactic mediators

5. 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.

6. 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.

7. 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 .

8. 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.

9. Recruitment of Leukocytes to Inflammation Site Multistep Model Tethering (Capture) Rolling Activation Firm Adhesion Transendothelial migration

10. 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.

11. 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.

12. 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.

13. 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.

14. 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 .

15. 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 .

16. 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).

17. 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.

18. 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 Ca 2+, 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.

19. An overview